CN117083303A

CN117083303A - Binding molecules with high affinity and/or specificity and methods of making and using the same

Info

Publication number: CN117083303A
Application number: CN202180094790.2A
Authority: CN
Inventors: 尹轶苑; 罗宇龄; A·格日博夫斯基
Original assignee: Alama Bioscience Co ltd
Current assignee: Alama Bioscience Co ltd
Priority date: 2020-12-31
Filing date: 2021-12-29
Publication date: 2023-11-17

Abstract

In some aspects, provided herein are binding molecules, including co-conjugates, having high affinity and/or high specificity for a target. In other aspects, provided herein are compositions, methods of making, and methods of using binding molecules taught herein, such as diagnostic and therapeutic methods of use involving co-conjugates taught herein.

Description

Binding molecules with high affinity and/or specificity and methods of making and using the same

Cross Reference to Related Applications

The present application claims priority and benefit from U.S. provisional patent application No. 63/133,005, filed on 31, 12, 2020, and U.S. provisional patent application No. 63/133,020, filed on 31, 12, 2020, each of which is incorporated herein by reference in its entirety.

Technical Field

In some aspects, the application relates to conjugate molecules, such as co-conjugates, having high affinity and/or high specificity for a target molecule. In other aspects, methods of making conjugate molecules, such as co-conjugates, methods of using the conjugate molecules, such as co-conjugates, e.g., diagnostic and therapeutic methods, and compositions comprising the conjugate molecules, such as co-conjugates, are also provided.

Background

Antibodies and other binding molecules are useful in a number of fields, including those involving molecular detection, diagnosis, and therapeutic methods. Producing such binding molecules with desirable characteristics (such as size and immunogenicity, let alone desirable binding affinity and specificity) remains a challenge in the art.

Disclosure of Invention

In some aspects, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein optionally the second binding moiety is a second antibody moiety comprising a variable domain having an N-terminal truncation ("N-terminal truncated variable domain") of the antibody, and wherein the first binding moiety is linked to the second binding moiety, optionally via a linker, through the N-terminus of the N-terminal truncated variable domain of the antibody. In some embodiments, the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain"). In some embodiments, the co-conjugate comprises a linker. In some embodiments, the co-conjugate comprises a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain"), and wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the N-terminal truncated antibody variable domain.

In some embodiments, the affinity of the co-conjugate for binding to the second target site is at least about 3-fold greater than the affinity of the control co-conjugate. In some embodiments, the control co-conjugate comprises an N-terminally truncated antibody variable domain that does not have an N-terminally truncated antibody variable domain (e.g., the N-terminally truncated antibody variable domain of the second binding portion of the co-conjugate described herein).

In some embodiments, the first target site and the second target site are non-overlapping binding sites on the target molecule. In some embodiments, the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold greater than the affinity of a control co-conjugate comprising an antibody variable domain that does not have an N-terminal truncation.

In some embodiments, the first binding moiety is a first antibody moiety. In some embodiments, the first antibody moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragments. In some embodiments, the first antibody moiety is a single domain antibody.

In some embodiments, the second antibody moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragments. In some embodiments, the N-terminal truncated antibody variable domain is a truncated VH or a truncated VL domain. In some embodiments, the second antibody moiety is Single domain antibodies. In some embodiments, the N-terminal truncated antibody variable domain is a truncated V _H H domain.

In some embodiments, the first binding moiety comprises a first V _H An H domain; wherein the second binding moiety comprises a second V having an N-terminal truncation _H H domain (truncated V) _H H domain "), wherein the first V _H The C-terminal end of the H domain is connected to the second V via a linker _H N-terminal ligation of H domains.

In some embodiments, the N-terminal truncation of the N-terminally truncated antibody variable domain is from about 1 to about 25 amino acids. In some embodiments, the N-terminal truncation of the N-terminal truncated antibody variable domain is 1 amino acid.

In some embodiments, the linker is a peptide linker. In some embodiments, the C-terminal amino acid of the peptide linker directly linked to the N-terminal truncated antibody variable domain is G.

In some embodiments, the three C-terminal amino acids of the peptide linker directly linked to the N-terminal truncated antibody variable domain are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G. In some embodiments, the three C-terminal amino acids of the peptide linker directly linked to the N-terminal truncated antibody variable domain are selected from the group consisting of: GVG, DSG, LLG, VSG, PPG, SCG, TLG and NPG.

In some embodiments, the linker comprises (G _x S _y ) _n Wherein x is 1 to 5, y is 0 to 5, and n is 1 or greater. In some embodiments, the linker comprises [ EAAAK] _n Wherein n is 1 or greater. In some embodiments, the linker is no more than about 40 amino acids in length. In some embodiments, the linker comprises [ EEEEKKKK] _n Wherein n is 1 or greater. In some embodiments, the linker comprises [ AP] _n Wherein n is 1 or greater.

In some embodiments, the truncated variable domain is from an antibody variable domain of either IgG, igA, igE, igM or IgD type.

In some embodiments, the co-conjugate further comprises a third binding moiety that specifically recognizes a third target site. In some embodiments, the third binding moiety is a third antibody moiety. In some embodiments, the third antibody portion comprises an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain"). In some embodiments, the third antibody moiety is linked to the second antibody moiety via a linker through the N-terminus of the N-terminal truncated antibody variable domain of the third antibody moiety.

In some embodiments, the third antibody portion is linked to the fourth binding portion via a linker through the N-terminus of the N-terminal truncated antibody variable domain of the third antibody portion.

In some embodiments, the co-conjugate is an antibody comprising an Fc region.

In some embodiments, the co-conjugate is a chimeric antigen receptor ("CAR").

In other aspects, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain; wherein the first binding moiety is linked to the second binding moiety via a peptide linker through the N-terminus of the N-terminally truncated antibody variable domain; wherein the three C-terminal amino acids of the peptide linker directly linked to the antibody variable domain of the second binding moiety are X ₁ -X ₂ -X ₃ Wherein X is ₁ Is any amino acid; x is X ₂ K, R, Y, M, G or N; and X is ₃ R, G, Y or P.

In some embodiments, the affinity of the co-conjugate to bind to the second target site is at least about 3-fold greater than the affinity of the linker control co-conjugate.

In some embodiments, the first target site and the second target site are non-overlapping binding sites on the target molecule.

In some embodiments, the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold greater than the affinity of the linker control co-conjugate.

In some embodiments, the second antibody moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragments. In some embodiments, the antibody variable domain is a VH or VL domain. In some embodiments, the second antibody moiety is a single domain antibody. In some embodiments, the antibody variable domain is V _H H domain.

In some embodiments, the first binding moiety comprises a first V _H An H domain; wherein the second binding moiety comprises a second V _H An H domain, wherein the first V _H The C-terminal end of the H domain is linked to the second V via a peptide linker _H N-terminal ligation of H domains.

In some embodiments, the three C-terminal amino acids of the peptide linker directly linked to the N-terminal truncated antibody variable domain are selected from the group consisting of: GVG, DSG, LLG, VSG, PPG, SCG, TLG and NPG.

In some embodiments, the co-conjugate further comprises a third binding moiety that specifically recognizes a third target site. In some embodiments, the third binding moiety is a third antibody moiety. In some embodiments, the third antibody portion comprises an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain"). In some embodiments, the third antibody moiety is linked to the second antibody moiety via a linker through the N-terminus of the N-terminal truncated antibody variable domain of the third antibody moiety. In some embodiments, the third antibody portion is linked to the fourth binding portion via a linker through the N-terminus of the N-terminal truncated antibody variable domain of the third antibody portion.

In some embodiments, the co-conjugate is an antibody comprising an Fc region.

In some embodiments, the co-conjugate is a chimeric antigen receptor ("CAR").

In other aspects, a library is provided comprising a plurality of co-binders or a plurality of polynucleotides encoding a plurality of co-binders, each co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, wherein the first binding moiety is linked to the second binding moiety via a peptide linker through the N-terminus of the antibody variable domain, wherein at least two co-binders in the library differ from each other in peptide linker sequence.

In some embodiments, the antibody variable domain has an N-terminal truncation ("N-terminally truncated antibody variable domain"). In some embodiments, at least two co-binders in the library differ from each other in terms of N-terminal truncation of the antibody variable domain.

In some embodiments, the diversity of the library is at least about 5000.

In some embodiments, substantially all of the plurality of co-conjugates comprise the same first binding moiety and second binding moiety.

In some embodiments, at least two of the plurality of co-conjugates comprise different first binding moieties and/or second binding moieties.

In other aspects, methods of screening for co-conjugates that specifically bind to a second target site with a desired affinity are provided, the methods comprising: (1) Contacting the library described herein with a target molecule comprising a second target site to form a complex between a co-conjugate that specifically binds the target molecule and the target molecule, and (2) identifying the co-conjugate that binds the second target site with a desired affinity.

In other aspects, methods of screening for co-conjugates that specifically bind a target molecule with a desired affinity are provided, the methods comprising: (1) Contacting the library described herein with a target molecule to form a complex between a co-conjugate that specifically binds the target molecule and the target molecule, and (2) identifying the co-conjugate that binds the target molecule with a desired affinity.

In other aspects, methods of increasing the binding affinity of a control co-binder for specifically binding a target molecule are provided, wherein the control co-binder comprises a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the antibody variable domain, wherein the control co-binder comprises a full-length antibody variable domain, wherein the binding affinity of the control co-binder to the second target site is lower than the affinity of the second antibody moiety in the free state, the method comprising obtaining a co-binder having an N-terminal truncation at the antibody variable domain of the second antibody moiety as compared to the control co-binder.

All applications, publications, patents, and other references cited herein are incorporated by reference in their entirety into the GenBank citations. In case of conflict, the specification, including definitions, will control.

Drawings

FIG. 1 depicts an exemplary algorithm for determining truncations or deletions of N-terminal residues in an antibody variable region.

FIG. 2 depicts another exemplary algorithm for determining truncations or deletions of N-terminal residues in an antibody variable region.

Figures 3A-3D depict exemplary sources of binding energy loss when two binding moieties are linked together. FIG. 3E depicts 7D12 and 9G 8V binding to EGFR _H H, wherein the cetuximab crystal structures overlap for comparison.

Fig. 4A depicts a strategy to improve the binding properties of a co-conjugate by modifying the linker attachment point between the linker and the antigen. FIGS. 4B-4C depict SDS-PAGE gels from purified proteins having a truncated (4B) HuL6-7D12 variant at the N-terminus of 7D12 and a truncated (4C) HuL6-9G8 variant at the N-terminus of 9G 8. All variants in fig. 4B and 4C were expressed and purified in the same manner except for the non-truncated co-conjugate.

FIG. 5 depicts a library design of co-binders with 3 amino acid randomization at the C-terminus of the linker with or without the first amino acid of the second binder.

FIG. 6A depicts the consensus sequence of each library, as described in Table 15 and the accompanying text, using Weblogo software for motif analysis of the first 20 most abundant sequences (shooks et al Genome Res.6 month 2004; 14 (6): 1188-90). FIG. 6B depicts affinity (K) between selected constructs with linker end modifications and human EGFR _D ) Yeast display and SPR measurement of (c).

FIG. 7A depicts a library design of co-binders with 3 amino acid randomization at the N-terminus and 2 amino acid randomization at the C-terminus of the linker, where the last C-terminal amino acid of the linker is glycine. Libraries utilized 4 different linker motifs: EAAAK and E4K4 repeats, AP repeats, and G3-4S repeats. FIG. 7B depicts the consensus sequence of each library, as described in Table 16 and the accompanying text, using Weblogo software for motif analysis of the first 20 most abundant sequences (shooks et al Genome Res.6 month 2004; 14 (6): 1188-90). FIG. 7C depicts linker length enrichment in screens as described in Table 16 and the accompanying text.

FIG. 8 depicts SPR affinity measurements of engineered co-binders for murine EGFR-Fc and human EGFR-Fc mutants (L325V, S340A).

Fig. 9 depicts a schematic of a method for discovering co-conjugates with synergistic co-binding.

FIG. 10A shows that anti-EGFR VHH yeast surface display library SB0 was constructed and single conjugate selection was performed using FACS. Fig. 10B depicts the selection of high affinity co-binders from a CB0 co-binder library using FACS.

Fig. 11 shows downregulation of EGF-induced EGFR signaling by the co-conjugate.

FIG. 12A shows a sensorgram of injection of 81nM 1E10 EGFR binder followed by injection of 81nM 15E2 EGFR binder on immobilized EGFR-Fc. FIG. 12B shows a sensorgram of injection of 81nM 7D12-9G8 EGFR conjugate on immobilized EGFR-Fc followed by injection of 81nM 15E2 EGFR conjugate. FIG. 12C shows a sensorgram of injection of 81nM 7D12-9G8 EGFR co-binder onto immobilized EGFR-Fc followed by injection of 81nM 1E10 EGFR binder.

FIG. 13 shows a graph of the distance between the N-terminal end of the VHH and Fab domains and the antigen surface. Each individual dot represents a unique structure selected from PDB.

Figure 14 shows a graph of affinity for anti-EGFR (filled circles) and anti-HIV p24 (open squares) co-binders and single binders.

Figure 15 shows a graph of the affinity of the co-binders for 14 different targets and the affinity of the conventional antibodies for the targets.

Detailed Description

Provided herein are conjugate molecules comprising a second binding moiety that specifically recognizes a target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncation"). The present disclosure is based on the unexpected discovery by the inventors that such conjugate molecules, such as co-conjugates, comprising a second binding moiety having an N-terminally truncated antibody variable domain provide a platform technology for conjugate molecules having high affinity and specificity. In addition, the second binding moiety having an N-terminally truncated antibody variable domain can be combined with various other features, including linkers, first binding moieties, labels, and/or drugs, to produce the desired conjugate molecule. Furthermore, the design of the conjugate molecules contemplated herein enables production, for example, via polypeptide expression, without the need for post-production synthetic steps that typically result in yield loss and contamination.

Thus, in some aspects, provided herein is a conjugate molecule, e.g., a target polypeptide, comprising a second binding moiety that specifically recognizes a target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain").

In other aspects, provided herein is a co-conjugate comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising a variable domain of an antibody having an N-terminal truncation ("N-terminal truncated variable domain of an antibody"), wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the N-terminal truncated variable domain of the antibody.

In other aspects, provided herein is a co-conjugate comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain; wherein the first binding moiety is linked to the second binding moiety via a peptide linker through the N-terminus of the N-terminally truncated antibody variable domain; wherein the three C-terminal amino acids of the peptide linker directly linked to the antibody variable domain of the second binding moiety are X ₁ -X ₂ -X ₃ Wherein X is ₁ Is any amino acid; x is X ₂ K, R, Y, M, G or N; and X is ₃ R, G, Y or P. In some embodiments, X ₁ -X ₂ -X ₃ X in (2) ₃ G.

In other aspects, provided herein is a library comprising a plurality of co-binders or a plurality of polynucleotides encoding a plurality of co-binders, each co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, wherein the first binding moiety is linked to the second binding moiety via a peptide linker through the N-terminus of the antibody variable domain, wherein at least two co-binders in the library differ from each other in peptide linker sequence.

In other aspects, provided herein are methods of screening for co-binders that specifically bind to a second target site with a desired affinity, the methods comprising: (1) Contacting the library described herein with a target molecule comprising a second target site to form a complex between a co-conjugate that specifically binds the target molecule and the target molecule, and (2) identifying the co-conjugate that binds the second target site with a desired affinity.

In other aspects, provided herein are methods of screening for co-binders that specifically bind to a target molecule with a desired affinity, the methods comprising: (1) Contacting the library described herein with a target molecule to form a complex between a co-conjugate that specifically binds the target molecule and the target molecule, and (2) identifying the co-conjugate that binds the target molecule with a desired affinity.

In other aspects, provided herein are methods of increasing the binding affinity of a control co-conjugate to a target molecule, wherein the control co-conjugate comprises a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the antibody variable domain, wherein the control co-conjugate comprises a full-length antibody variable domain, wherein the binding affinity of the control co-conjugate to the second target site is lower than the affinity of the second antibody moiety in the free state, the method comprising obtaining a co-conjugate having an N-terminal truncation at the antibody variable domain of the second antibody moiety as compared to the control co-conjugate.

I. Definition of the definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For purposes of explaining the present specification, the following description of terms will apply, and terms used in the singular will also include the plural, if appropriate, and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. If any description of a stated term conflicts with any document incorporated by reference herein, the description of the term set forth below controls.

Techniques and procedures described or referenced herein include those techniques and procedures generally well understood and/or commonly used by those skilled in the art using conventional methods, such as, for example, sambrook et al Molecular Cloning: A Laboratory Manual (3 rd edition 2001); current Protocols in Molecular Biology (Ausubel et al, 2003); therapeutic Monoclonal Antibodies: from Bench to Clinic (An et al 2009); monoclonal Antibodies: methods and Protocols (Albitar 2010); and Antibody Engineering volumes 1 and 2 (Kontermann and Dubel, 2 nd edition 2010).

The term "co-binder/co-binders/binders" means a molecule having at least two binding moieties (i.e., a first binding moiety comprising a first paratope and a second binding moiety comprising a second paratope) that bind to a non-overlapping epitope of a target molecule or a target complex (e.g., a protein complex). In some embodiments, the first binding moiety and the second binding moiety bind simultaneously to a non-overlapping epitope of a target molecule or a target complex (e.g., a protein complex). In some embodiments, at least two binding moieties bind to non-overlapping epitopes of one target molecule or one target complex (e.g., a protein complex) simultaneously. The co-conjugates described herein comprise at least two binding moieties, e.g., any of 2, 3, 4, 5, 6, or 7 or more binding moieties. In some embodiments, two or more binding moieties on one binding molecule are identical. In some embodiments, two or more binding moieties on one binding molecule are different. In some embodiments, the co-conjugate has two binding moieties, and the two epitopes recognized by the co-conjugate are non-overlapping and different. In some embodiments, the co-conjugate has two binding moieties, and the two epitopes recognized by the co-conjugate are located close to each other, yet still allow sufficient space to accommodate the linker of the co-conjugate. In some embodiments, the co-conjugate has two binding moieties and the first epitope and the second epitope have a distance of no more than 150 angstroms. In some embodiments, the co-conjugate has two binding moieties and the first epitope and the second epitope have a distance of no more than 100 angstroms, no more than 50 angstroms, no more than 40 angstroms, no more than 30 angstroms, no more than 20 angstroms, no more than 15 angstroms, no more than 10 angstroms, or no more than 5 angstroms. For linear epitopes on the target peptide or target protein, the distance between any two epitopes can be within 200 amino acids of each other. In some embodiments, the co-conjugate has two binding moieties and the distance between the two epitopes can be within 200 amino acids, 150 amino acids, 100 amino acids, 50 amino acids, 40 amino acids, 30 amino acids, 20 amino acids, 15 amino acids, or 10 amino acids of each other. In some embodiments, the co-conjugate has two binding moieties, and the two epitopes recognized by the co-conjugate are selected such that the two binding interactions are cooperative and synergistic, and do not interfere with each other. The co-binders have a higher binding affinity and a higher binding specificity than typical bivalent antibodies, because for example there is a superposition of two paratope-epitope binding interactions.

As used herein, the term "binding moiety" refers to a molecule or portion of a molecule that binds to a specific target molecule. The binding moiety may include a protein, peptide, nucleic acid, carbohydrate, lipid, or small molecular weight compound. In some embodiments, the binding moiety comprises an antibody. In some embodiments, the binding moiety comprises an antigen binding fragment of an antibody. In some embodiments, the binding moiety comprises an antibody or antigen binding fragment thereof. In some embodiments, the binding moiety comprises a heavy chain variable region of an antibody. In some embodiments, the binding moiety comprises a light chain variable region of an antibody. In some embodiments, the binding moiety comprises a variable region of an antibody. In some embodiments, the binding moiety comprises an antibody mimetic. In some embodiments, the binding moiety comprises a small molecular weight component. In some embodiments, the binding molecule has only one binding moiety. In some embodiments, the binding molecule has two binding moieties. In some embodiments, the binding molecule has three or more binding moieties. In some embodiments, two or more binding moieties on one binding molecule are identical. In some embodiments, two or more binding moieties on one binding molecule are different. For example, a binding molecule may have two binding moieties, both of which are antigen binding fragments, such as VHH. For another example, the binding molecule may also have two binding moieties, one being a VHH and the other being a scFv.

As used herein, the term "paratope" is a portion of a binding moiety that recognizes and binds a target molecule. The paratopes of antibodies, also known as "antigen binding sites", can be identified by conventional methods for epitopes and paratopes of a given target molecule/binding molecule pair (e.g., ag/Ab). For example, the target molecule and the binding molecule may bind to form a complex, which may crystallize. The crystal structure of the complex can be determined, for example, by X-ray diffraction and used to identify specific interaction sites between the target molecule/binding molecule, i.e. epitope/paratope.

An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds, e.g., a localized region of the surface of an antigen (e.g., EGFR), which is capable of binding to one or more antigen binding regions of an antibody and has antigenic or immunogenic activity in an animal, such as a mammal (e.g., a human), capable of eliciting an immune response. An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. Epitopes having antigenic activity are part of an antibody-binding polypeptide, as determined by any method well known in the art, including, for example, by an immunoassay. An epitope is not necessarily immunogenic. Epitopes are generally composed of chemically active surface groups of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural features as well as specific charge characteristics. The antibody epitope may be a linear epitope or a conformational epitope. Linear epitopes are formed by contiguous amino acid sequences in proteins. Conformational epitopes are formed by discrete amino acids in the protein sequence, but they are clustered together when the protein is folded into its three-dimensional structure. An induced epitope is formed when the three-dimensional structure of a protein is in an altered conformation, e.g., upon activation or binding of another protein or ligand. Typically, an antigen has several or many different epitopes and may respond to many different antibodies.

The term "binding protein" refers to a protein comprising: a moiety that binds to a target antigen (e.g., EGFR) (e.g., one or more binding regions such as CDRs) and optionally a scaffold or framework moiety (e.g., one or more scaffold or framework regions) that allows the binding moiety to adopt a conformation that promotes binding of the binding protein to the target polypeptide, fragment, or epitope thereof. Examples of such binding proteins include antibodies, such as human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, single chain antibodies, diabodies, triabodies, tetrabodies, fab fragments, F (ab') ₂ Fragments, igD antibodies, igE antibodies, igM antibodies, igG1 antibodies, igG2 antibodies, igG3 antibodies, or IgG4 antibodies, and fragments thereof. The binding protein may comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced, for example, to stabilize the three-dimensional structure of the binding protein, as well as fully synthetic scaffolds comprising, for example, biocompatible polymers. See, e.g., korndorfer et al 2003,Proteins:Structure,Function,and Bioinformatics 53 (1): 121-29; and Roque et al, 2004, biotechnol. Prog.20:639-54. In addition, peptide antibody mimics ("PAMs") may be used, as well as scaffolds based on antibody mimics that utilize fibronectin components as scaffolds. In the context of the present disclosure, a binding protein is considered to specifically bind or selectively bind to a target, e.g., when the dissociation constant (K _D ) Is less than or equal to 10 ^-5 M. In some embodiments, the binding protein (e.g., co-conjugate and antibody) may be about 10 ^-7 M to about 10 ^-12 K of M _D Specifically binds to the target. In some embodiments of the present invention, in some embodiments,when K is _D Is less than or equal to 10 ^-8 M or K _D Is less than or equal to 10 ^-9 M, binding proteins (e.g., co-binders and antibodies) can specifically bind to the target with high affinity. In one embodiment, such as byThe binding proteins (e.g., co-conjugates and antibodies) can be 1x10 as measured ^-9 M to 10x10 ^-9 K of M _D Specifically bind to purified human targets. In another embodiment, binding proteins (e.g., co-conjugates and antibodies) can specifically bind to purified human targets, K _D Is 0.1X10 × 10 ^-9 M to 1X10 ^- ⁹ M, e.g. by KinExA ^TM (Sapidyne, boise, ID). In yet another embodiment, binding proteins (e.g., co-conjugates and antibodies) specifically bind to a target expressed on a cell, K _D Is 0.1X10 × 10 ^-9 M to 10X10 ^-9 M. In certain embodiments, the binding moiety (e.g., co-conjugate and antibody) specifically binds to a target expressed on a cell, K _D Is 0.1X10 × 10 ^- ⁹ M to 1X10 ^-9 M. In some embodiments, binding proteins (e.g., co-conjugates and antibodies) specifically bind to a target expressed on a cell, K _D Is 1X 10 ^-9 M to 10X 10 ^-9 M. In certain embodiments, binding proteins (e.g., co-conjugates and antibodies) specifically bind to a target expressed on a cell, K _D Is about 0.1X10 × 10 ^-9 M, about 0.5X10) ^-9 M, about 1X 10 ^-9 M, about 5X 10 ^-9 M, about 10X 10 ^-9 M or any range or interval thereof.

The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense and specifically covers, for example, a single monoclonal antibody (including agonists, antagonists, neutralizing antibodies, full length or intact monoclonal antibodies), an antibody composition having a multi-epitope or mono-epitope specificity, a polyclonal antibody, a monovalent antibody, a multivalent antibody, a multispecific antibody (e.g., bispecific antibodies so long as they exhibit the desired biological activity) The multispecific antibodies are formed from at least two intact antibodies, single chain antibodies, and antibody fragments, as described below. Antibodies can be human, humanized, chimeric and/or affinity matured, as well as antibodies from other species such as mice and rabbits, and the like. Thus, the term "antibody" includes a variety of antibody structures, including, but not limited to, polyclonal antibodies, recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, double antigen site antibodies, bispecific antibodies, multispecific antibodies, diabodies, triabodies, tetrabodies, single chain Fv (scFv) antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity. The term "antibody" is intended to include a polypeptide product of a B cell within an immunoglobulin-type polypeptide that is capable of binding a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair of polypeptide chains has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain comprises a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain comprises a constant region. See, e.g., antibody Engineering (Borrebaeck, 2 nd edition 1995); and Kuby, immunology (3 rd edition 1997). The term "whole antibody" or "full length antibody" refers to an antibody having a structure substantially similar to the structure of a natural antibody. This includes, for example, antibodies comprising two light chains and two heavy chains, each light chain comprising a variable region and a light chain constant region (CL), each heavy chain comprising a variable region and at least heavy chain constant regions CH1, CH2 and CH3. In particular embodiments, the specific molecular antigen may be bound by an antibody provided herein. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments (e.g., antigen binding fragments) of any of the foregoing, which refer to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), fab fragments, F (ab') fragments, F (ab) ₂ Fragments, F (ab') ₂ Fragment, disulfide-linked Fv (dsFv), disulfide-linkedThe attached scFv (dsscFv), fd fragments, fv fragments, diabodies, triabodies, tetrabodies and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen binding domains or molecules that contain an antigen binding site that binds an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found in Harlow and Lane antibodies: ALaboratory Manual (1989); mol. Biology and Biotechnology: AComprehensive Desk Reference (Myers, 1995); huston et al, 1993,Cell Biophysics 22:189-224; pluckthun and Skerra,1989, meth. Enzymol.178:497-515; and Day, advanced Immunochemistry (2 d edition 1990). Antibodies provided herein can be of any class (e.g., igG, igE, igM, igD and IgA) or subclass (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) of immunoglobulin molecules. The antibody may be an agonistic antibody or an antagonistic antibody. Provided herein are antagonistic antibodies against a target antigen, such as EGFR.

An "antigen" is a predetermined antigen to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide.

The terms "antigen binding fragment," "antigen binding domain," "antigen binding region," "antibody fragment," and similar terms refer to a portion of an antibody that comprises amino acid residues that interact with an antigen and confer specificity and affinity to the antigen (e.g., CDR) to the binding agent. Examples of antigen binding fragments include, but are not limited to, fab ', F (ab') 2, fv, single chain antibody molecules (e.g., scFv), disulfide-linked Fv (dsFv), disulfide-linked scFv (dsscFv), fd fragments, diabodies, triabodies, tetrabodies, minibodies, diabody (DVD), single variable domain antibodies (e.g., camelbody, alpaca antibody), single variable domain of heavy chain antibodies (VHH), and multispecific antibodies formed from antibody fragments.

As used herein, a light chain variable region (VLAb) encompasses all light chain variable region subtypes, including, for example, kappa (kappa) light chain variable region (kappa VLAb) and/or lambda (lambda) light chain variable region (lambda VLAb), unless specifically indicated otherwise herein. Heavy chain variable region (VHAb) as used herein encompasses all heavy chain variable region subtypes, including, for example, gamma, delta, alpha, mu and/or epsilon heavy chain variable regions, unless specifically stated otherwise herein. In some embodiments, the VLAb is followed by an arabic number to label the different VLAb. In some embodiments, the VHAb is followed by an arabic number to tag a different VHAb.

As used herein, the term "antibody mimetic" refers to a molecule that can specifically bind to an antigen as an antibody, but is structurally unrelated to an antibody. The antibody mimetic is typically an artificial peptide having a molecular weight of about 2 to 20 kDa. Nucleic acids and small molecules are also sometimes referred to as antibody mimics. Antibody mimics known in the art include affibodies (affibodies), affilin, affimer, affitin, alpha bodies (alphabodies), anticalin, aptamers, affibodies (avimers), DARPin, fynomer, kunitz domain peptides, monomers (monobodies), and nanocamp.

As used herein, the term "antagonist," when used in reference to the function of an antigen, is intended to mean a molecule capable of inhibiting, reducing, attenuating, reducing, or completely blocking one or more biological activities or functions of the antigen. Antagonists of antigen function include molecules that block, inhibit, attenuate or reduce antigen-mediated or antigen-dependent signaling in cells expressing the antigen. Antagonists of antigen function also include molecules that block, inhibit, attenuate or reduce antigen signaling, including downstream signaling induced by the linkage or engagement between an antigen and its ligand. In some examples, antagonists of an antigen also include molecules that block, inhibit, attenuate, or reduce binding of the antigen to the native antigen binding molecule. In other examples, antagonists of the antigen additionally include molecules that block, inhibit, or reduce binding of the antigen to the antigen ligand. An "antagonist" of an antigen is "antagonistic" to the function of the antigen. In some embodiments, provided herein are antagonistic co-conjugates. In some embodiments, provided herein are co-conjugates that are EGFR antagonists.

When used in reference to the function of an antigen, "blocking" or "neutralizing" or "antagonizing" a co-conjugate is intended to mean a co-conjugate that binds to an antigen and acts as an antagonist of the activity or function of the antigen. For example, a blocking co-conjugate or antagonist co-conjugate may substantially or completely inhibit the biological activity of an antigen or binding of an antigen to its ligand. In some embodiments, provided herein are blocking co-conjugates. In some embodiments, provided herein are EGFR blocking co-conjugates.

The term "binding" refers to interactions between molecules, including, for example, interactions between a binding molecule (e.g., a co-conjugate or binding moiety) and a target molecule to form a complex. The interactions may be, for example, non-covalent interactions, including hydrogen bonding, ionic bonding, hydrophobic interactions, and/or van der Waals interactions. A complex may also include two or more molecules that are bound together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interaction between a single binding molecule and a single epitope of a target molecule is the affinity of the binding molecule or binding moiety for that epitope. Dissociation rate (k) of binding molecules for monovalent antigens _off ) And binding rate (k) _on ) Ratio (k) _off /k _on ) Is the dissociation constant K _D Which is inversely proportional to the affinity. K (K) _D The lower the value, the higher the affinity of the antibody. K (K) _D The value of (2) varies depending on the different complexes of the binding molecule and the target molecule and depends on k _on And k _off . Dissociation constant K of the binding molecules provided herein _D May be determined using any of the methods provided herein or any other method known to those of skill in the art. The affinity at one binding site does not always reflect the actual strength of interaction between the binding molecule and the target molecule. When a target molecule containing multiple epitopes is contacted with a binding molecule containing multiple binding moieties that bind the target molecule, the interaction of the binding molecule with the target molecule at one site will increase the likelihood of a reaction at a second site. The strength of such multiple interactions between multivalent binding molecules and target molecules is referred to as affinity. Affinity of binding molecules compared to the affinity of a single binding siteThe force may be a better measure of its binding capacity. For example, high avidity may compensate for low affinity, as sometimes found in pentameric IgM antibodies, which may have lower affinity than IgG due to their multivalent nature, but have high avidity of IgM, enabling them to bind antigen effectively.

As used herein, the term "specific binding" refers to more frequent, more rapid, longer duration, greater affinity, greater strength of interaction, less frequent dissociation, slower dissociation rate, or shorter dissociation duration of a binding molecule or binding moiety with a particular epitope or target molecule, or some combination or permutation of the foregoing, as compared to the alternative. Binding molecules (e.g., co-conjugates, binding moieties, antibodies, or antigen binding fragments thereof) that specifically bind to a target molecule (e.g., an antigen) can be identified by, for example, an immunoassay, a Radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), SPR (e.g., biacore), or other techniques known to those of skill in the art. Typically, the specific response will be at least twice the background signal or noise and may be more than 10 times the background. See, e.g., paul et al, 1989,Fundamental Immunology Second Edition,Raven Press,New York, pages 332-336 for discussion of antibody specificity. Binding molecules (e.g., co-conjugates, binding moieties, antibodies, or antigen binding fragments thereof) that specifically bind to a target molecule may bind to the target molecule with a higher affinity than it does to a different molecule. In some embodiments, a binding molecule (e.g., a co-conjugate, binding moiety, antibody, or antigen binding fragment thereof) that specifically binds a target molecule may bind the target molecule with an affinity that is at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, or at least 100-fold greater than its affinity for a different molecule. In some embodiments, the binding agent that specifically binds to a particular target molecule binds to a different molecule with such low affinity that binding cannot be detected using the assays described herein or known in the art. Specific binding can be measured, for example, by determining binding of a molecule as compared to binding of a control molecule In amounts, the control molecule is typically a similarly structured molecule that does not have binding activity. For example, specific binding can be determined by competition with a control molecule (e.g., excess unlabeled target) that is similar to the target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term "specifically binds" or "specifically binds to" or "is specific for" a particular target molecule or an epitope on a particular target molecule, which may be expressed, for example, as a K of the molecule to the target _D At least about 10 ^-5 M, alternatively at least about 10 ^-6 M, alternatively at least about 10 ^-7 M, alternatively at least about 10 ^-8 M, alternatively at least about 10 ^-9 M, alternatively at least about 10 ^-10 M, alternatively at least about 10 ^-11 M, alternatively at least about 10 ^-12 M, alternatively at least about 10 ^-13 M, alternatively at least about 10 ^-14 M, alternatively at least about 10 ^-15 M or lower. In one embodiment, the term "specific binding" refers to the following binding: the binding molecule binds to a specific target molecule or an epitope on a specific target molecule, but does not substantially bind to any other polypeptide or polypeptide epitope.

As used herein, the term "bispecific antibody" refers to an antibody that is at least bispecific, i.e., capable of binding two different antigens or target molecules. Bispecific antibodies have at least two different antigen binding sites, wherein a first antigen binding site binds a first antigen or target molecule and a second antigen binding site binds a second antigen or target molecule. Bispecific antibodies can bind, among other things, different surface molecules of two different cells, bringing these cells into close proximity. For example, bispecific antibodies that recognize an antigen on a target cell (e.g., FLT3 or CD19 on leukemia cells, CSPG 4-antigen on melanoma cells, or EGFR on glioblastoma cells) and an antigen-specific T Cell Receptor (TCR)/CD 3-complex can target tumor cells for T cell mediated lysis.

As used herein, the term "linker" refers to a molecule that connects two binding moieties by covalent or non-covalent bonding. Thus, a peptide linker is an intervening peptide sequence that does not include an amino acid residue from the C-terminus of the variable region of the first binding moiety (e.g., variable light chain or variable heavy chain) or the N-terminus of the variable region of the second binding moiety (e.g., variable light chain or variable heavy chain). As a linker "linking" two binding moieties, the linkage to each binding moiety may be covalent or non-covalent. In particular, the two linkages of the linker to the two binding moieties may be covalent and covalent, covalent and non-covalent, or non-covalent and non-covalent. In some embodiments, the linker of the co-conjugate facilitates the co-conjugate to achieve binding interactions with its target molecule. In some embodiments, the linker does not interfere with the binding interaction of the first binding moiety and the second binding moiety with their corresponding epitopes in the antigen. In some embodiments, the length of the linker is minimized to reduce or minimize entropy loss at the time of binding. In some embodiments, the rigidity of the joint is enhanced or maximized to reduce or minimize the loss of entropy upon bonding. The linker may be a "non-cleavable" linker. The linker may be a "cleavable linker" which can cleave under a variety of physiological or non-physiological conditions. Such cleavable linkers include, but are not limited to, acid labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers comprising amino acids, such as valine and/or citrulline, e.g., citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers (see, e.g., chari et al, 1992,Cancer Res.52:127-31; and U.S. Pat. No. 5,208,020), thioether linkers, or hydrophilic linkers designed to evade multidrug transporter mediated resistance (see, e.g., kovtun et al, 2010,Cancer Res.70:2528-37). The linker may be made of different compositions or chemical compositions. In some embodiments, the linker is a polypeptide linker, a nucleic acid linker, and/or a chemical linker. In some embodiments, the linker is not antigenic and does not elicit an immune response. The linker may connect the variable region of the first antibody as part of the first binding moiety and the variable region of the second antibody as part of the second binding moiety by covalent bonds. The linker may also connect the variable region of the first antibody as part of the first binding moiety and the variable region of the second antibody as part of the second binding moiety by non-covalent binding. Some examples of polypeptide linkers are described in Chen et al, adv Drug Deliv rev.2013, 10 months 15; 65 1357-1369, which is incorporated herein by reference in its entirety.

An "isolated" antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source and/or other contaminating components from which the antibody is derived, or substantially free of chemical precursors or other chemicals upon chemical synthesis. The language "substantially free of cellular material" includes preparations of antibodies in which the antibody is isolated from cellular components of the cells from which it was isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include antibody preparations having less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). In certain embodiments, when the antibody is recombinantly produced, it is substantially free of culture medium, e.g., culture medium represents less than about 20%, 15%, 10%, 5%, or 1% of the volume of the protein preparation. In certain embodiments, when the antibody is produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, e.g., it is separated from chemical precursors or other chemicals that are involved in protein synthesis. Thus, such antibody formulations have less than about 30%, 25%, 20%, 15%, 10%, 5% or 1% (by dry weight) of chemical precursors or compounds other than the antibody of interest. The contaminating components may also include, but are not limited to, substances that interfere with the therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody will be purified to (1) greater than 95% by weight of the antibody, as determined by the Lowry method (Lowry et al, 1951, j.bio.chem.193:265-75), e.g., 96%, 97%, 98% or 99%; (2) To a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotary cup sequencer; or (3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies include in situ antibodies within recombinant cells because at least one component of the natural environment of the antibody will not be present. However, isolated antibodies are typically prepared by at least one purification step. In particular embodiments, the antibodies provided herein are isolated.

The 4-chain antibody unit is a heterologous tetralin protein, consisting of two identical light chains (L) and two identical heavy chains (H). In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) of the alpha and gamma chains and four CH domains of the mu and epsilon isoforms. The N-terminus of each L chain has a variable domain (VL) followed by a constant domain (CL) at the other end. VL is aligned with VH, and CL is aligned with the first constant region (CH 1) of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. The VH and VL pairs together form a single antigen binding site. See, e.g., basic and Clinical Immunology (Stites et al, 8 th edition 1994) for the structure and properties of different classes of antibodies.

The terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody's light or heavy chain that is typically at the amino terminus of the light or heavy chain, and the heavy chain is about 110 to 140 amino acids long by about 100 to 110 amino acids long, and is used for the binding and specificity of each particular antibody for its particular antigen. The variable region of a heavy chain may be referred to as the variable region of a "VH" light chain may be referred to as the "VL" term "variable" refers to the fact that certain segments of the variable region vary greatly in sequence between antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 amino acid span of the variable region. In contrast, the V region consists of fragments of about 15-30 amino acids called Framework Regions (FR) that are less variable (e.g., relatively unchanged), which fragments are separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions", each of about 9-12 amino acids in length. The variable regions of the heavy and light chains each comprise four FR, principally in the beta sheet configuration, joined by three hypervariable regions that form loops connecting the beta sheet structure, and in some cases form part of the beta sheet structure. The hypervariable regions in each chain are held together tightly by the FR and together with the hypervariable regions from the other chain contribute to the formation of the antigen binding site of the antibody (see, e.g., kabat et al, sequences of Proteins of Immunological Interest (5 th edition 1991)). The constant region is not directly involved in binding of an antibody to an antigen, but exhibits various effector functions, such as antibody involvement in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable region sequences vary widely from antibody to antibody. In a specific embodiment, the variable region is a human variable region.

The term "variable region residue number as in Kabat" or "amino acid position number as in Kabat" and variants thereof refers to the numbering system of the heavy chain variable region or the light chain variable region used in antibody assembly, as in Kabat et al. Using such numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which correspond to shortening of the variable region FR or CDR or insertion therein. For example, the heavy chain variable domain may comprise a single amino acid insertion following residue 52 (residue 52a according to Kabat) and three inserted residues following residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). For a given antibody, the Kabat numbering of residues may be determined by aligning regions of homology of the antibody sequence with "standard" Kabat numbering sequences. When referring to residues in the variable domain (about residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., kabat et al, supra). When referring to residues in the immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" is generally used (e.g., kabat et al, EU index as reported above). "EU index as in Kabat" refers to the residue numbering of the human IgG 1EU antibody. Other numbering systems have been described by, for example, abM, chothia, contact, IMGT and AHo.

An "intact" antibody is an antibody comprising an antigen binding site, CL and at least heavy chain constant regions CH1, CH2 and CH 3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In certain embodiments, the intact antibody has one or more effector functions.

An "antibody fragment" comprises a portion of an intact antibody, such as an antigen-binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, fab ', F (ab') ₂ And Fv fragments; diabodies and diabodies (see, e.g., holliger et al, 1993, proc. Natl. Acad. Sci.90:6444-48; lu et al, 2005, J. Biol. Chem.280:19665-72; hudson et al, 2003, nat. Med.9:129-34; WO 93/11161; and U.S. Pat. Nos. 5,837,242 and 6,492,123); single chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778;5,260,203;5,482,858; and 5,476,786); a double variable domain antibody (see, e.g., U.S. patent No. 7,612,181); single variable domain antibodies (sdAbs) (see, e.g., woolven et al, 1999,Immunogenetics 50:98-101; and Streltsov et al, 2004,Proc Natl Acad Sci USA.101:12444-49); and multispecific antibodies formed from antibody fragments.

A "functional fragment," "binding fragment," or "antigen-binding fragment" of a therapeutic antibody will exhibit at least one, if not some or all, biological function possessed by the intact antibody, including at least binding to a target antigen.

When used in reference to an antibody, the term "heavy chain" refers to a polypeptide chain of about 50-70kDa, wherein the amino-terminal portion comprises a variable region of about 120-130 or more amino acids and the carboxy-terminal portion comprises a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types (e.g., isoforms), referred to as alpha (α), delta (δ), epstein (epsilon), gamma (γ), and spurious (μ). The different heavy chains vary in size: alpha, delta and gamma contain about 450 amino acids, while mu and epsilon contain about 550 amino acids. When combined with light chains, these different types of heavy chains produce five well-known antibody classes (e.g., isotypes), igA, igD, igE, igG and IgM, respectively, including the four subclasses of IgG, namely IgG1, igG2, igG3, and IgG4. The heavy chain may be a human heavy chain.

When used in reference to an antibody, the term "light chain" refers to a polypeptide chain of about 25kDa, wherein the amino-terminal portion comprises a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion comprises a constant region. The approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa (kappa) or lambda (lambda). The light chain amino acid sequences are well known in the art. The light chain may be a human light chain.

As used herein, the term "host" refers to an animal, such as a mammal (e.g., a human).

As used herein, the term "host cell" refers to a cell of a particular subject that can be transfected with a nucleic acid molecule, as well as the progeny or potential progeny of such a cell. The progeny of such a cell may differ from the parent cell transfected with the nucleic acid molecule due to mutations or environmental effects that may occur in subsequent generations or integration of the nucleic acid molecule into the host cell genome.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody typically recognizes a single epitope on an antigen. In particular embodiments, as used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell, wherein the antibody binds only to an epitope of a target, as determined by, for example, ELISA or other antigen binding or competitive binding assays known in the art. The term "monoclonal" is not limited to any particular method for producing antibodies. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma method described first by Kohler et al, 1975,Nature 256:495, or may be prepared using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al, 1991,Nature 352:624-28 and Marks et al, 1991, J.mol.biol. 222:581-97. Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are well known in the art. See, e.g., short Protocols in Molecular Biology (Ausubel et al, 5 th edition 2002). Exemplary methods of producing monoclonal antibodies are provided in the examples herein.

The term "native" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to those substances that are found in nature and that have not been manipulated, modified and/or altered (e.g., isolated, purified, selected) by humans.

Antibodies provided herein may include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al, 1984,Proc.Natl.Acad.Sci.USA 81:6851-55).

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody comprising a human immunoglobulin (e.g., recipient antibody) in which the natural CDR residues are replaced with corresponding CDR residues of the desired specificity, affinity, and capacity from a non-human species (e.g., donor antibody), such as a mouse, rat, rabbit, or non-human primate. In some cases, one or more FR region residues of a human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are not present in the recipient antibody or in the donor antibody. These modifications were made to further improve antibody performance. The humanized antibody heavy or light chain may comprise substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al, 1986,Nature 321:522-25; riechmann et al 1988,Nature 332:323-29; presta,1992, curr.op. Struct. Biol.2:593-96; carter et al, 1992,Proc.Natl.Acad.Sci.USA 89:4285-89; U.S. patent No. 6,800,738;6,719,971;6,639,055;6,407,213; and 6,054,297.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of a human-produced antibody, and/or an antibody prepared using any of the techniques for preparing human antibodies as disclosed herein. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter,1991, J. Mol. Biol.227:381; marks et al, 1991, J. Mol. Biol. 222:581) and yeast display libraries (Chao et al, 2006,Nature Protocols 1:755-68). Methods which can also be used for the preparation of human monoclonal antibodies are described in Cole et al, monoclonal Antibodies and Cancer Therapy 77 (1985); boerner et al, 1991, J.Immunol.147 (1): 86-95; and van Dijk and van de Winkel,2001, curr. Opin. Pharmacol.5:368-74. Human antibodies can be prepared by administering an antigen to a transgenic animal, such as a mouse, that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous locus has been disabled (see, e.g., jakobovits,1995, curr. Opin. Biotechnol.6 (5): 561-66; bruggemann and Taussing,1997, curr. Opin. Biotechnol.8 (4): 455-58; and in relation to XENOMOUSE) ^TM U.S. Pat. nos. 6,075,181 and 6,150,584 to the technology). See also, e.g., li et al, 2006,Proc.Natl.Acad.Sci.USA 103:3557-62 for human antibodies produced by human B cell hybridoma technology.

"CDR" refers to one of the three hypervariable regions (H1, H2 or H3) within the non-framework regions of the immunoglobulin (Ig or antibody) VH β -sheet framework or one of the three hypervariable regions (L1, L2 or L3) within the non-framework regions of the antibody VL β -sheet framework. Thus, CDRs are variable region sequences interspersed with framework region sequences. CDR regions are well known to those skilled in the art and have been defined, for example, by Kabat as hypervariable regions within the variable (V) domain of an antibody (Kabat et al, 1997, J. Biol. Chem.252:6609-16; kabat,1978, adv. Prot. Chem. 32:1-75). CDR region sequences are also structurally defined by Chothia as those residues that are not part of the conserved β -sheet framework and are therefore able to adapt to different conformations (Chothia and Lesk,1987, j.mol.biol.196:901-17). Both terms are well known in the art. CDR region sequences have also been defined by AbM, contact and IMGT. The position of the CDRs within the variable region of a standard antibody has been determined by comparison of various structures (Al-Lazikani et Al 1997, J. Mol. Biol.273:927-48; morea et Al 2000,Methods 20:267-79). Because the number of residues within the hypervariable region varies among antibodies, additional residues relative to standard positions are typically numbered a, b, c, etc., alongside the residue numbers in the standard variable region numbering scheme (Al-Lazikani et Al, supra). This naming is also well known to those skilled in the art.

The term "hypervariable region", "HVR" or "HV" as used herein refers to a region of an antibody variable region that is hypervariable in sequence and/or forms a structurally defined loop. Typically, an antibody comprises six hypervariable regions, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). The description of many hypervariable regions is in use and is included herein. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are most commonly used (see, e.g., kabat et al, supra). In contrast, chothia refers to the position of the structural loop (see, e.g., chothia and Lesk,1987, J.mol. Biol. 196:901-17). When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop (since the Kabat numbering scheme places insertions at H35A and H35B; loops end at 32 if neither 35A nor 35B is present; loops end at 33 if only 35A is present; loops end at 34 if both 35A and 35B are present). The AbM hypervariable region represents a compromise between Kabat CDRs and Chothia structural loops and is used by Oxford Molecular AbM antibody modeling software (see, e.g., antibody Engineering vol.2 (Kontermann and dubel, 2 nd edition 2010)). The "contact" hypervariable region is based on analysis of existing complex crystal structures. Residues of each of these hypervariable regions or CDRs are shown below.

Recently, a general numbering system was developed and widely adopted, imMunoGeneTics (IMGT) Information (Lafranc et al, 2003, dev. Comp. Immunol.27 (1): 55-77). IMGT is an integrated information system, specifically studying Immunoglobulins (IG), T Cell Receptors (TCR) and Major Histocompatibility Complex (MHC) in humans and other vertebrates. Herein, CDR refers to the amino acid sequence and position in the light or heavy chain. Since the "position" of CDRs within an immunoglobulin variable domain structure is conserved across species and exists in a structure called a loop, CDRs and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from immunoglobulins of one species into the acceptor framework typically derived from human antibodies. Honyger and Pluckthun, 2001, J.mol.biol.309:657-70 developed an additional numbering system (AHo). The correspondence between numbering systems (including, for example, the Kabat numbering and IMGT unique numbering systems) is well known to those skilled in the art (see, for example, kabat, supra; chothia and Lesk, supra; martin, supra; lefranc et al, supra).

	IMGT	Kabat	AbM	Chothia	Contact
						V _H CDR1	27-38	31-35	26-35	26-32	30-35
V _H CDR2	56-65	50-65	50-58	53-55	47-58
						V _H CDR3	105-117	95-102	95-102	96-101	93-101
V _L CDR1	27-38	24-34	24-34	26-32	30-36
						V _L CDR2	56-65	50-56	50-56	50-52	46-55
V _L CDR3	105-117	89-97	89-97	91-96	89-96

The hypervariable region may comprise the following "extended hypervariable region": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. As used herein, the terms "HVR" and "CDR" are used interchangeably.

The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding an antibody to an antigen, but that exhibit various effector functions, such as interactions with Fc receptors. The term refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of the immunoglobulin, i.e., the variable region that contains the antigen binding site. The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

The term "framework" or "FR" refers to those variable region residues flanking the CDRs. FR residues are present in, for example, chimeric, humanized, human, domain, diabodies, linear and bispecific antibodies. FR residues are variable domain residues other than hypervariable region residues or CDR residues.

An "affinity matured" antibody is an antibody that has one or more alterations (e.g., amino acid sequence variations, including alterations, additions, and/or deletions) in one or more of its HVRs, which results in an improved affinity of the antibody for the antigen as compared to the parent antibody without those alterations. Affinity matured antibodies may have nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For reviews, see Hudson and Sourau, 2003,Nature Medicine 9:129-34; hoogenboom,2005,Nature Biotechnol.23:1105-16; quiroz and Sinclair,2010,Revista Ingeneria Biomedia 4:39-51.

"binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a binding protein, such as an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibodies and antigens). The affinity of a binding molecule X for its binding partner Y can generally be determined by the dissociation constant (K _D ) And (3) representing. Affinity can be measured by conventional methods known in the art, including those described herein. Low affinity antibodies typically bind antigen slowly and tend to dissociate easily, while high affinity antibodies typically bind antigen faster and tend to remain bound for longer periods of time. A variety of methods of measuring binding affinity are known in the art, any of which may be used for the purposes of this disclosure. Specific illustrative embodiments include the following. In one embodiment, "K _D "or" K _D The value "may be measured by assays known in the art, for example by binding assays. K (K) _D Measurements can be made in RIA, for example with Fab versions of the antibodies of interest and their antigens (Chen et al, 1999,J.Mol Biol 293:865-81). K (K) _D Or K _D The value may also be obtained by usingFor example +.>Or->Or by using, for example +.>The biological layer interferometry of the system. "association Rate (on-rate/rate of association/association rate/k) _on ) "can also be usedThe same surface plasmon resonance or biological layer interferometry techniques described above, e.g. using +.>Or-> Or->The system was used for measurement. Dissociation rate (off-rate/rate of dissociation/dissociation rate/k) _off ) "the same surface plasmon resonance or biological layer interferometry techniques described above can also be used, e.g., usingOr->Or->The system was used for measurement.

As used herein, the term "effective amount" refers to an amount of a co-conjugate or pharmaceutical composition provided herein sufficient to produce a beneficial or desired result. An effective amount may be administered by one or more administrations, applications or administrations. Such delivery depends on many variables including the time of use of the individual dosage units, the bioavailability of the agent, the route of administration, and the like.

As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent (e.g., a co-conjugate provided herein) sufficient to reduce and/or ameliorate the severity and/or duration of a given disease and/or symptom associated therewith. A therapeutically effective amount of a therapeutic agent may be an amount necessary to reduce or ameliorate the progression or progression of a given disease, reduce or ameliorate the recurrence, progression or onset of a given disease, and/or improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than administration of a co-conjugate provided herein).

The term "variant" when used in connection with a polypeptide refers to a polypeptide that comprises one or more (e.g., from about 1 to about 50, from about 1 to about 45, from about 1 to about 40, from about 1 to about 35, from about 1 to about 30, from about 1 to about 25, from about 1 to about 20, from about 1 to about 18, from about 1 to about 15, from about 1 to about 10, or from about 1 to about 5) amino acid sequence substitutions, deletions, and/or additions as compared to the native or unmodified sequence of the polypeptide. For example, variants of a co-conjugate may result from one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 18, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes in the amino acid sequence of the natural or previously unmodified co-conjugate. Variants may be constructed by molecular cloning techniques known to those of ordinary skill in the art, such as random mutagenesis or site-directed mutagenesis. Variants can be prepared from the corresponding nucleic acid molecules encoding the variants. In particular embodiments, variants of the co-conjugate retain the functional properties or activity (e.g., binding, agonist, antagonist, blocking, neutralizing, and/or activating activity/properties) of the co-conjugate. In particular embodiments, the variants are encoded by nucleic acid molecules comprising one or more Single Nucleotide Polymorphisms (SNPs) in one or more regions or subregions, e.g., one or more CDRs, of the co-conjugate.

The term "vector" refers to a material used to carry or contain a nucleic acid sequence, including, for example, nucleic acid sequences encoding a co-conjugate as described herein, in order to introduce the nucleic acid sequence into a host cell. Vectors suitable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, exosomes and artificial chromosomes, which may comprise selection sequences or markers operable for stable integration into a host cell chromosome. In addition, the vector may comprise one or more selectable marker genes and appropriate expression control sequences. For example, selectable marker genes may be included to provide resistance to antibiotics or toxins, to supplement auxotrophs, or to supply critical nutrients not present in the medium. Expression control sequences may include constitutive and inducible promoters, transcriptional enhancers, transcriptional terminators, and the like, as are well known in the art. When two or more nucleic acid molecules are co-expressed (e.g., antibody heavy and light chains or antibody VH and VL), the two nucleic acid molecules may be inserted into, for example, a single expression vector or into separate expression vectors. For single vector expression, the coding nucleic acids may be operably linked to one common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of a nucleic acid molecule into a host cell may be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as northern blot or Polymerase Chain Reaction (PCR) amplification of mRNA, immunoblotting of gene product expression, or other suitable analytical methods to test the expression of the introduced nucleic acid sequence or its corresponding gene product. It will be appreciated by those skilled in the art that the nucleic acid molecules are expressed in amounts sufficient to produce the desired product (e.g., a co-conjugate as described herein), and that the expression levels may be optimized to obtain adequate expression using methods well known in the art.

As used herein, the term "conservative substitution" refers to an amino acid substitution known to those skilled in the art, and can generally be made without altering the biological activity of the resulting molecule. Those skilled in The art recognize that in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., watson et al, MOLECULAR BIOLOGY OF THE GENE, the Benjamin/Cummings pub. Co., page 224 (4 th edition 1987)). Such exemplary substitutions may be made according to those listed in table 1 and the description below. In conservative amino acid substitutions, the amino acid residue is replaced with an amino acid residue that contains a side chain with a similar charge or a side chain with similar properties. Families of amino acid residues comprising side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Amino acids can also be grouped according to their similarity in side chain properties (see, e.g., lehninger, biochemistry 73-75 (2 nd edition 1975)): (1) nonpolar: ala (A), val (V), leu (L), ile (I), pro (P), phe (F), trp (W), met (M); (2) uncharged polarity: gly (G), ser (S), thr (T), cys (C), tyr (Y), asn (N), gln (Q); (3) acidity: asp (D), glu (E); and (4) alkaline: lys (K), arg (R), his (H). Alternatively, naturally occurring residues can be grouped into several groups based on common side chain properties: (1) hydrophobicity: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr, asn, gln; (3) acidity: asp, glu; (4) alkaline: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

For example, any cysteine residue that does not participate in maintaining the proper conformation of the antibody may also be substituted with, for example, another amino acid such as alanine or serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. In certain embodiments, conservative substitutions include substitution of any of these hydrophobic amino acids with any of isoleucine (I), valine (V), and leucine (L); substitution of aspartic acid (D) for glutamic acid (E) and vice versa; substitution of asparagine (N) with glutamine (Q) and vice versa; serine (S) replaces threonine (T) and vice versa. Other substitutions may also be considered conservative, depending on the context of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (a) may be interchangeable, as may alanine (a) and valine (V). Methionine (M), which is relatively hydrophobic, can be exchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) may be interchangeable in positions where the significant feature of the amino acid residues is their charge and the different pK of the two amino acid residues are not significant. Still other changes may be considered "conservative" in certain circumstances (see, e.g., table 1, pages 13-15, "Biochemistry" 2 nd edition Lubert Stryer edition (Stanford University), henikoff et al, PNAS1992, volume 89 10915-10919; lei et al, J Biol Chem 1995, month 19; 270 (20): 11882-11886). Other substitutions are also permissible and may be determined empirically or based on known conservative substitutions.

Table 1: amino acid substitutions or similarity matrices

The amino acid substitution matrix (block substitution matrix) was adapted from GCG software 9.0BLOSUM62.

The higher the value, the greater the likelihood of substitution being found in the relevant native protein.

The term "homology" or "homologous" refers to sequence similarity between two polynucleotides or between two polypeptides. Similarity may be determined by comparing the positions in each sequence aligned for comparison purposes. If a given position of two polypeptide sequences is not identical, the similarity or conservation of that position can be determined by assessing the similarity of the amino acids at that position, e.g., according to Table 1, according to the similarity of the side chain charge as described above, or according to the similarity of the side chain properties as described above. The degree of similarity between sequences is a function of the number of matched (identical) or homologous positions that the sequences share. Alignment of the two sequences to determine their percent sequence similarity can be performed using software programs known in the art, such as those described, for example, in Ausubel et al, current Protocols in Molecular Biology, john Wiley and Sons, baltimore, MD (1999). Preferably, the alignment is performed using default parameters, examples of which are described below. One alignment program known in the art that may be used is BLAST, which is set as a default parameter. Specifically, the programs are BLASTN and BLASTP using the following default parameters: genetic code = standard; filter = none; chain = two; cut-off value = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sorting basis = high score; database = non-redundant, genBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + spldate + PIR. Details of these procedures can be found in the national center for biotechnology information.

The term "homologue" of a given amino acid sequence or nucleic acid sequence is intended to indicate that the corresponding sequence of the "homologue" has substantial identity or homology to the given amino acid sequence or nucleic acid sequence.

The term "identity" refers to the relationship between sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megasign (DNAStar, inc.) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the compared sequences.

Determination of the percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms. Non-limiting examples of mathematical algorithms for comparing two sequences are those of Karlin and Altschul,1990, proc. Natl. Acad. Sci. U.S. A.87:22642268, as adapted in Karlin and Altschul,1993,Proc.Natl.Acad.Sci.U.S.A.90:5873 5877. Such algorithms are incorporated in the NBLAST and XBLAST programs of Altschul et al, 1990, J.mol. Biol. 215:403. BLAST nucleotide searches can be performed using the NBLAST nucleotide program parameter set (e.g., score=100, word length=12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program parameter set (e.g., to score 50, word length=3) to obtain amino acid sequences homologous to protein molecules described herein. To obtain a gap alignment for comparison purposes, gap BLAST as described in Altschul et al, 1997,Nucleic Acids Res.25:3389 3402 may be utilized. Alternatively, PSI BLAST can be used to conduct an iterative search to detect the distance relationship (id.) between molecules. When using BLAST, gapped BLAST, and PSI BLAST programs, default parameters for each program (e.g., XBLAST and NBLAST default parameters) can be used (see, e.g., national Center for Biotechnology Information (NCBI) on the world Wide Web NCBI. Nlm. Nih. Gov). Another non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller,1988,CABIOS 4:11 17. This algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When amino acid sequences are compared using the ALIGN program, PAM120 weight residue table, gap length penalty 12, and gap penalty 4 can be used.

The percent identity between two sequences may be determined using techniques similar to those described above, with or without gaps allowed. In calculating the percent identity, only exact matches are typically calculated.

As used herein, the term "truncated" when used in the context of a polypeptide/protein, means that the amino acid sequence of the polypeptide is shortened from either end of the polypeptide sequence, the algorithm used to determine the shortening is provided further below, and in certain embodiments of the co-conjugates provided herein in the sentence "[ i ], the disclosure indicates that the truncation or deletion of VR2, VLAb2, VHAb2, or the second binding moiety is provided in several paragraphs after the" beginning paragraph "as determined by, for example, the following exemplary methods. Similarly, the term "truncated" when used in the context of a nucleic acid refers to the shortening of the nucleotide sequence of the nucleic acid from the 5 'or 3' end of the nucleotide sequence. N-terminal truncation or truncation from the N-terminal of a polypeptide/protein refers to shortening the polypeptide/protein sequence from the N-terminus (i.e., N-terminal) of the polypeptide/protein. Similarly, a truncated C-terminal truncation or truncation from the C-terminal refers to shortening the polypeptide/protein sequence from the C-terminus (i.e., C-terminal) of the polypeptide/protein. Truncations may be a shortening of one or more amino acids at either or both ends of the polypeptide/protein. For example, the truncation may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids from the N-or C-terminus of the polypeptide/protein. For example, truncations may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids from the N-and C-termini of the polypeptide/protein. Protein truncations may be the result of truncations of the nucleic acid sequence encoding the protein, substitutions or other mutations that produce premature stop codons without shortening the nucleic acid sequence, or alternatively from splicing of RNA, wherein substitutions or other mutations that do not themselves cause truncations result in aberrant RNA processing. "truncation mutant" or "truncation mutation" refers to a variant having one or more amino acid truncations in the case of a polypeptide/protein or one or more nucleotide truncations in the case of a nucleic acid.

As used herein, the term "deletion" when used in the context of a polypeptide/protein refers to the removal of one or more amino acids from the sequence of the polypeptide/protein. The removed one or more amino acids may be a continuous sequence, i.e. a continuous portion, of the polypeptide/protein or may be interspersed with the sequence of the polypeptide/protein. Deletions may be internal deletions in which none of the removed amino acids are the N-terminal or C-terminal amino acids of the polypeptide/protein sequence. Deletions may also be deletions from the N-terminus (N-terminal deletion) or from the C-terminus (C-terminal deletion), wherein one or more amino acid sequences contiguous from the N-terminus or C-terminus of the polypeptide/protein are removed. Deletions may also be deletions including internal deletions, N-terminal deletions, and/or C-terminal deletions. It is clear from the description that the N-terminal deletion is also an N-terminal truncation and the C-terminal deletion is also a C-terminal truncation. Sequences that meet the definition of internal deletions may also be considered to be N-terminally truncated, if the criteria for N-terminally truncated are met by application of the algorithms described herein.

"modification" of an amino acid residue/position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, wherein the change is caused by a sequence change involving the amino acid residue/position. For example, typical modifications include substitution of a residue with another amino acid (e.g., conservative or non-conservative substitutions), insertion of one or more (e.g., typically less than 5, 4, or 3) amino acids near the residue/position, and/or deletion of the residue/position.

When two antibodies recognize identical, overlapping or adjacent epitopes in three-dimensional space, the antibodies bind to an "epitope," an epitope that is substantially identical to a reference antibody, or an "identical epitope. The most widely used and rapid method for determining whether two antibodies bind to the same, overlapping or adjacent epitope in three-dimensional space is a competition assay, which can be configured in many different forms, for example using a labeled antigen or a labeled antibody. In some assays, the antigen is immobilized on a 96-well plate, or expressed on the surface of cells, and the ability of the unlabeled antibody to block binding of the labeled antibody is measured using a radiolabel, fluorescent label, or enzymatic label.

"epitope mapping" is the process of identifying the binding site or epitope of an antibody on its target antigen. "Epitope clustering" is the process of grouping antibodies according to their recognized epitopes. More specifically, epitope clustering includes methods and systems for differentiating epitope recognition properties of different antibodies using competition assays, combined with computational methods for clustering antibodies based on their epitope recognition properties and identifying antibodies with different binding specificities.

As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers which are non-toxic to the cells or mammals exposed thereto at the dosages and concentrations employed. Typically, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid; a low molecular weight (e.g., less than about 10 amino acid residues) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; single sheetSugars, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, e.g. TWEEN ^TM Polyethylene glycol (PEG) and PLURONICS ^TM . The term "carrier" may also refer to a diluent, adjuvant (e.g., freund's adjuvant (complete or incomplete)), excipient or vehicle. Such carriers, including pharmaceutical carriers, can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the composition (e.g., pharmaceutical composition) is administered intravenously, water is an exemplary carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The composition can be in the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release preparation, etc. Oral compositions, including formulations, may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable drug carriers are described in Remington and Gennaro, remington' sPharmaceutical Sciences (18 th edition 1990). The compositions, including pharmaceutical compounds, may contain the co-conjugate, for example, in isolated or purified form, and an appropriate amount of carrier.

As used herein, the term "pharmaceutically acceptable" refers to approval by a regulatory agency of the federal or a state government or by inclusion in the U.S. pharmacopeia, the european pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

As used herein, "polyclonal antibody" refers to a population of antibodies produced in an immunogenic response to a protein having a number of epitopes, and thus includes a plurality of different antibodies directed against the same or different epitopes within the protein. Methods for producing polyclonal antibodies are known in the art (see, e.g., short Protocols in Molecular Biology (Ausubel et al, 5 th edition 2002)).

An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or a mixture of nucleic acids, that is substantially isolated from other genomic DNA sequences, and proteins or complexes that naturally accompany the native sequence, such as ribosomes and polymerases. An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that are found in the natural source of the nucleic acid molecule. In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding an antibody as described herein are isolated or purified. The term includes nucleic acid sequences that have been removed from their naturally occurring environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized by heterologous systems. Substantially pure molecules may include isolated forms of the molecule.

"Polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length and includes DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and/or an analogue thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. As used herein, "oligonucleotide" refers to a short, typically single stranded, synthetic polynucleotide, typically, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides applies equally and entirely to oligonucleotides. Cells producing the co-conjugates of the present disclosure may include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acids encoding antibodies have been introduced. Suitable host cells are disclosed below.

Unless otherwise indicated, the left-hand end of any single stranded polynucleotide sequence disclosed herein is the 5' end; the left hand orientation of the double stranded polynucleotide sequence is referred to as the 5' orientation. The direction of 5 'to 3' addition of nascent RNA transcripts is referred to as the transcription direction; the region of the DNA strand having the same sequence as the region of the RNA transcript, i.e., the 5' -end region of the RNA transcript, is referred to as the "upstream sequence"; the region of the DNA strand having the same sequence as the RNA transcript, i.e., the 3 '-3' end of the RNA transcript, is referred to as the "downstream sequence"

The term "recombinant antibody", "recombinant co-conjugate" or "recombinant polypeptide/protein" refers to an antibody, co-conjugate, polypeptide/protein that is produced, expressed, produced or isolated by recombinant means. For example, the recombinant co-conjugate may be a co-conjugate expressed using a recombinant expression vector transfected into a host cell; a co-conjugate isolated from a recombinant combinatorial library; or by any other method involving splicing immunoglobulin gene sequences into other DNA sequences. For another example, the recombinant polypeptide/protein may be a polypeptide/protein expressed using a recombinant expression vector transfected into a host cell; polypeptides/proteins isolated from recombinant combinatorial libraries; or by any other means involving splicing of immunoglobulin gene sequences to other DNA sequences. For another example, the recombinant antibody may be an antibody expressed using a recombinant expression vector transfected into a host cell; an antibody isolated from a recombinant combinatorial antibody library; antibodies isolated from animals (e.g., mice or cattle) that are transgenic and/or transchromosomal human immunoglobulin genes (see, e.g., taylor et al, 1992,Nucl.Acids Res.20:6287-95); or antibodies produced, expressed, produced or isolated by any other method involving splicing immunoglobulin gene sequences into other DNA sequences. Such recombinant antibodies may have variable and constant regions, including those derived from human germline immunoglobulin sequences (see Kabat et al, supra). However, in certain embodiments, such recombinant antibodies may be subjected to in vitro mutagenesis (or, when transgenic animals directed against human Ig sequences are used, in vivo somatic mutagenesis), and thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are those sequences that, while derived from and associated with human germline VH and VL sequences, may not naturally occur in the human germline repertoire of antibodies.

As used herein, the term "therapeutic agent" refers to an agent that can be used to treat, manage, or ameliorate a disease and/or symptoms associated therewith. In certain embodiments, the therapeutic agent comprises a co-conjugate as described herein.

As used herein, the term "diagnostic agent" refers to a substance that aids in diagnosing a disease. The diagnostic agent may be used in vitro or in vivo. In some embodiments, the diagnostic agent is used in an in vitro assay. In some embodiments, a diagnostic agent is administered to a subject. Such agents may be used to reveal, pinpoint, and/or determine the location of pathogenic processes. In some embodiments, the diagnostic agent, when administered to a subject or contacted with a sample from the subject, aids in diagnosing cancer or tumor formation. In certain embodiments, the diagnostic agent comprises a co-conjugate as described herein.

The terms "subject" and "patient" may be used interchangeably. As used herein, in certain embodiments, the subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). In particular embodiments, the subject is a human.

"substantially all" means at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

The term "detectable agent" or "detectable molecule" is used interchangeably herein and refers to a substance that can be used to determine the presence or absence of a desired molecule in a sample or subject, such as a co-conjugate as described herein. The detectable agent may be a substance that can be visualized or a substance that can be determined and/or measured (e.g., by quantification).

When used in reference to a nucleic acid molecule, the term "encoding nucleic acid" or grammatical equivalents thereof refers to a nucleic acid molecule in its natural state or when manipulated by methods well known to those of skill in the art that can be transcribed to produce mRNA and then translated into a polypeptide and/or fragment thereof. The antisense strand is the complementary strand of such a nucleic acid molecule, and the coding sequence can be deduced therefrom.

The term "excipient" refers to an inert substance that is commonly used as a diluent, vehicle, preservative, binder, or stabilizer, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylates, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), sugars (e.g., sucrose, maltose, trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). See also Remington and Gennaro, remington's Pharmaceutical Sciences (18 th edition 1990), which is incorporated herein by reference in its entirety.

As used herein, the term "compound" includes small organic molecules and inorganic chemicals having a molecular weight of less than about 5kD, less than about 4kD, less than about 3kD, less than about 2kD, less than about 1kD, or less than about 0.5kD, including but not limited to all analogs, derivatives, salts, and solvates (e.g., hydrates) thereof. In some examples, the compound may include a nucleic acid, a peptide, a peptidomimetic, a peptoid, other small organic compounds or drugs, or the like. Libraries of chemical and/or biological mixtures (such as fungal, bacterial or algal extracts) are known in the art and can be screened using any of the assays provided herein. Examples of methods for synthesis of libraries of compounds can be found in: (Carell et al, 1994a; carell et al, 1994b; cho et al, 1993; deWitt et al, 1993; gallop et al, 1994; zuckermann et al, 1994).

In the context of peptides or polypeptides, the term "fragment" as used herein refers to a peptide or polypeptide comprising less than the full-length amino acid sequence. Such fragments may result from, for example, an amino-terminal truncation, a carboxy-terminal truncation, and/or an internal deletion of amino acid sequence residues.

The terms "about" and "approximately" mean within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a given value or range.

"administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., a co-conjugate as described herein) present in vitro into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other physical delivery method described herein or known in the art.

The term "composition" is intended to include products comprising optionally specified ingredients (e.g., antibodies provided herein) in specified amounts.

The term "and/or" as used herein in phrases such as "a and/or B" is intended to include both a and B; a or B; a (alone); and B (alone). Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide sequence" includes a plurality of such sequences, and the like.

As used herein, numerical values are generally expressed in a range format throughout the document. The use of range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure unless the context clearly indicates otherwise. Thus, the use of a range includes, unless the context clearly indicates otherwise, all possible sub-ranges, all individual values within the range, and all values or ranges of values, including integers within such range and fractions of values or integers within the range. This structure applies to all contexts of this patent document regardless of the breadth of the range.

For simplicity, certain abbreviations are used herein. One example is a single letter abbreviation representing an amino acid residue. Amino acids and their corresponding three-letter and one-letter abbreviations are as follows:

alanine Ala (A)

Arginine Arg (R)

Asparagine Asn (N)

Asp (D)

Cysteine Cys (C)

Glutamic acid Glu (E)

Glutamine Gln (Q)

Glycine Gly (G)

Histidine His (H)

Isoleucine Ile (I)

Leucine Leu (L)

Lysine Lys (K)

Met methionine (M)

Phe (F)

Proline Pro (P)

Serine Ser (S)

Threonine Thr (T)

Trp tryptophan (W)

Tyrosine Tyr (Y)

Valine Val (V)

II. conjugate molecules-their composition and configuration

In some aspects, provided herein is a conjugate molecule, e.g., a target polypeptide, comprising a second binding moiety that specifically recognizes a target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain"). As described herein, the second binding portion of the conjugate molecules described herein enables a high affinity binding platform that can include various other components to provide a variety of configurations useful for various applications. It is to be understood that the term "second binding moiety" does not mean that there is a separate first binding moiety. In other words, the conjugate molecule may comprise: 1) A single binding moiety that is a second binding moiety, 2) a first moiety and a second binding moiety that are not binding moieties; or it may comprise a first binding moiety and a second binding moiety. Similar reasoning applies to other aspects of the description provided herein, e.g. describing the co-conjugate as comprising a second antibody moiety does not mean that there is a separate first antibody moiety.

For example, in some embodiments, the conjugate molecule comprises a co-conjugate comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an N-terminal truncated antibody variable domain ("N-terminal truncated antibody variable domain"), wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the N-terminal truncated antibody variable domain. In some embodiments, the first binding moiety comprises a first V _H An H domain, wherein the second binding moiety comprises a second V having an N-terminal truncation _H H domain (truncated V) _H H domain "), and wherein the first V _H The C-terminal end of the H domain is connected to the second V via a linker _H N-terminal ligation of H domains.

In some embodiments, the conjugate molecule comprises a first moiety, such as an enzyme, drug or toxin, wherein the first moiety is linked to a second binding moiety via a linker through the N-terminus of an N-terminally truncated antibody variable domain.

In some embodiments, the conjugate molecule comprises a linker, wherein the second binding moiety is linked to the linker through the N-terminus of the N-terminally truncated antibody variable domain. In some embodiments, the conjugate molecule does not comprise a linker.

In the following sections, additional descriptions of various aspects of the conjugate molecules are provided. Such description as being made in a modular fashion is not intended to limit the scope of the present disclosure, and based on the teachings provided herein, one of ordinary skill in the art will readily appreciate that certain modules may be integrated, at least partially. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

In some embodiments, one or more characteristics of the conjugate molecule, such as one or more of FR1, CDR1, VH or VL, are determined according to the IMGT numbering scheme or Kabat numbering scheme.

A. Second binding portion

The conjugate molecules, e.g., co-conjugates, provided herein comprise a second binding moiety that is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncation"). As provided herein, the second antibody portion can take a variety of forms, and includes descriptions that determine its N-terminal truncation. In some embodiments, the second binding moiety further comprises another moiety, such as a conjugate label or a drug.

1. Antibody portion of the second binding moiety

Provided herein are antibody portions comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain"). The antibody portion of the second binding moiety specifically recognizes a target site, such as a polypeptide epitope.

In some embodiments, the antibody portion of the second binding moiety is a variable region (in some embodiments, referred to herein as VR, and optionally, having a numerical designation thereof, e.g., VR 2). In some embodiments, the antibody portion of the second binding moiety is a heavy chain variable region (in some embodiments, referred to herein as a VHAb or VH domain). In some embodiments, the heavy chain variable region is associated with a light chain variable region. In some embodiments, wherein the heavy chain variable region associated with the light chain variable region is single chain, such as scFv. In some embodiments, the heavy chain variable region is linked to at least one constant region and/or the light chain variable region is linked to at least one constant region, such as Fab or scFab. In some embodiments, wherein the heavy chain variable region is associated with a light chain variable region, the heavy chain variable region and the light chain variable region are from the same antibody or antigen binding fragment. In some embodiments, the heavy chain variable region associated with the light chain variable region forms a stable complex. In some embodiments, the heavy chain variable region and the light chain variable region associate with each other to form an antigen binding domain.

In some embodiments, the antibody portion of the second binding moiety is a light chain variable region (in some embodiments, referred to herein as a VLAb or VL domain). In some embodiments, the light chain variable region is the light chain variable region of the human lambda (λ) light chain. In some embodiments, the light chain variable region is a light chain variable region of a human kappa (kappa) light chain. In some embodiments, the light chain variable region is associated with a heavy chain variable region. In some embodiments, wherein the light chain variable region associated with the heavy chain variable region is single chain, e.g., scFv. In some embodiments, the light chain variable region is linked to at least one constant region and/or the heavy chain variable region is linked to at least one constant region, e.g., fab or scFab. In some embodiments, wherein the light chain variable region is associated with a heavy chain variable region, the light chain variable region and the heavy chain variable region are from the same antibody or antigen binding fragment. In some embodiments, the light chain variable region associated with the heavy chain variable region forms a stable complex. In some embodiments, the light chain variable region and the heavy chain variable region associate with each other to form an antigen binding domain.

In some embodiments, the antibody portion of the second binding moiety further comprises one or more constant domains, such as any one or more of CH1, CH2, CH3, or CL.

In some embodiments, the antibody portion of the second binding moiety is V _H H domain. In some embodiments, the antibody portion of the second binding moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'and (Fab') 2 fragments. In some embodiments, the antibody portion of the second binding moiety is a single domain antibody.

In some embodiments, the N-terminal truncated antibody variable region of the second binding moiety is a truncated variable region. In some embodiments, the second binding moietyThe N-terminal truncated antibody variable region of (a) is a truncated heavy chain variable region. In some embodiments, the N-terminal truncated antibody variable region of the second binding moiety is a truncated heavy chain variable region associated with a light chain variable region. In some embodiments, the N-terminal truncated antibody variable region of the second binding moiety is a truncated light chain variable region. In some embodiments, the N-terminal truncated antibody variable region of the second binding moiety is a truncated light chain variable region associated with a heavy chain variable region. In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety is a truncated V _H H domain. In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety is a truncated Fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragment. In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety is a truncated single domain antibody.

The second binding moiety provided herein, or at least a portion thereof, may be obtained or derived from a variety of sources. For example, in some embodiments, the second binding moiety, or at least a portion thereof, is obtained or derived from a camelid, such as a camelid single chain V _H H。

In some embodiments, the second binding moiety or at least a portion thereof is obtained or derived from an affibody, affilin, affimer, affitin, alpha body, anticalin, aptamer, affibody, DARPin, fynomer, kunitz domain peptide, monomer, nanobody (also referred to as a single domain antibody, sdAb), or nanoCLAMP. In some embodiments, the second binding moiety or at least a portion thereof is obtained or derived from IgG, igA, igE, igM or IgD.

In some embodiments, the second binding moiety or at least a portion thereof is obtained or derived from a mammal, including a camel, a human, a non-human primate (such as a monkey), a domestic animal, a farm or a zoo animal, such as a dog, a horse, a rabbit, a cow, a pig, a hamster, a gerbil, a mouse, a ferret, a rat or a cat. In some embodiments, the second binding moiety, or at least a portion thereof, is obtained or derived from a synthetic source.

The antibody portion of the second binding moiety provided herein specifically recognizes the target site. The target sites include a variety of epitopes including epitopes on polypeptides, nucleic acids and small molecules.

2. Truncations and determination thereof

In certain aspects, the second binding moiety described herein is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain").

In some embodiments, the truncation of the second binding moiety is a truncation of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the N-terminal truncation of the second binding moiety is a truncation in framework region 1 (FR 1) of the second binding moiety. In some embodiments, the second binding moiety comprises V _H H comprising an N-terminal truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in framework region 1 (FR 1) of the second binding moiety.

In some embodiments, the second antibody portion (or the N-terminal amino acid of the second antibody portion) has a polypeptide linker X ₃ Amino acids and the origin of complementarity determining region 1 (CDR 1) (as characterized by the first amino acid of CDR1 on the N-terminal amino acid side of CDR 1) are separated by no more than 25 amino acids, such as no more than any one of 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids, 4 amino acids, or 3 amino acids.

N-terminal truncation or truncation from the N-terminus of a polypeptide/protein refers to shortening the polypeptide/protein sequence from the N-terminus (i.e., N-terminus) of the polypeptide/protein. For antibody variable domains (e.g., second antibody portions) included in the conjugate molecule, the N-terminal truncation of the antibody variable domain is determined based on comparison to the full-length antibody variable domain. The FR1 region of the antibody variable region is very conserved,and whether a polypeptide comprises an antibody variable region with an N-terminal truncation can be readily determined by methods known in the art. For example, the corresponding position of an amino acid ("numbered amino acid") in a polypeptide comprising an antibody variable domain may be determined by first aligning the polypeptide sequence with the full-length antibody variable region, or according to any established variable region residue numbering system, such as Kabat, IMGT, EU numbering system, abM, chothia, contact, and AHo. Many computer algorithms have been developed and those algorithms are available to those of ordinary skill in the art from the internet to input sequences and obtain sequences numbered according to any of the specific numbering schemes provided herein. Such exemplary tools include: antigen receptor numbering and receptor classification (ANARCI, opt.stats.ox.ac.uk/webapps/newsabdab/sabred/ANARCI/; described in Dunbar et al, bioinformation.2016, 1/15; 32 (2): 298-300), which is incorporated herein by reference in its entirety; ysis on-line or stand-alone tools developed by prof. Andrew C.R.Martin (bionf. Org. Uk/abs/; abysis. Org /); AHo's Amazing Atlas of Antibody Anatomy (AAAAA; bioc. Uzh. Ch/anti; described in A.Honyger and A.Pluckthun. J.mol. Biol,309 (2001) 657-670, which are incorporated herein by reference in their entirety). Second, each numbered amino acid of the co-conjugate (comprising a portion of the antibody variable domain sequence and possibly the linker sequence) is compared to an amino acid naturally occurring at a frequency at the corresponding numbered position under the same numbering scheme. If the amino acid at position 1 of the numbered amino acids of the co-conjugate occurs no more frequently than about 3% of the naturally occurring antibody variable region, the antibody variable region in the co-conjugate is considered to have a truncation at the first N-terminal amino acid, and the amino acid at position 1 of the numbered amino acids will be considered to be part of the linker sequence. Similarly, if the amino acids at positions 1 and 2 of the numbered amino acids of the co-conjugate occur no more frequently than about 3% of the naturally occurring antibody variable region, then the antibody variable region in the co-conjugate is considered to have truncations at the first N-terminal amino acid and the second N-terminal amino acid (i.e., the N-terminal truncations of the N-terminal truncated antibody variable region are 2 amino acids), and the positions 1 and 2 of the numbered amino acids Amino acids will be considered part of the linker sequence. If the amino acids at positions 1, 2 and 3 of the numbered amino acids of the co-conjugate occur no more frequently than about 3% of the naturally occurring antibody variable region, the antibody variable region in the co-conjugate is considered to have truncations at the first, second and third N-terminal amino acids (i.e., the N-terminal of the N-terminally truncated antibody variable region is truncated to 3 amino acids), and the amino acids at positions 1, 2 and 3 of the numbered amino acids will be considered to be part of the linker sequence. This comparison was repeated for the N-terminal N-position of the amino acid. If the amino acids at positions 1, 2, 3, … … and N of the numbered amino acids of the co-conjugate occur no more frequently than about 3% of the naturally occurring antibody variable region, the antibody variable region in the co-conjugate is considered to have truncations at the first N-terminal amino acid, the second N-terminal amino acid, the third N-terminal amino acid and the N-terminal amino acid (i.e., the N-terminal truncations of the N-terminal truncated antibody variable region are N-amino acids), and the amino acids at positions 1-N of the numbered amino acids will be considered to be part of the linker sequence. In some embodiments, the N-terminal truncation is determined using the ANARCI program (see Dunbar et al, nucleic Acids Res,44,2016). In some embodiments, the N-terminal truncation is determined using the abYsis program (e.g., version 3.4.1; see also Swindex et al, J Mol Biol,429,2017). In some embodiments, the N-terminal truncation is determined using the AAAAA program (see honeygger and plockthun, J Mol Biol,309,2001). Alternatively or additionally, the N-terminal truncation of the antibody variable domain (e.g., the second antibody portion) contained in the conjugate molecule can be determined (or confirmed) by mimicking the tertiary structure of the second binding portion and optionally adjacent residues. Relative to the corresponding full-length FR1 region in the wild-type antibody portion (e.g., V _H H) The shortened β -sheet structure indicates the presence of an N-terminal truncation. Various computer programs for modeling antibody tertiary structure are well known in the art, such as Alphafold (see Jumper et al, nature,596,2021).

Because the second binding moiety is typically located before other amino acid sequences (e.g., linker sequences), the presence of an N-terminal truncation in the second binding moiety may be less apparent by visual inspection of the amino acid sequence alignment. In such cases, the truncation in the second binding moiety may be determined, for example, by the following exemplary method. First, the amino acid sequence of the conjugate molecule (or a portion thereof comprising the second binding moiety and an adjacent amino acid residue) is aligned with the amino acid sequence of an immunoglobulin, such as an isotype of the immunoglobulin (Ig) family to which the second binding moiety belongs. Next, each amino acid of the second binding moiety sequence is then numbered according to the position numbering of the Ig isotype amino acids aligned with the second binding moiety (FIG. 1). Each numbered amino acid is then compared to the amino acids of the Ig family that naturally occur at that numbered position or naturally occur at a frequency. In some embodiments, such a comparison is made at the same numbered position of the Ig family with a frequency of naturally occurring amino acids that is more than 1%, such as more than any of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%. This comparison was repeated for the N-terminal N-position of the amino acid in the second binding portion of the conjugate molecule (FIG. 1). In some embodiments, N is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

Based on the comparison done to determine truncations, if the amino acid at the numbered position is different from the natural amino acid at the corresponding position in the Ig family, the numbered position is a mismatch in the second binding portion of the conjugate molecule, and the mismatched amino acid is defined as the amino acid that is absent or absent in the second binding portion of the conjugate molecule (because the natural amino acid is absent at that position). The number of mismatches or deletions in the first N amino acids is calculated as m=the number of positions in the first N amino acids that do not match the naturally occurring residues. The percent mismatch ("percent mismatch") is calculated as (M/N) ×100%, which is the percentage converted from the ratio of the number of positions within the first N amino acids that do not match the naturally occurring amino acids to the number N. When the% mismatch of the N-terminal N amino acids exceeds a certain threshold, the N-terminal N amino acids have been truncated according to the disclosure provided herein. In some embodiments, the specific threshold for% mismatch is at least about 50%, e.g., at least about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the disclosure indicates that the N-terminal N-amino acids have been truncated when the% mismatch of the N-terminal N-amino acids is at least 50%, e.g., any of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the present disclosure indicates that the N-terminal N amino acids have been truncated when the% mismatch of the N-terminal N amino acids is 100%. In some embodiments, the N-terminal N amino acids have been truncated when the% mismatch of the N-terminal N amino acids is 50% or more. Without being bound by theory, the inventors believe that a mismatch of 50% or more of the N-terminal N-amino acids is associated with disruption of the β -sheet structure at the N-terminal N-amino acid of the second binding moiety. Thus, by structural analysis, the presence of an N-terminal truncation relative to the corresponding full-length FR1 region in the wild-type antibody portion can be confirmed by shortened β -sheet.

The flow chart in fig. 1 shows an iterative process for determining the total number of amino acids missing, deleted and/or truncated from the second binding moiety of the conjugate molecule. In some embodiments, to determine amino acid truncations, an alignment is made between the sequence of the conjugate molecule or a portion thereof (e.g., the sequence of the second binding moiety) and one or more sequences of framework 1 regions (FR 1, framework region 1) of isotype Ig as set forth in tables 3, 4, and 5 (which can be found in the portions entitled certain tables). In some embodiments, to determine amino acid truncations, an alignment is made between the sequence of the conjugate molecule or a portion thereof (e.g., the sequence of the second binding moiety) and the sequence of one or more isotypes Ig, which are disclosed in the database according to the database identifiers listed in the left column of tables 3, 4, and 5, and which are incorporated herein by reference. In some embodiments, to determine amino acid truncations, an alignment is made between the sequence of the conjugate molecule or portion thereof (e.g., the sequence of the second binding moiety) and one or more of the sequences of framework 1 regions (FR 1, framework region 1) of isotype Ig listed in tables 3, 4 and 5, based on the isotype of the conjugate molecule or portion thereof.

The N-terminal truncation in the second binding moiety can also be determined, for example, by the following additional exemplary methods. First, the sequence of the second binding moiety is numbered according to any known antibody numbering scheme, including, for example, kabat, chothia, abM, contact, IMGT or AHo numbering (fig. 2) known to one of ordinary skill in the art and provided herein. Many computer algorithms have been developed and those algorithms are available to those of ordinary skill in the art from the internet to input sequences and obtain sequences numbered according to any of the specific numbering schemes provided herein. Such exemplary tools include: antigen receptor numbering and receptor classification (ANARCI, opt.stats.ox.ac.uk/webapps/newsabdab/sabred/ANARCI/; described in Dunbar et al, bioinformation.2016, 1/15; 32 (2): 298-300), which is incorporated herein by reference in its entirety; ysis on-line or stand-alone tools developed by prof. Andrew C.R.Martin (bionf. Org. Uk/abs/; abysis. Org /); AHo's Amazing Atlas ofAntibody Anatomy (AAAAA; bioc. Uzh. Ch/anti; described in A.Honyger and A.Pluckthun. J.mol. Biol,309 (2001) 657-670, which are incorporated herein by reference in their entirety). Second, each numbered amino acid of the second binding moiety sequence is compared to amino acids naturally occurring or naturally occurring at a frequency at the same numbering position (under the same numbering scheme) in the same Ig family to which the second binding moiety belongs. In some embodiments, such a comparison is made with an amino acid naturally occurring at a frequency that exceeds any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% at the same numbered position of the same Ig family to which the second binding moiety belongs. This comparison was repeated for the N-terminal N position of the amino acid in the second binding moiety (fig. 1). In some embodiments, N is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

Based on the comparisons described herein, a numbered position is a mismatch in a second binding moiety if the second binding moiety is different from the native amino acid at the corresponding position in the Ig family, and a mismatched amino acid is defined as an amino acid that is absent or absent from the second binding moiety (because the native amino acid is absent at that position). The number of mismatches or deletions in the first N amino acids is calculated as m=the number of positions in the first N a.a. that do not match the naturally occurring residues. The percent mismatch ("percent mismatch") is calculated as (M/N) ×100%, which is the percentage converted from the ratio of the number of positions within the first N amino acids that do not match the naturally occurring amino acids to the number N. When the% mismatch of the N-terminal N amino acids exceeds a certain threshold, the present disclosure indicates that the N-terminal N amino acids have been truncated. In one embodiment, the disclosure indicates that an N-terminal N-amino acid has been truncated when the% mismatch of the N-terminal N-amino acid is at least 20%, such as any of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Thus, the total number of amino acids that are missing, deleted, and/or truncated can be determined as described herein. The flow chart in fig. 2 shows an iterative process for determining the total number of amino acids missing, deleted and/or truncated from the second binding moiety to classify the second binding moiety as having an "N-terminally truncated antibody variable domain".

In some embodiments, the natural frequency of occurrence of an amino acid is determined based on any one or more of the sequences provided in tables 3, 4, and 5, such as in the second binding moiety. In some embodiments, the natural frequency of occurrence of an amino acid is determined based on table 7, table 9, and/or table 11, e.g., in the second binding moiety.

In some embodiments, the naturally occurring amino acids at each position in the variable heavy chain at a frequency of greater than 1% according to an antibody numbering scheme, e.g., according to the IMGT numbering scheme, are listed in table 6.

Table 6: naturally occurring amino acids in the variable heavy chain (frequency > 1%).

In some embodiments, the amino acids naturally occurring at each position of the variable heavy chain and their frequency of occurrence according to an antibody numbering scheme, e.g., according to the IMGT numbering scheme, are listed in table 7.

Table 7: naturally occurring amino acids in the variable heavy chain framework 1 (FR 1) region and their frequency of occurrence.

In some embodiments, the naturally occurring amino acids at each position of the variable kappa light chain at a frequency of greater than 2% according to an antibody numbering scheme, e.g., according to the IMGT numbering scheme, are listed in table 8.

Table 8: naturally occurring amino acids in variable kappa light chains (frequency > 2%).

In some embodiments, naturally occurring amino acids at each position of the variable kappa light chain and their frequency of occurrence according to an antibody numbering scheme, e.g., according to the IMGT numbering scheme, are listed in table 9.

Table 9: naturally occurring amino acids in the variable kappa light chain frame 1 (FR 1) region and frequency of occurrence thereof.

In some embodiments, naturally occurring amino acids at each position of the variable lambda light chain at a frequency of more than 2% according to an antibody numbering scheme, e.g., according to the IMGT numbering scheme, are listed in table 10.

Table 10: naturally occurring amino acids in the variable lambda light chain (frequency > 2%).

In some embodiments, naturally occurring amino acids at each position of the variable lambda light chain and their frequency of occurrence according to an antibody numbering scheme, e.g., according to the IMGT numbering scheme, are listed in table 11.

Table 11: naturally occurring amino acids in the variable lambda light chain framework 1 (FR 1) region and frequency of occurrence thereof.

In some embodiments, the second binding moiety may be considered to comprise an "internal" deletion and/or insertion. Such internal deletions and/or insertions may also be considered N-terminally truncated based on the N-terminal truncation determination process described herein. In this case, an "internal" deleted and/or truncated N-terminal sequence will be considered to be part of the linker sequence, but not part of the second binding moiety. The presence of an N-terminal truncation in the second binding moiety can be further confirmed by mimicking the tertiary structure of the conjugate molecule.

In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 1 of VHAb2, is not E or Q. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 1 of VHAb2, is not E, Q or R. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 2 of VHAb2, is not I, L, M or V. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 3 of VHAb2, is not Q or T. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 3 of VHAb2, is not Q, T, H or R. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 4 of VHAb2, is not L or V. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 4 of VHAb2, is not L, V or R. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 5 th amino acid of VHAb2, is not K, L, Q, R or V. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 6 th amino acid of VHAb2, is not E or Q. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 6 of VHAb2, is not E, K, Q or D. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 7 th amino acid of VHAb2, is not P, S or W. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 7 th amino acid of VHAb2, is not P, S, W, L or T. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 8 of VHAb2, is not G. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 8 of VHAb2, is not G, A or V. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 9 of VHAb2, is not A, E, G, P or S. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 11 th amino acid of VHAb2, is not A, E, G, T or V. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 12 of VHAb2, is not L or V. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 13 th amino acid of VHAb2, is not I, K, L, R or V. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 14 of VHAb2, is not K, Q or R. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 14 th amino acid of VHAb2, is not K, Q, R or N. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 15 th amino acid of VHAb2, is not a or P. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 15 th amino acid of VHAb2, is not A, P, D, L or T. In some embodiments, the truncated second binding moiety, e.g., the N-terminal amino acid 16 of VHAb2, is not G, P, S or T. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 17 th amino acid of VHAb2, is not A, D, E, G, Q, R or S. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 17 th amino acid of VHAb2, is not A, D, E, G, Q, R, S, P, T or V. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 18 th amino acid of VHAb2, is not S or T. In some embodiments, the truncated second binding moiety, e.g., the N-terminal 18 th amino acid of VHAb2, is not S, T, A, L or M.

In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety further comprises 1 to 18 amino acid substitutions, e.g., in the framework 1 (FR 1) region.

In some embodiments, the conjugate molecule comprising the second binding moiety comprises the N-terminal amino acid a ₁ Wherein A is ₁ Is any amino acid other than E or Q. In some embodiments, the conjugate molecule comprising the second binding moiety comprises the N-terminal amino acid a ₁ -A ₂ Wherein A is ₁ Is any amino acid other than E or Q, and wherein A ₂ Is any amino acid other than I, L, M or V. In some embodiments, the conjugate molecule comprising the second binding moiety comprises the N-terminal amino acid a ₁ -A ₂ -A ₃ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, and wherein A ₃ Is any amino acid other than Q or T. In some embodiments, the conjugate molecule comprising the second binding moiety comprises the N-terminal amino acid a ₁ -A ₂ -A ₃ -A ₄ Wherein A is ₁ Is any amino group other than E or QAcids, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T, and wherein A ₄ Is any amino acid other than L or V. In some embodiments, the conjugate molecule comprising the second binding moiety comprises the N-terminal amino acid a ₁ -A ₂ -A ₃ -A ₄ -A ₅ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T, wherein A ₄ Is any amino acid other than L or V, and wherein A ₅ Is any amino acid other than K, L, Q, R or V.

3. Other features associated with the second binding moiety

In some embodiments, the second binding moiety is associated with another feature useful for the description provided herein. In some embodiments, the second binding moiety is associated with a drug, and such second binding moiety is covalently conjugated to the drug. In some embodiments, the second binding moiety is associated with a label, such as the second binding moiety is covalently conjugated to an affinity label (e.g., biotin) or a visual label (such as a fluorescent label). In some embodiments, the second binding moiety is associated with an enzyme, e.g., the second binding moiety is covalently conjugated to an enzyme. In some embodiments, the second binding moiety is associated with the toxin, e.g., the second binding moiety is covalently conjugated to the toxin. In some embodiments, the second binding moiety is associated with the nucleic acid, e.g., the second binding moiety is covalently conjugated to the nucleic acid. In some embodiments, the second binding moiety is associated with albumin, such as human serum albumin.

B. Co-conjugate

In certain aspects, provided herein is a co-conjugate comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein optionally the second binding moiety is a second antibody moiety comprising an N-terminal truncated antibody variable domain ("N-terminal truncated antibody variable domain"), and wherein the first binding moiety is linked to the second binding moiety through the N-terminus of the N-terminal truncated antibody variable domain, optionally via a linker. In some embodiments, the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain"). In some embodiments, the first binding moiety is linked to the second binding moiety via a linker (e.g., a polypeptide linker) through the N-terminus of the N-terminally truncated antibody variable domain. In some embodiments, the co-conjugate comprises a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain"), and wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the N-terminal truncated antibody variable domain. In some embodiments, the co-conjugate is a single amino acid chain.

In some embodiments, the co-conjugate specifically recognizes two target sites (epitopes) on a single target antigen, such as a polypeptide. As described herein, the co-conjugate is configured to increase affinity and specificity for a target antigen by specifically recognizing two target sites (epitopes). In some embodiments, the co-conjugate is a multi-specific co-conjugate, such as a dual-specific co-conjugate. In some embodiments, the bispecific co-conjugate recognizes two target antigens in spatial proximity, e.g., in a complex. In some embodiments, the bispecific co-conjugate recognizes two of the same target antigens, e.g., target antigens present in a homodimer.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety comprises a second V comprising an N-terminal truncation _H H domain (' N-terminally truncated V) _H H domain "), wherein the first binding moiety comprises a first V _H An H domain wherein the first binding moiety is truncated N-terminally via a linker to the second binding moiety by N-terminal truncation of the N-terminal antibody variable domain And (5) connection. In some embodiments, the N-terminally truncated V _H The H domain comprises V _H Truncations in the FR1 region of the H domain. In some embodiments, the N-terminally truncated V _H The H domain comprises a truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ Wherein A is ₁ Is any amino acid other than E or Q. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ Wherein A is ₁ Is any amino acid other than E or Q, and wherein A ₂ Is any amino acid other than I, L, M or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, and wherein A ₃ Is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T, and wherein A ₄ Is any amino acid other than L or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ -A ₅ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T, wherein A ₄ Is any amino acid other than L or V, and wherein A ₅ Is any amino acid other than K, L, Q, R or V. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises a C-terminus from N-terminus to C-terminus that forms a polypeptide linkerThree amino acids X of a consecutive series of (2) ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is comprised in V _H The FR1 region of the H domain comprises a N-terminally truncated second V _H H domain (' N-terminally truncated V) _H H domain "), wherein the first binding moiety comprises a first V _H An H domain, wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the N-terminally truncated antibody variable domain. In some embodiments, the N-terminally truncated V _H The H domain comprises a truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ Wherein A is ₁ Is any amino acid other than E or Q. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ Wherein A is ₁ Is any amino acid other than E or Q, and wherein A ₂ Is any amino acid other than I, L, M or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, and wherein A ₃ Is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T,and wherein A is ₄ Is any amino acid other than L or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ -A ₅ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T, wherein A ₄ Is any amino acid other than L or V, and wherein A ₅ Is any amino acid other than K, L, Q, R or V. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises a continuous series of three amino acids X from the N-terminal to the C-terminal direction that form the C-terminal of the polypeptide linker ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is comprised in V _H A second V comprising an N-terminal truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in the FR1 region of the H domain _H H domain (' N-terminally truncated V) _H H domain "), wherein the first binding moiety comprises a first V _H An H domain, wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the N-terminally truncated antibody variable domain. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ Wherein A is ₁ Is any amino acid other than E or Q. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ Wherein A is ₁ Is any amino acid other than E or Q, and wherein A ₂ Is any amino acid other than I, L, M or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, and wherein A ₃ Is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T, and wherein A ₄ Is any amino acid other than L or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ -A ₅ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T, wherein A ₄ Is any amino acid other than L or V, and wherein A ₅ Is any amino acid other than K, L, Q, R or V. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises a continuous series of three amino acids X from the N-terminal to the C-terminal direction that form the C-terminal of the polypeptide linker ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is comprised in V _H A second V comprising an N-terminal truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in the FR1 region of the H domain _H H domain (' N-terminally truncated V) _H H domain "), wherein the first binding moiety comprises a first V _H An H domain in which the N-terminal truncates V _H The H domain comprises the N-terminal amino acid A ₁ Wherein A is ₁ Is any amino acid other than E or Q, and wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the N-terminally truncated antibody variable domain. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ Wherein A is ₁ Is any amino acid other than E or Q, and wherein A ₂ Is any amino acid other than I, L, M or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, and wherein A ₃ Is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T, and wherein A ₄ Is any amino acid other than L or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ -A ₅ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T, wherein A ₄ Is any amino acid other than L or V, and wherein A ₅ Is any amino acid other than K, L, Q, R or V. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises a continuous series of three amino acids X from the N-terminal to the C-terminal direction that form the C-terminal of the polypeptide linker ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K is a,M, C, F, T, P or E; and X is ₃ Is G.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is comprised in V _H A second V comprising an N-terminal truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in the FR1 region of the H domain _H H domain (' N-terminally truncated V) _H H domain "), wherein the first binding moiety comprises a first V _H An H domain in which the N-terminal truncates V _H The H domain comprises the N-terminal amino acid A ₁ Wherein A is ₁ Is any amino acid other than E or Q, wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the N-terminally truncated antibody variable domain, and wherein the linker comprises a contiguous series of three amino acids X forming the C-terminus of the polypeptide linker in the N-terminal to C-terminal direction ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ Wherein A is ₁ Is any amino acid other than E or Q, and wherein A ₂ Is any amino acid other than I, L, M or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, and wherein A ₃ Is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is to divide Q or T byAny amino acid outside, and wherein A ₄ Is any amino acid other than L or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ -A ₅ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T, wherein A ₄ Is any amino acid other than L or V, and wherein A ₅ Is any amino acid other than K, L, Q, R or V. In some embodiments, the linker is a polypeptide linker.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein optionally the second binding moiety is a second antibody moiety comprising a variable domain having an N-terminal truncated antibody ("N-terminal truncated antibody variable domain"), wherein the first binding moiety is linked to the second binding moiety through the N-terminus of the N-terminal truncated antibody variable domain, optionally via a linker. In some embodiments, the affinity of the co-conjugate for binding to the second target site is at least about 3-fold greater than the affinity of a control co-conjugate comprising an antibody variable domain without an N-terminal truncation. In some embodiments, the first target site and the second target site are non-overlapping binding sites on the target molecule. In some embodiments, the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold greater than the affinity of a control co-conjugate comprising an antibody variable domain that does not have an N-terminal truncation. In some embodiments, the first antibody moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragments. In some embodiments, the N-terminal truncated antibody variable domain is a truncated VH or a truncated VL domain. In some embodiments, the second antibody moiety is a single domain antibody. In some embodiments, the N-terminal truncation of the N-terminally truncated antibody variable domain is from about 1 to about 25 amino acids. In some embodiments, the N-terminal truncation of the N-terminal truncated antibody variable domain is 1 amino acid.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein optionally the second binding moiety is a second antibody moiety comprising an N-terminal truncated antibody variable domain ("N-terminal truncated antibody variable domain"), wherein the first binding moiety is the first antibody moiety, and wherein the first binding moiety is linked to the second binding moiety, optionally via a linker, through the N-terminus of the N-terminal truncated antibody variable domain. In some embodiments, the affinity of the co-conjugate for binding to the second target site is at least about 3-fold greater than the affinity of a control co-conjugate comprising an antibody variable domain without an N-terminal truncation. In some embodiments, the first target site and the second target site are non-overlapping binding sites on the target molecule. In some embodiments, the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold greater than the affinity of a control co-conjugate comprising an antibody variable domain that does not have an N-terminal truncation. In some embodiments, the first antibody moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragments. In some embodiments, the N-terminal truncated antibody variable domain is a truncated VH or a truncated VL domain. In some embodiments, the second antibody moiety is a single domain antibody. In some embodiments, the N-terminal truncation of the N-terminally truncated antibody variable domain is from about 1 to about 25 amino acids. In some embodiments, the N-terminal truncation of the N-terminal truncated antibody variable domain is 1 amino acid.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety comprises a second V having an N-terminal truncation _H H domain (' N-terminally truncated V) _H H domain "), wherein the first binding moiety comprises a first V _H An H domain, and wherein the first V _H The C-terminal end of the H domain is connected to the second V via a linker _H N-terminal ligation of H domains. In some embodiments, the affinity of the co-conjugate for binding to the second target site is at least about 3-fold greater than the affinity of a control co-conjugate comprising an antibody variable domain without an N-terminal truncation. In some embodiments, the first target site and the second target site are non-overlapping binding sites on the target molecule. In some embodiments, the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold greater than the affinity of a control co-conjugate comprising an antibody variable domain that does not have an N-terminal truncation. In some embodiments, the first antibody moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragments. In some embodiments, the N-terminal truncation of the N-terminally truncated antibody variable domain is from about 1 to about 25 amino acids.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety comprises a second V having an N-terminal truncation _H H domain (' N-terminally truncated V) _H H domain "), wherein V is truncated at the N-terminus _H N-terminal truncation of H is 1 amino acid, wherein the first binding moiety comprises a first V _H An H domain, and wherein the first V _H The C-terminal end of the H domain is connected to the second V via a linker _H N-terminal ligation of H domains. In some embodiments, the affinity of the co-conjugate for binding to the second target site is at least about 3-fold greater than the affinity of a control co-conjugate comprising an antibody variable domain without an N-terminal truncation. In some embodiments, the first target site and the second target site are non-overlapping binding sites on the target molecule. In some embodiments, the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold greater than the affinity of a control co-conjugate comprising an antibody variable domain that does not have an N-terminal truncation. In some embodiments, the first antibody moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragments. In some embodiments, the C-terminal amino acid of the peptide linker directly linked to the N-terminal truncated antibody variable domain is G. In some embodiments, the three C-terminal amino acids of the peptide linker directly linked to the N-terminal truncated antibody variable domain are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain; wherein the first binding moiety is linked to the second binding moiety via a peptide linker through the N-terminus of the N-terminally truncated antibody variable domain; wherein the three C-terminal amino acids of the peptide linker directly linked to the antibody variable domain of the second binding moiety are X ₁ -X ₂ -X ₃ Wherein X is ₁ Is any amino acid; x is X ₂ K, R, Y, M, G or N; and X is ₃ R, G, Y or P. In some embodiments, the affinity of the co-conjugate to bind to the second target site is at least about 3-fold greater than the affinity of the linker control co-conjugate. In some embodiments, the first target site and the second target site are non-overlapping binding sites on the target molecule. In some embodiments, the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold greater than the affinity of the linker control co-conjugate.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain; wherein the first binding moiety is linked to the second binding moiety via a peptide linker through the N-terminus of the N-terminally truncated antibody variable domain; wherein the three C-terminal amino acids of the peptide linker directly linked to the antibody variable domain of the second binding moiety are X ₁ -X ₂ -X ₃ Wherein X is ₁ Is any amino acid; x is X ₂ K, R, Y, M, G or N; and X is ₃ R, G, Y or P, andand wherein the first binding moiety is a first antibody moiety. In some embodiments, the affinity of the co-conjugate to bind to the second target site is at least about 3-fold greater than the affinity of the linker control co-conjugate. In some embodiments, the first target site and the second target site are non-overlapping binding sites on the target molecule. In some embodiments, the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold greater than the affinity of the linker control co-conjugate. In some embodiments, the antibody variable domain is a VH or VL domain. In some embodiments, the second antibody moiety is a single domain antibody.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the first binding moiety comprises a first V _H An H domain; wherein the second binding moiety comprises a second V _H An H domain, wherein the first V _H The C-terminus of the H domain is linked to the second V via a peptide linker _H N-terminal linkage of H domain, wherein the three amino acids at the C-terminal of the peptide linker directly linked to the antibody variable domain of the second binding moiety are X ₁ -X ₂ -X ₃ Wherein X is ₁ Is any amino acid; x is X ₂ K, R, Y, M, G or N; and X is ₃ R, G, Y or P. In some embodiments, the affinity of the co-conjugate to bind to the second target site is at least about 3-fold greater than the affinity of the linker control co-conjugate. In some embodiments, the first target site and the second target site are non-overlapping binding sites on the target molecule. In some embodiments, the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold greater than the affinity of the linker control co-conjugate.

In some embodiments, a co-conjugate is provided comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety comprises a second V that does not comprise an N-terminal truncation _H An H domain, wherein the first binding moiety comprises a first V _H An H domain, wherein the first binding moiety is variable via a linker by N-terminally truncated antibodiesThe N-terminus of the domain is linked to a second binding moiety, and wherein the three N-terminal amino acids of the second binding moiety are selected from the group consisting of: HKR, FKR, MKR, CKR, QKR, VKR, RKR, LKR, KKR, WKR, SKR, KRG, EKR, YKR, IKR, TKR, NKR, FRR, YRR, AKR, ZLE, ZHQ, MZL, AMV, EHY, TYP, WAP, YMY, IYK, YTY, YYP, QNY, DKR and SGY.

In some embodiments, the N-terminally truncated V _H The H domain comprises V _H Truncations in the FR1 region of the H domain. In some embodiments, the N-terminally truncated V _H The H domain comprises a truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ Wherein A is ₁ Is any amino acid other than E or Q. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ Wherein A is ₁ Is any amino acid other than E or Q, and wherein A ₂ Is any amino acid other than I, L, M or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, and wherein A ₃ Is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is any amino acid other than I, L, M or V, wherein A ₃ Is any amino acid other than Q or T, and wherein A ₄ Is any amino acid other than L or V. In some embodiments, the N-terminally truncated V _H The H domain comprises the N-terminal amino acid A ₁ -A ₂ -A ₃ -A ₄ -A ₅ Wherein A is ₁ Is any amino acid other than E or Q, wherein A ₂ Is to removeI. L, M or any amino acid other than V, wherein A ₃ Is any amino acid other than Q or T, wherein A ₄ Is any amino acid other than L or V, and wherein A ₅ Is any amino acid other than K, L, Q, R or V. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises a continuous series of three amino acids X from the N-terminal to the C-terminal direction that form the C-terminal of the polypeptide linker ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G.

In some embodiments, the affinity of the co-conjugate for binding to the second target site is at least about 3-fold, e.g., at least about 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, or 50-fold, that of a control co-conjugate comprising an antibody variable domain that does not have an N-terminal truncation. In some embodiments, the first target site and the second target site are non-overlapping binding sites on the target molecule. In some embodiments, the first antibody moiety and the second antibody moiety specifically bind to different targets, such as the first antibody moiety specifically binds to a first polypeptide target and the second antibody moiety specifically binds to a second polypeptide target that is different from the first polypeptide target. In some embodiments, the first target site and the second target site are on different target molecules, including homologous targets and heterologous target complexes. In some embodiments, the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold, e.g., at least about 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, or 50-fold, the affinity of a control co-conjugate comprising an antibody variable domain that does not have an N-terminal truncation.

In some aspects, provided herein are co-conjugates (such as high affinity and/or high specificity co-conjugates) that specifically bind to a target and complexes thereof with a target. In some embodiments, the co-conjugate has a first binding moiety, a second binding moiety, and a linker connecting the first binding moiety and the second binding moiety. In some embodiments, the complex comprises a co-conjugate and a target, such as a target molecule, wherein the co-conjugate comprises a first binding moiety, a second binding moiety, and a linker connecting the first binding moiety and the second binding moiety. In some embodiments, the first binding moiety and the second binding moiety bind to a non-overlapping epitope on the target, such as a polypeptide or polypeptide complex. In some embodiments, the first binding moiety and the second binding moiety bind simultaneously to a non-overlapping epitope on the target, such as a polypeptide or polypeptide complex. In some embodiments, the affinity of the co-conjugate for the target is at least 50-fold greater than the affinity of the first binding moiety and/or the second binding moiety, such as any of at least 100-fold greater, 200-fold greater, 500-fold greater, 1000-fold greater, 2000-fold greater, 5000-fold greater, or 10000-fold greater. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker is a nucleic acid linker. In some embodiments, the linker is a chemical linker.

1. First binding portion

The co-conjugates provided herein comprise a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site. Details of the second binding moiety are provided in the section above.

In some embodiments, the first binding moiety is a first antibody moiety. In some embodiments, the first binding moiety is a non-truncated antibody moiety, e.g., a non-truncated form having an N-terminally truncated second binding moiety as described herein. In some embodiments, the first binding moiety is a first antibody moiety comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain"). In some embodiments, the first binding moiety is a first antibody moiety comprising an antibody variable domain having a C-terminal truncation. In some embodiments, the first binding moiety is another molecule that provides affinity for the target site. For example, in some embodiments, the first binding moiety is a ligand that recognizes the receptor or a portion thereof. In some embodiments, the first binding moiety is a receptor or a portion thereof that recognizes a ligand, such as an extracellular domain of a receptor. In some embodiments, the first binding moiety is an aptamer. In some embodiments, the first binding moiety is a non-protein binding moiety, such as biotin or a nucleic acid. In some embodiments, the first binding moiety is a non-immunoglobulin binding agent.

In some embodiments, the antibody portion of the first binding moiety comprises a variable region (in some embodiments, referred to herein as VR, and optionally, having a numerical designation thereof, e.g., VR1 or VR 2). In some embodiments, the antibody portion of the first binding portion comprises a heavy chain variable region (in some embodiments, referred to herein as a VHAb or VH domain). In some embodiments, the heavy chain variable region is associated with a light chain variable region. In some embodiments, wherein the heavy chain variable region is associated with a light chain variable region, the heavy chain variable region and the light chain variable region are from the same antibody or antigen binding fragment. In some embodiments, the heavy chain variable region associated with the light chain variable region forms a stable complex.

In some embodiments, the antibody portion of the first binding portion comprises a light chain variable region (in some embodiments, referred to herein as a VLAb or VL domain). In some embodiments, the light chain variable region is the light chain variable region of the human lambda (λ) light chain. In some embodiments, the light chain variable region is a light chain variable region of a human kappa (kappa) light chain. In some embodiments, the light chain variable region is associated with a heavy chain variable region. In some embodiments, wherein the light chain variable region is associated with a heavy chain variable region, the light chain variable region and the heavy chain variable region are from the same antibody or antigen binding fragment. In some embodiments, the light chain variable region associated with the heavy chain variable region forms a stable complex.

In some embodiments, the antibody portion of the first binding moiety further comprises one or more constant domains, such as any one or more of CH1, CH2, CH3, or CL.

In some embodiments, the antibody portion of the first binding portion comprises V _H H domain. In some embodiments, the antibody portion of the first binding moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'and (Fab') 2 fragments. In some embodiments, the antibody portion of the first binding moiety is a single domain antibody.

In some embodiments, the first binding moiety is a truncated first binding moiety, e.g., comprising an N-terminal and/or C-terminal truncation. In some embodiments, the truncated antibody variable region of the first binding moiety is a truncated variable region. In some embodiments, the truncated antibody variable region of the first binding moiety is a truncated heavy chain variable region. In some embodiments, the truncated antibody variable region of the first binding moiety is a truncated heavy chain variable region associated with a light chain variable region. In some embodiments, the truncated antibody variable region of the first binding moiety is a truncated light chain variable region. In some embodiments, the truncated antibody variable region of the first binding moiety is a truncated light chain variable region associated with a heavy chain variable region. In some embodiments, the truncated antibody variable domain of the first binding moiety is a truncated V _H H domain. In some embodiments, the truncated antibody variable domain of the first binding moiety is a truncated Fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragment. In some embodiments, the truncated antibody variable domain of the first binding moiety is a truncated single domain antibody.

The first binding moiety provided herein, or at least a portion thereof, may be obtained or derived from a variety of sources. For example, in some embodiments, the first binding moiety or at least a portion thereof is obtained or derived from a camelid, e.g., camelid single chain V _H H。

In some embodiments, the first binding moiety or at least a portion thereof is obtained or derived from an affibody, affilin, affimer, affitin, alpha body, anticalin, aptamer, affibody, DARPin, fynomer, kunitz domain peptide, monomer, nanobody (also referred to as a single domain antibody, sdAb), or nanoCLAMP. In some embodiments, the first binding moiety or at least a portion thereof is obtained or derived from IgG, igA, igE, igM or IgD.

In some embodiments, the truncation of the first binding moiety, e.g., N-terminal truncation, is a truncation of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the first The N-terminal truncation of the binding moiety is the truncation in framework region 1 (FR 1) of the second binding moiety. In some embodiments, the first binding moiety comprises V _H H comprising an N-terminal truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in framework region 1 (FR 1) of the first binding moiety.

In some embodiments, the first binding moiety or at least a portion thereof is obtained or derived from a mammal, including a camel, a human, a non-human primate (such as a monkey), a domestic animal, a farm or a zoo animal, such as a dog, a horse, a rabbit, a cow, a pig, a hamster, a gerbil, a mouse, a ferret, a rat or a cat. In some embodiments, the first binding moiety, or at least a portion thereof, is obtained or derived from a synthetic source.

The antibody portion of the first binding moiety provided herein specifically recognizes the target site. The target sites include a variety of epitopes including epitopes on polypeptides, nucleic acids and small molecules.

2. Certain co-conjugate configurations

In certain aspects, the co-conjugates described herein comprise a first antibody portion of a first binding moiety and a second antibody portion of a second binding moiety, wherein the first antibody portion and the second antibody portion independently comprise one of: a Variable Region (VR), a heavy chain variable region (VH or VHAb) or a light chain variable region (VL or VLAb). Those of ordinary skill in the art will readily appreciate that many combinations of first binding moiety and second antibody moiety pairing are possible, including but not limited to any of the following first and second antibody moiety pairing: (i) VR1 and VR2; (ii) VHAb1 and VHAb2; (iii) VHAb1 and VLAb2; (iv) VLAb1 and VHAb2; (v) VLAb1 and VLAb2; (vi) VR1 and VHAb2; (vii) VHAb1 and VR2; (viii) VR1 and VLAb2; and (vii) VLAb1 and VR2. In some embodiments, a heavy chain variable region (e.g., VHAb1 or VHAb 2) is associated with a light chain variable region. In some embodiments, a light chain variable region (e.g., VLAb1 or VLAb 2) is associated with a heavy chain variable region.

In one aspect, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) A first binding moiety comprising a first variable region (VR 1) of a first antibody; (ii) A second binding moiety comprising a second variable region (VR 2) of a second antibody, said second variable region comprising an N-terminal truncation; and (iii) a polypeptide linker that links the VR 1C-terminal amino acid to the N-terminal amino acid of truncated VR 2.

In one aspect, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) A first binding moiety comprising a first variable region (VR 1) of a first antibody; (ii) A second binding moiety comprising a second variable region (VR 2) of a second antibody, said second variable region comprising an N-terminal truncation of 1 to 18 amino acids; and (iii) a polypeptide linker linking the VR 1C-terminal amino acid to the N-terminal amino acid of truncated VR 2; wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

In one aspect, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) A first binding moiety comprising a first variable region (VR 1) of a first antibody; (ii) A second binding moiety comprising a second variable region (VR 2) of a second antibody comprising a truncation of 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VR 1C-terminal amino acid to the N-terminal amino acid of truncated VR 2; wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

In some aspects, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) A first binding moiety comprising a first variable region (VR 1) of a first antibody; (ii) A second binding moiety comprising a second variable region (VR 2) of a second antibody, said second variable region comprising an N-terminal truncation in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VR 1C-terminal amino acid to the N-terminal amino acid of truncated VR 2; wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

In some embodiments, provided herein is a co-conjugate comprising: (i) A first binding moiety comprising a first antibody moiety that specifically recognizes a first target site; (ii) A second binding moiety comprising a second antibody moiety that specifically recognizes a second target site, wherein the second antibody moiety comprises an N-terminally truncated antibody variable domain having from 1 to 18 amino acids; and (iii) a polypeptide linker linking the C-terminal amino acid of the first antibody moiety to the N-terminal amino acid of the second antibody moiety.

In some embodiments, the N-terminal truncation of 1 to 18 amino acids of the second antibody portion is located in the framework 1 (FR 1) region of the second antibody portion. In some embodiments, the second antibody portion of the polypeptide linker of X ₃ The amino acids and the start of complementarity determining region 1 (CDR 1), as characterized by the first amino acid of CDR1 on the N-terminal amino acid side of CDR1, are 5 to 25 amino acids apart. In some embodiments, the second antibody portion of the polypeptide linker of X ₃ The amino acids and the origin of complementarity determining region 1 (CDR 1) are no more than 25 amino acids apart.

The linkers are described in more detail in the section entitled "linkers" provided herein. In some embodiments, the polypeptide linker comprises a continuous series of three amino acids X from the N-terminus to the C-terminus that form the C-terminus of the polypeptide linker ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G.

In one aspect, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) a first variable region (VR 1) of a first antibody; (ii) a second variable region (VR 2) of a second antibody; and (iii) a polypeptide linker linking the VR 1C-terminal amino acid to the N-terminal amino acid of VR 2. In some embodiments, the three amino acids at the C-terminus of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G. In some embodiments, VR1 and VR2 bind to non-overlapping epitopes on the target. In some embodiments, VR2 comprises an N-terminal truncation of 1 to 18 amino acids.

In one aspect, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) Of primary antibodiesA first variable region (VR 1); (ii) A second variable region (VR 2) of a second antibody comprising an N-terminal truncation of 1 to 18 amino acids; and (iii) a polypeptide linker that links the VR 1C-terminal amino acid to the N-terminal amino acid of truncated VR 2. In some embodiments, the three amino acids at the C-terminus of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G. In some embodiments, VR1 and VR2 bind to non-overlapping epitopes on the target.

In some aspects, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) a first variable region (VR 1) of a first antibody; (ii) A second variable region (VR 2) of a second antibody comprising an N-terminal truncation of 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VR 1C-terminal amino acid to the N-terminal amino acid of truncated VR 2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

In some aspects, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) a first variable region (VR 1) of a first antibody; (ii) A second variable region (VR 2) of a second antibody comprising a truncation of 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VR 1C-terminal amino acid to the N-terminal amino acid of truncated VR 2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

In another aspect, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i)A first variable region (VR 1) of a first antibody; (ii) A second variable region (VR 2) of a second antibody comprising a truncation in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VR 1C-terminal amino acid to the N-terminal amino acid of truncated VR 2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; wherein X of the polypeptide linker ₃ Amino acids and VR2 complementarity determining region 1 (CDR 1) are 5 to 25 amino acids apart; and wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

In another aspect, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) a first variable region (VR 1) of a first antibody; (ii) A second variable region (VR 2) of a second antibody comprising a truncation in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VR 1C-terminal amino acid to the N-terminal amino acid of truncated VR 2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; wherein X of the polypeptide linker ₃ Amino acids and VR2 complementarity determining region 1 (CDR 1) are no more than 25 amino acids apart; and wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

In another aspect, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) a first variable region (VR 1) of a first antibody; (ii) A second variable region (VR 2) of a second antibody comprising a truncation of 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VR 1C-terminal amino acid to the N-terminal amino acid of truncated VR 2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; wherein X of the polypeptide linker ₃ Amino groupThe acid and VR2 complementarity determining region 1 (CDR 1) are 5 to 25 amino acids apart; and wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

In another aspect, provided herein is a co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) a first variable region (VR 1) of a first antibody; (ii) A second variable region (VR 2) of a second antibody comprising a truncation of 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VR 1C-terminal amino acid to the N-terminal amino acid of truncated VR 2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; wherein X of the polypeptide linker ₃ Amino acids and VR2 complementarity determining region 1 (CDR 1) are no more than 25 amino acids apart; and wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

In some embodiments, VR1 is a light chain variable region. In some embodiments, VR1 is a heavy chain variable region. In some embodiments, VR2 is a light chain variable region. In some embodiments, VR2 is a heavy chain variable region. In some embodiments, VR1 is a light chain variable region and VR2 is a light chain variable region. In some embodiments, VR1 is a light chain variable region and VR2 is a heavy chain variable region. In some embodiments, VR1 is a heavy chain variable region and VR2 is a light chain variable region. In some embodiments, VR1 is a heavy chain variable region and VR2 is a heavy chain variable region. In some embodiments, VR1 is V _H H. In some embodiments, VR2 is V _H H. In some embodiments, VR1 is V _H H and VR2 is V _H H

In some embodiments, the N-terminal truncation of the second binding moiety is a truncation of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the second binding moiety comprises X of a polypeptide linker ₃ Amino acids and CDR1 are separated by no more than 25 amino acids, such as no more than 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids,Any of 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids, 4 amino acids, or 3 amino acids. In some embodiments, the second binding moiety comprises X of a polypeptide linker ₃ Amino acids and CDR1 are separated by any one of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, or 25 amino acids. In some embodiments, the N-terminal truncation of the second binding moiety is a truncation in framework region 1 (FR 1) of the second binding moiety.

In some embodiments, the variable region, e.g., the N-terminally truncated antibody variable domain of the second binding moiety, is V _H H. In some embodiments, the first binding moiety comprises a first V _H An H domain; wherein the second binding moiety comprises a second V having an N-terminal truncation _H H domain (truncated V) _H H domain "), wherein the first V _H The C-terminal end of the H domain is connected to the second V via a linker _H N-terminal ligation of H domains.

Thus, in some aspects, provided herein is a co-conjugate comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second V having an N-terminal truncation _H H domain (truncated V) _H H domain "), wherein the first binding moiety comprises a first V _H An H domain, and wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the N-terminally truncated antibody variable domain. In some embodiments, the truncated V of the second binding moiety _H H domains are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17. A truncation of any of 18, 19 or 20 amino acids. In some embodiments, the second binding moiety comprises X of a polypeptide linker ₃ Amino acids and CDR1 are separated by no more than 25 amino acids, such as any one of no more than 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids, 4 amino acids, or 3 amino acids. In some embodiments, the second binding moiety comprises X of a polypeptide linker ₃ Amino acids and CDR1 are separated by any one of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, or 25 amino acids. In some embodiments, the N-terminal truncation of the second binding moiety is a truncation in framework region 1 (FR 1) of the second binding moiety. In some embodiments, the three amino acids at the C-terminus of the linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G.

In some embodiments, the co-conjugate has only a first binding moiety and a second binding moiety that bind non-overlapping and different epitopes on the target molecule. In some embodiments, the co-conjugate may also have a third binding moiety that binds a third, non-overlapping and different epitope on the target molecule. In some embodiments, the co-conjugate may also have a third binding moiety and a fourth binding moiety, each of which binds to a third and fourth non-overlapping and different epitope on the target molecule. These third binding moiety and/or fourth binding moiety may or may not be N-terminally truncated as described for the second binding moiety.

The co-conjugate may be a monomeric or multimeric complex. In some embodiments, the co-conjugate is a monomer molecule having a set of binding moieties. In some embodiments, the co-conjugate is a monomer molecule having a set of a first binding moiety and a second binding moiety. In some embodiments, the co-conjugate is a monomeric molecule having a set of a first binding moiety, a second binding moiety, and a third binding moiety. In some embodiments, the co-conjugate is a monomeric molecule having a set of a first binding moiety, a second binding moiety, a third binding moiety, and a fourth binding moiety.

In some embodiments, the co-conjugate is a multimeric complex having at least two sets of binding moieties. In some embodiments, the co-conjugate is a multimeric complex having at least three sets of binding moieties. In some embodiments, the co-conjugate is a multimeric complex having at least four sets of binding moieties. In some embodiments, the co-conjugate is a multimeric complex having two sets of binding moieties. In some embodiments, the co-conjugate is a multimeric complex having two sets of a first binding moiety and a second binding moiety. In some embodiments, the co-conjugate is a multimeric complex having two sets of a first binding moiety, a second binding moiety, and a third binding moiety. In some embodiments, the co-conjugate is a multimeric complex having two sets of a first binding moiety, a second binding moiety, a third binding moiety, and a fourth binding moiety. In some embodiments, the co-conjugate is a multimeric complex having three sets of binding moieties. In some embodiments, the co-conjugate is a multimeric complex having three sets of a first binding moiety and a second binding moiety. In some embodiments, the co-conjugate is a multimeric complex having three sets of a first binding moiety, a second binding moiety, and a third binding moiety. In some embodiments, the co-conjugate is a multimeric complex having three sets of a first binding moiety, a second binding moiety, a third binding moiety, and a fourth binding moiety. In some embodiments, the co-conjugate is a multimeric complex having four sets of binding moieties. In some embodiments, the co-conjugate is a multimeric complex having four sets of a first binding moiety and a second binding moiety. In some embodiments, the co-conjugate is a multimeric complex having four sets of a first binding moiety, a second binding moiety, and a third binding moiety. In some embodiments, the co-conjugate is a multimeric complex having four sets of a first binding moiety, a second binding moiety, a third binding moiety, and a fourth binding moiety.

In some embodiments, the co-conjugates that are multimeric complexes may have different orientations of the various sets of binding moieties. In some embodiments, the binding moieties of each set are arranged in sequence. For example, two sets of a first binding moiety (containing paratope P1) and a second binding moiety (containing paratope P2) may be arranged as P1-P2-P1-P2. For another example, two sets of first binding moieties (containing paratope P1), second binding moieties (containing paratope P2) and their binding moieties (containing paratope P3) may be arranged as P1-P2-P3-P1-P2-P3. In some embodiments, the binding moieties of each set are arranged in a reverse orientation. For example, two sets of a first binding moiety (containing paratope P1) and a second binding moiety (containing paratope P2) may be arranged as P1-P2-P2-P1. For another example, two sets of first binding moieties (containing paratope P1), second binding moieties (containing paratope P2) and their binding moieties (containing paratope P3) may be arranged as P1-P2-P3-P3-P2-P1. In some embodiments, the sets of binding moieties are arranged in a staggered fashion. For example, two sets of a first binding moiety (containing paratope P1) and a second binding moiety (containing paratope P2) may be arranged as P1-P1-P2-P2. For another example, two sets of first binding moieties (containing paratope P1), second binding moieties (containing paratope P2) and their binding moieties (containing paratope P3) may be arranged as P1-P1-P2-P2-P3-P3. The binding moieties of the multimeric co-conjugates described herein can be arranged in any order, as will be appreciated by those of ordinary skill in the art. In some embodiments, the order of the binding moieties of the multimeric co-conjugates is optimized to maximize binding affinity to the target molecule and/or minimize any non-specific binding.

In some embodiments, the co-conjugates disclosed herein have a first binding moiety and a second binding moiety that bind two distinct and non-overlapping epitopes in a target molecule. Two different and non-overlapping epitopes in a target molecule can be in relative proximity to each other. In some embodiments, the two epitopes recognized by the co-conjugate are close to each other, but there is still sufficient space to accommodate the linker of the co-conjugate. In some embodiments, the first epitope and the second epitope have a distance of no more than 150 angstroms, such as no more than about any one of 120 angstroms, 100 angstroms, 80 angstroms, 50 angstroms, 40 angstroms, 30 angstroms, 15 angstroms, 10 angstroms, or 5 angstroms.

For linear epitopes on the target peptide or target protein, the distance between the two epitopes may be within 200 amino acids of each other, for example within any of about 150 amino acids of each other, 120 amino acids of each other, 100 amino acids of each other, 80 amino acids of each other, 50 amino acids of each other, 40 amino acids of each other, 30 amino acids of each other, 20 amino acids of each other, 15 amino acids of each other, 10 amino acids of each other, or 5 amino acids of each other. In some embodiments, the two epitopes recognized by the co-conjugate are selected such that the two binding interactions are cooperative and synergistic and do not interfere with each other.

In some embodiments, the co-conjugate comprises a first antibody moiety (VR 1) as a variable region and a second antibody moiety (VR 2) as a variable region. In some embodiments, VR1 binds to an epitope of the target, thereby producing a desired biological effect on the target, but with insufficient binding affinity to the target for the VR1 itself to be used for therapeutic or diagnostic purposes; VR2 binds with sufficient affinity to different epitopes of the target; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy. In some embodiments of the co-conjugates provided herein, VR2 binds to an epitope of the target, thereby producing a desired biological effect on the target, but does not bind to the target with sufficient affinity for the VR2 itself for therapeutic or diagnostic use; VR1 binds with sufficient affinity to different epitopes of the target; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy. In some embodiments of the co-conjugates provided herein, the first binding moiety binds to an epitope of the target, thereby producing a desired biological effect on the target, but does not bind to the target with sufficient affinity for the first binding moiety itself for therapeutic or diagnostic use; the second binding moiety binds to a different epitope of the target with sufficient affinity; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy. In some embodiments of the co-conjugates provided herein, the second binding moiety binds to an epitope of the target, thereby producing a desired biological effect on the target, but does not bind to the target with sufficient affinity for the second binding moiety itself for therapeutic or diagnostic use; the first binding moiety binds to a different epitope of the target with sufficient affinity; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy.

In some embodiments, the co-conjugate comprises a first antibody moiety that is a heavy chain variable region of a first antibody (VHAb 1) and a second antibody moiety that is a heavy chain variable region of a second antibody (VHAb 2). In some embodiments, VHAb1 binds to an epitope of the target, thereby producing a desired biological effect on the target, but does not bind to the target with sufficient affinity for VHAb1 itself for therapeutic or diagnostic use; VHAb2 binds with sufficient affinity to different epitopes of the target; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy. In some embodiments of the co-conjugates provided herein, VHAb2 binds to an epitope of the target, thereby producing a desired biological effect on the target, but does not bind with sufficient affinity to the target for VHAb2 itself for therapeutic or diagnostic use; VHAb1 binds with sufficient affinity to different epitopes of the target; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy.

In some embodiments, the co-conjugate comprises a first antibody moiety that is a light chain variable region of a first antibody (VLAb 1) and a second antibody moiety that is a heavy chain variable region of a second antibody (VHAb 2). In some embodiments, VLAb1 binds to an epitope of the target, thereby producing a desired biological effect on the target, but does not bind to the target with sufficient affinity for the VLAb1 itself to be used for therapeutic or diagnostic purposes; VHAb2 binds with sufficient affinity to different epitopes of the target; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy. In some embodiments of the co-conjugates provided herein, VHAb2 binds to an epitope of the target, thereby producing a desired biological effect on the target, but does not bind with sufficient affinity to the target for VHAb2 itself for therapeutic or diagnostic use; VLAb1 binds with sufficient affinity to different epitopes of the target; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy.

In some embodiments, the co-conjugate comprises a first antibody moiety that is a heavy chain variable region of a first antibody (VHAb 1) and a second antibody moiety that is a light chain variable region of a second antibody (VLAb 2). In some cases, VHAb1 binds to an epitope of the target, thereby producing a desired biological effect on the target, but does not bind with sufficient affinity to the target for therapeutic or diagnostic use of VHAb1 itself; VLAb2 binds with sufficient affinity to different epitopes of the target; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy. In some embodiments of the co-conjugates provided herein, VLAb2 binds to an epitope of the target, thereby producing a desired biological effect on the target, but with insufficient affinity to bind the target for use by the VLAb2 itself in therapeutic or diagnostic use; VHAb1 binds with sufficient affinity to different epitopes of the target; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy.

In some embodiments, the co-conjugate comprises a first antibody moiety that is a light chain variable region of a first antibody (VLAb 1) and a second antibody moiety that is a light chain variable region of a second antibody (VLAb 2). In some embodiments of the co-conjugates provided herein, VLAb1 binds to an epitope of the target, thereby producing a desired biological effect on the target, but with insufficient affinity to bind the target for use by the VLAb1 itself in therapeutic or diagnostic use; VLAb2 binds with sufficient affinity to different epitopes of the target; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy. In some embodiments of the co-conjugates provided herein, VLAb2 binds to an epitope of the target, thereby producing a desired biological effect on the target, but with insufficient affinity to bind the target for use by the VLAb2 itself in therapeutic or diagnostic use; VLAb1 binds with sufficient affinity to different epitopes of the target; and the resulting co-conjugate binds the target with sufficient affinity and produces the desired biological efficacy.

In some embodiments of the co-conjugates provided herein, VR1 binds to an epitope of the target, thereby producing a desired biological effect on the target, and binds to an epitope other than the target, thereby producing an undesired side effect; VR2 binds with high affinity to a different epitope of the target, but not with sufficient affinity to bind to a non-target; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects. In some embodiments of the co-conjugates provided herein, VR2 binds to an epitope of the target, thereby producing a desired biological effect on the target, but also binds to an epitope other than the target, thereby producing an undesired side effect; VR1 binds with high affinity to a different epitope of the target, but not with sufficient affinity to bind to a non-target; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects.

In some embodiments of the co-conjugates provided herein, the first binding moiety binds to an epitope of the target, thereby producing a desired biological effect on the target, and also binds to a non-target epitope, thereby producing an undesired side effect; the second binding moiety binds to a different epitope of the target with high affinity, but does not bind to the non-target with sufficient affinity; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects. In some embodiments of the co-conjugates provided herein, the second binding moiety binds to an epitope of the target, thereby producing a desired biological effect on the target, but also binds to a non-target epitope, thereby producing an undesired side effect; the first binding moiety binds to a different epitope of the target with high affinity, but does not bind to the non-target with sufficient affinity; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects.

In some embodiments of the co-conjugates provided herein, VHAb1 binds to an epitope of the target, thereby producing a desired biological effect on the target, but also binds to a non-target epitope, thereby producing an undesired side effect; VHAb2 binds with high affinity to a different epitope of the target, but not with sufficient affinity to bind to a non-target; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects. In some embodiments of the co-conjugates provided herein, VHAb2 binds to an epitope of the target, thereby producing a desired biological effect on the target, but also binds to a non-target epitope, thereby producing an undesired side effect; VHAb1 binds with high affinity to a different epitope of the target, but not with sufficient affinity to bind to a non-target; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects.

In some embodiments of the co-conjugates provided herein, VLAb1 binds to an epitope of the target, thereby producing a desired biological effect on the target, but also binds to an epitope other than the target, thereby producing an undesired side effect; VHAb2 binds with high affinity to a different epitope of the target, but not with sufficient affinity to bind to a non-target; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects. In some embodiments of the co-conjugates provided herein, VHAb2 binds to an epitope of the target, thereby producing a desired biological effect on the target, but also binds to a non-target epitope, thereby producing an undesired side effect; VLAb1 binds to a different epitope of the target with high affinity, but not to a non-target with sufficient affinity; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects.

In some embodiments of the co-conjugates provided herein, VHAb1 binds to an epitope of the target, thereby producing a desired biological effect on the target, but also binds to a non-target epitope, thereby producing an undesired side effect; VLAb2 binds to a different epitope of the target with high affinity, but not to a non-target with sufficient affinity; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects. In some embodiments of the co-conjugates provided herein, VLAb2 binds to an epitope of the target, thereby producing a desired biological effect on the target, but also binds to an epitope other than the target, thereby producing an undesired side effect; VHAb1 binds with high affinity to a different epitope of the target, but not with sufficient affinity to bind to a non-target; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects.

In some embodiments of the co-conjugates provided herein, VLAb1 binds to an epitope of the target, thereby producing a desired biological effect on the target, but also binds to an epitope other than the target, thereby producing an undesired side effect; VLAb2 binds to a different epitope of the target with high affinity, but not to a non-target with sufficient affinity; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects. In some embodiments of the co-conjugates provided herein, VLAb2 binds to an epitope of the target, thereby producing a desired biological effect on the target, but also binds to an epitope other than the target, thereby producing an undesired side effect; VLAb1 binds to a different epitope of the target with high affinity, but not to a non-target with sufficient affinity; and the resulting co-conjugates bind the target with sufficient affinity and produce the desired biological efficacy, but do not bind non-targets with sufficient affinity to produce undesirable side effects.

The present disclosure demonstrates that various VH or VL can be used to construct the co-conjugates provided herein, and obtain the affinity and/or specificity improvements provided for such co-conjugates. In one embodiment, the antigen-binding fragments (e.g., VH and/or VL) contained in the second binding portion of the co-conjugates provided herein can be selected based on the proximity of the paratope of the antigen-binding fragment to the N-terminus of the antigen-binding fragment. In certain embodiments, the co-binders can be constructed with VH, with the paratope of VH immediately adjacent to the N-terminus of VH, and produce the affinity and/or specificity improvements provided for the co-binders of the present disclosure. In some embodiments, the co-conjugate can be constructed with VH as part of the second binding moiety, and produce the affinity and/or specificity improvements provided for the co-conjugate of the present disclosure, wherein the paratope of VH is immediately adjacent to the N-terminus of VH. In other embodiments, the co-binders can be constructed with VHAb2, with an epitope of VHAb2 immediately adjacent to the N-terminus of VHAb2, and produce an improvement in affinity and/or specificity provided for the co-binders of the present disclosure. In certain embodiments, the co-conjugates can be constructed with a VL, with the paratope of the VL immediately adjacent to the N-terminus of the VL, and produce an improvement in affinity and/or specificity provided for the co-conjugates of the present disclosure. In some embodiments, the co-conjugates can be constructed with VL as part of the second binding moiety and produce an improvement in affinity and/or specificity provided for the co-conjugates of the present disclosure, wherein the paratope of VL is immediately adjacent to the N-terminus of VL. In other embodiments, the co-conjugates can be constructed with VLAb2 and produce an improvement in affinity and/or specificity provided for the co-conjugates of the present disclosure, wherein the paratope of VLAb2 is immediately adjacent to the N-terminus of VLAb 2. In some embodiments, the co-conjugates can be constructed with Variable Regions (VR) and result in improved affinity and/or specificity provided for the co-conjugates of the present disclosure, wherein the paratope of the VR is immediately adjacent to the N-terminus of the VR. In certain embodiments, the co-conjugates can be constructed with a variable region as part of the second binding moiety (VR 2) and result in an improvement in affinity and/or specificity provided for the co-conjugates of the present disclosure, wherein the paratope of VR2 is immediately adjacent to the N-terminus of VR 2. VH, VH, VHAb, VHAb, VL as part of the second binding moiety, VL, VLAb, VLAb, VR as part of the second binding moiety, and VR2 of this paragraph can be any of the corresponding embodiments described herein.

The proximity between the paratope of an antigen binding fragment (e.g., VH, VL, VHAb, VHAb2, VLAb2, VR, and VR 2) and the N-terminus of such an antigen binding fragment can be determined based on the structure of the antigen binding fragment and/or the structure of the complex of such an antigen binding fragment with its target antigen. In some embodiments, the antigen binding fragment and its targetIn the structure of the complex of antigens, the nearest non-hydrogen atom on the antigen surface can be used as a representation of the paratope for determining the proximity of the paratope to the N-terminus of the antigen binding fragment. Thus, proximity may be determined based on the distance between the first C alpha atom (N-terminal) of such antigen binding fragment and the nearest non-hydrogen atom on the antigen surface in the structure of the complex of the antigen binding fragment and its target antigen. In one embodiment, antigen binding fragments (e.g., VH, VL, VHAb, VHAb2, VLAb2, VR and VR 2) as described herein are suitable for use in constructing the co-conjugates provided herein and result in improved affinity and/or specificity for the co-conjugates of the present disclosure if the proximity determined by the distance between the first C alpha atom (N-terminus) of the antigen binding fragment and the nearest non-hydrogen atom on the antigen surface does not exceed or is about a threshold, where such threshold is required to provide sufficient space for linking the two binding moieties of the co-conjugate without interfering with binding to the target antigen. In another embodiment, an antigen binding fragment as described herein (e.g., VH, VL, VHAb, VHAb2, VLAb2, VR and VR 2) is suitable for use as a second binding moiety or part of a second binding moiety if the proximity determined by the distance between the first C alpha atom (N-terminus) of the antigen binding fragment and the nearest non-hydrogen atom on the antigen surface does not exceed or is about a threshold that is required to provide sufficient space for linking the two binding moieties of the co-conjugate without interfering with binding to the target antigen to construct the co-conjugate provided herein and to produce the affinity and/or specificity improvements provided for the co-conjugate of the present disclosure. In a particular embodiment, the proximity referred to herein is no more than about Such as no more than about-> Or (b)

The present disclosure further demonstrates that the proximity between the N-terminus of an antigen binding fragment and the paratope of the antigen binding fragment (e.g., using the nearest non-hydrogen atom on the surface of the binding antigen) can be determined by looking at such proximity from existing structures in the database (e.g., PDB). Such proximity may also be determined via homologous structure modeling using many structures available in the structure database, as practiced by those skilled in the art. Furthermore, such proximity may be determined by other structure modeling software or methods such as Molecular Dynamics or Molecular Mechanics (e.g., CHARMM, AMBER, and NAMD) and other structures determined from computational protein modeling (e.g., rosetta), as practiced by those skilled in the art. Accordingly, the present disclosure demonstrates and one of ordinary skill in the art will appreciate that the structure of an antigen to which an antigen-binding fragment binds and the proximity between the N-terminus of the antigen-binding fragment and the paratope (e.g., using the nearest non-hydrogen atom on the surface of the binding antigen as a representation) can be determined without having to experimentally determine any structure.

Alternatively, the proximity between the N-terminus of the antigen binding fragment and the paratope (e.g., using the nearest non-hydrogen atom on the surface of the binding antigen as a proxy, for example) may be determined using the functional effect of placing a linkage at the N-terminus of the antigen binding fragment as a reporter of such proximity. As described above, the proximity between the N-terminus of the antigen binding fragment and the paratope is used to determine whether there is sufficient space to link the two binding portions of the co-conjugate without interfering with binding to the target antigen. The present disclosure further shows that if such proximity between the N-terminus of the antigen binding fragment and the paratope is below a certain threshold that is needed to provide sufficient space to connect the two binding moieties of the co-conjugate without interfering with binding to the target antigen, the affinity of the antigen binding fragment will be negatively affected when inserting or connecting the linker to the N-terminus of the antigen binding fragment. Thus, the present disclosure shows that the change in affinity after insertion or ligation of a linker to the N-terminus of an antigen binding fragment can be correlated with determining whether the proximity between the N-terminus of the antigen binding fragment and the paratope is below a certain threshold sufficient to ligate the two binding moieties of the co-conjugate without interfering with binding to the target antigen.

Thus, the present disclosure demonstrates that antigen binding fragments (e.g., VH, VL, VHAb, VHAb, VLAb2, VR and VR 2) as described herein are suitable for use in constructing the co-conjugates provided herein and result in improved affinity and/or specificity for the co-conjugates of the present disclosure if the affinity of the antigen binding fragment for the antigen changes by more than a certain threshold upon insertion or ligation of a linker to the N-terminus of the antigen binding fragment. Briefly, in some embodiments, antigen binding fragments of the target ("ABFs") may be synthesized via (GGGS) ₄ The linker is fused to a reference immunoglobulin domain (refIg) that does not specifically bind to the target to produce a refIg-GS-ABF construct. If the fusion construct refIg-GS-ABF has a weaker affinity for the target than ABF has for the target by a certain threshold, such ABF is suitable for use in constructing the co-conjugates provided herein and results in an improvement in affinity and/or specificity provided for the co-conjugates of the present disclosure. In a specific embodiment, the fusion construct refIg-GS-ABF has an affinity for the target that is at least 2-fold weaker than the affinity of ABF for the target, e.g., at least any one of the following: 3. 4, 5, 6, 7, 8, 9, 10, 15 or 20.

Similarly, since affinity can be determined by dissociation equilibrium constants (K _D ) The present disclosure shows that the antigen binding fragments described herein (e.g., VH, VL, VHAb, VHAb2, VLAb2, VR and VR 2) are suitable for use in constructing the co-conjugates provided herein and result in improved affinity and/or specificity for the co-conjugates of the present disclosure if the antigen binding fragment is K to the antigen _D The linker is inserted or ligated into the N-terminus of the antigen binding fragment by a change above a certain threshold. Briefly, in some embodiments, antigen binding fragments of the target ("ABFs") may be synthesized via (GGGS) ₄ The linker is fused to a reference immunoglobulin domain (refIg) that does not specifically bind to the target to produce a refIg-GS-ABF construct. If the fusion construct refIg-GS-ABF targets K _D K to target than ABF _D Above a certain threshold, such ABFs are suitable for use in constructing the co-conjugates provided herein and result in improved affinity and/or specificity provided for the co-conjugates of the present disclosure. In a specific embodiment, the fusion construct refIg-GS-ABF pairs target K _D K to target than ABF _D At least 2 times greater, for example at least any of the following: 3. 4, 5, 6, 7, 8, 9, 10, 15 or 20 times.

Furthermore, in some embodiments provided herein, an antigen binding fragment of a target ("ABF") can be fused to a reference immunoglobulin domain (refIg) that does not specifically bind to the target via a (GGGS) x linker, where x can be 1, 2, 3, 4, 5, 6, 7, or 8, to produce a refIg-GS-ABF construct. In one embodiment, the ABF of the target may be fused via a GGGS linker to refIg that does not specifically bind to the target to produce the refIg-GS-ABF constructs described herein. In another embodiment, the ABF of the target may be fused via a (GGGS) 2 linker to refIg that does not specifically bind to the target to produce the refIg-GS-ABF construct described herein. In another embodiment, the ABF of the target may be fused via a (GGGS) 3 linker to refIg that does not specifically bind to the target to produce the refIg-GS-ABF construct described herein. In yet another embodiment, the ABF of the target may be fused via a (GGGS) 4 linker to refIg that does not specifically bind to the target to produce the refIg-GS-ABF constructs described herein. In one embodiment, the ABF of the target may be fused via a (GGGS) 5 linker to refIg that does not specifically bind to the target to produce the refIg-GS-ABF constructs described herein. In another embodiment, the ABF of the target may be fused via a (GGGS) 6 linker to refIg that does not specifically bind to the target to produce the refIg-GS-ABF construct described herein. In another embodiment, the ABF of the target may be fused via a (GGGS) 7 linker to refIg that does not specifically bind to the target to produce the refIg-GS-ABF construct described herein. In yet another embodiment, the ABF of the target may be fused via a (GGGS) 8 linker to refIg that does not specifically bind to the target to produce the refIg-GS-ABF constructs described herein.

The present disclosure shows that refIg described herein can be any immunoglobulin domain (e.g., VH, VL, scFv, VHH) as long as refIg does not specifically bind to the same antigen to which the co-conjugate is specifically constructed to bind. For example and as described herein, when co-binders are constructed to bind EGFR, anti-human lysozyme VHH HuL6 can be used as such refIg. Similarly, in some embodiments, refIg may be an antigen binding domain or fragment of an isotype immunoglobulin (e.g., VH, VL, scFv or VHH). In certain embodiments, refIg may be an antigen binding domain or fragment (e.g., VH, VL, scFv or VHH) that binds an antigen that is different from the antigen to which the co-conjugate is specifically constructed to bind.

C. Joint

In certain aspects of the conjugate molecules described herein, the conjugate molecules comprise a linker. Typically, the linkers described herein are associated, e.g., covalently associated, with one or more components of the conjugate molecules described herein. For example, in some embodiments, the co-conjugate comprises a second binding moiety and a linker, wherein the linker is attached to the second binding moiety via the N-terminus of the second binding moiety. In some embodiments, the co-conjugate comprises a linker connecting the first binding moiety and the second binding moiety, wherein the linker is attached to the second binding moiety via the N-terminus of the second binding moiety, and wherein the linker is attached to the first binding moiety via the C-terminus of the first binding moiety. In some embodiments, the second binding moiety is an N-terminally truncated antibody variable domain. In some embodiments, the linker connects the first binding moiety and the second binding moiety via a covalent bond. In some embodiments, the linker connects the first binding moiety and the second binding moiety via a combination of covalent and non-covalent bonds, e.g., the linker is covalently bound to the second binding moiety or the first binding moiety and non-covalently bound to the other binding moiety. In some embodiments, the linker of the co-conjugate facilitates the co-conjugate to achieve a cooperative and/or synergistic binding interaction with its target molecule. As described herein, the linker may take a variety of forms and may be selected based on a variety of characteristics.

In some embodiments, the linker comprises a polypeptide. In some embodiments, the linker is a polypeptide. In some embodiments, the linker comprises a polypeptide complex, such as a polypeptide complex comprising two or more polypeptide subunits. In some embodiments, the linker comprises a polynucleotide. In some embodiments, the linker is a polynucleotide. In some embodiments, the linker is a polynucleotide complex, such as a first polynucleotide strand and a second polynucleotide strand having complementary regions. In some embodiments, the linker is a chemical or synthetic linker, such as a polymer-based linker.

In some embodiments, the linker is covalently attached to the binding moiety of the conjugate molecule. For example, in some embodiments, the conjugate molecule comprises a second binding moiety and a linker, wherein the second binding moiety and the linker are a single polypeptide. In some embodiments, the linker is non-covalently associated with the binding moiety of the conjugate molecule.

In some embodiments, the linker is associated, such as covalently attached, with the N-terminus of the second binding moiety. In some embodiments, the terminal portion of the linker associated (e.g., covalently attached) to the N-terminus of the second binding moiety comprises three amino acids X ₁ -X ₂ -X ₃ . For example, in some embodiments, the linker comprises a polypeptide in which the C-terminal portion of the linker associated (e.g., covalently attached) with the N-terminus of the second binding moiety comprises three amino acids X in the N-terminal to C-terminal direction ₁ -X ₂ -X ₃ . In some embodiments, X ₃ For example covalently attached via a peptide bond to the N-terminal residue of the second binding moiety.

In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ G, R or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G or R. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is R or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G. In some embodimentsIn the scheme, the C-terminal portion X of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is R. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is Y.

In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₁ Is V, L, W, P, S, G, K, D, F, M, T, N or R. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; and X of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₃ G, R or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G or R. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is R or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is R. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is Y.

In certain embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₃ G, R or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G or R. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is ROr Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is R. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is Y.

In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₃ G, R or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G or R. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is R or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is R. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is Y.

In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₁ Is any amino acid; x of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₂ Is any amino acid; and X of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₃ G, R or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₁ Is any amino acid; x of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₂ Is any amino acid; and X of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G or R. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₁ Is any amino acid; x of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₂ Is any amino acid; and X of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₁ Is any amino acid; x of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₂ Is any amino acid; and X of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is R or Y. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₁ Is any amino acid; x of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₂ Is any amino acid; and X of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is G. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₁ Is any amino acid; x of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₂ Is any amino acid; and X of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is R. In some embodiments, X of the C-terminal portion of the linker ₁ -X ₂ -X ₃ X of (2) ₁ Is any amino acid; x of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₂ Is any amino acid; and X of the C-terminal part of the linker ₁ -X ₂ -X ₃ X of (2) ₃ Is Y.

In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is VVG, VAG, VLG, VSG, VGG, VRG, VKG, VMG, VCG, VFG, VTG, VPG or VEG. In some embodiments, the graftingThe C-terminal part of the head (X ₁ -X ₂ -X ₃ ) Is LVG, LAG, LLG, LSG, LGG, LRG, LKG, LMG, LCG, LFG, LTG, LPG or LEG. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) WVG, WAG, WLG, WSG, WGG, WRG, WKG, WMG, WCG, WFG, WTG, WPG or WEG. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) PVG, PAG, PLG, PSG, PGG, PRG, PKG, PMG, PCG, PFG, PTG, PPG or PEG. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is SVG, SAG, SLG, SSG, SGG, SRG, SKG, SMG, SCG, SFG, STG, SPG or SEG. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is GVG, GAG, GLG, GSG, GGG, GRG, GKG, GMG, GCG, GFG, GTG, GPG or GEG. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is KVG, KAG, KLG, KSG, KGG, KRG, KKG, KMG, KCG, KFG, KTG, KPG or KEG. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is DVG, DAG, DLG, DSG, DGG, DRG, DKG, DMG, DCG, DFG, DTG, DPG or DEG. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) FVG, FAG, FLG, FSG, FGG, FRG, FKG, FMG, FCG, FFG, FTG, FPG or FEG. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is MVG, MAG, MLG, MSG, MGG, MRG, MKG, MMG, MCG, MFG, MTG, MPG or MEG. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is TVG, TAG, TLG, TSG, TGG, TRG, TKG, TMG, TCG, TFG, TTG, TPG or TEG. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is NVG, NAG, NLG, NSG, NGG, NRG, NKG, NMG, NCG, NFG, NTG, NPG or NEG. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is RVG, RAG, RLG, RSG, RGG, RRG, RKG, RMG, RCG, RFG, RTG, RPG or REG.

In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is VVR, VAR, VLR, VSR, VGR, VRR, VKR, VMR, VCR, VFR, VTR, VPR or VER. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is LVR, LAR, LLR, LSR, LGR, LRR, LKR, LMR, LCR, LFR, LTR, LPR or LER. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) WVR, WAR, WLR, WSR, WGR, WRR, WKR, WMR, WCR, WFR, WTR, WPR or WER. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is PVR, PAR, PLR, PSR, PGR, PRR, PKR, PMR, PCR, PFR, PTR, PPR or PER. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) SVR, SAR, SLR, SSR, SGR, SRR, SKR, SMR, SCR, SFR, STR, SPR or SER. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) GVR, GAR, GLR, GSR, GGR, GRR, GKR, GMR, GCR, GFR, GTR, GPR or GERs. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is KVR, KAR, KLR, KSR, KGR, KRR, KKR, KMR, KCR, KFR, KTR, KPR or KER. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is DVR, DAR, DLR, DSR, DGR, DRR, DKR, DMR, DCR, DFR, DTR, DPR or DER. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) FVR, FAR, FLR, FSR, FGR, FRR, FKR, FMR, FCR, FFR, FTR, FPR or FER. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is MVR, MAR, MLR, MSR, MGR, MRR, MKR, MMR, MCR, MFR, MTR, MPR or MER. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) TVR, TAR, TLR, TSR, TGR, TRR, TKR, TMR, TCR, TFR, TTR, TPR or TER. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) NVR, NAR, NLR is a,NSR, NGR, NRR, NKR, NMR, NCR, NFR, NTR, NPR or NER. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) RVR, RAR, RLR, RSR, RGR, RRR, RKR, RMR, RCR, RFR, RTR, RPR or RER.

In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) VVY, VAY, VLY, VSY, VGY, VRY, VKY, VMY, VCY, VFY, VTY, VPY or VEY. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) LVY, LAY, LLY, LSY, LGY, LRY, LKY, LMY, LCY, LFY, LTY, LPY or LEY. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) WVY, WAY, WLY, WSY, WGY, WRY, WKY, WMY, WCY, WFY, WTY, WPY or WEY. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) PVY, PAY, PLY, PSY, PGY, PRY, PKY, PMY, PCY, PFY, PTY, PPY or PEY. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) SVY, SAY, SLY, SSY, SGY, SRY, SKY, SMY, SCY, SFY, STY, SPY or SEY. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) GVY, GAY, GLY, GSY, GGY, GRY, GKY, GMY, GCY, GFY, GTY, GPY or GEY. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is KVY, KAY, KLY, KSY, KGY, KRY, KKY, KMY, KCY, KFY, KTY, KPY or KEY. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) DVY, DAY, DLY, DSY, DGY, DRY, DKY, DMY, DCY, DFY, DTY, DPY or DEY. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) FVY, FAY, FLY, FSY, FGY, FRY, FKY, FMY, FCY, FFY, FTY, FPY or FEY. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) MVY, MAY, MLY, MSY, MGY, MRY, MKY, MMY, MCY, MFY, MTY, MPY or MEY. In some embodiments, the C-terminal portion of the linker(X ₁ -X ₂ -X ₃ ) Is TVY, TAY, TLY, TSY, TGY, TRY, TKY, TMY, TCY, TFY, TTY, TPY or TEY. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is NVY, NAY, NLY, NSY, NGY, NRY, NKY, NMY, NCY, NFY, NTY, NPY or NEY. In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) RVY, RAY, RLY, RSY, RGY, RRY, RKY, RMY, RCY, RFY, RTY, RPY or REY.

In some embodiments, the C-terminal portion (X ₁ -X ₂ -X ₃ ) Is selected from any one of Table 2.

Table 2: exemplary sequences of three amino acids at the C-terminus of the linker.

In some embodiments, the linker comprises a polypeptide comprising (EAAAK) _n Wherein n is an integer from 1 to 25 (e.g., 1 to 20 or 1 to 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker comprises a polypeptide comprising (XP) _n 、(XPP) _n Or (XPPP) _n Wherein X is any amino acid, and wherein n is an integer from 1 to 25 (e.g., 1 to 20 or 1 to 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker comprises a polypeptide comprising (XP) _n 、(XPP) _n Or (XPPP) _n Wherein each X is G, A, P or S, and wherein n is an integer from 1 to 25 (e.g., 1 to 20 or 1 to 10), including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21. 22, 23, 24 or 25. In some embodiments, the linker comprises a polypeptide comprising (AP) _n Or (APAP) _n Wherein n is an integer from 1 to 25 (e.g., 1 to 20 or 1 to 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker comprises a polypeptide comprising (EEEEKKK K) _n Wherein n is an integer from 1 to 25 (e.g., 1 to 20 or 1 to 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker comprises a peptide sequence comprising (G _x S _y ) _n Wherein x is 1 to 5, wherein y is 1 to 5, and wherein n is an integer from 1 to 25 (e.g., 1 to 20 or 1 to 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker comprises a polypeptide comprising (GGGGS) _n Wherein n is an integer from 1 to 25 (e.g., 1 to 20 or 1 to 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker comprises a peptide sequence disclosed herein and X of the C-terminal portion of the linker disclosed herein ₁ -X ₂ -X ₃ 。

In some embodiments, the joint is a rigid joint. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

In some embodiments, the rigidity of the rigid linker is maximized to increase the affinity of the co-binding moiety. In some embodiments, the rigid joint is only capable of bending or flexing no more than 90 degrees, such as no more than any one of 75 degrees, 60 degrees, 45 degrees, 30 degrees, 15 degrees, or 5 degrees. In some embodiments, the rigid joint can only bend or flex no more than 45 degrees and can twist no more than 30 degrees. In some embodiments, the joint can only twist less than 360 degrees, such as less than any one of 300 degrees, 240 degrees, 180 degrees, 150 degrees, 120 degrees, 90 degrees, 60 degrees, 30 degrees, 15 degrees, or 5 degrees. In some embodiments, the joint can only twist less than 5 degrees. In some embodiments, the rigid joint can only bend or flex no more than 90 degrees, such as no more than any of 75 degrees, 60 degrees, 45 degrees, 30 degrees, 15 degrees, or 5 degrees, and can twist less than any of 360 degrees, 300 degrees, 240 degrees, 180 degrees, 150 degrees, 120 degrees, 90 degrees, 60 degrees, 30 degrees, 15 degrees, or 5 degrees.

In some embodiments, the rigid joint has a rigid middle portion and a tip that is less rigid at one or more ends that is connected to the binding portion. In some embodiments, the rigid joint has a rigid middle portion and a tip that is less rigid at one or more ends that is connected to the binding portion. For example, such rigid linkers can facilitate simultaneous binding of the binding moiety to non-overlapping epitopes on the target molecule.

In some embodiments, the linker associates the first binding moiety and the second binding moiety via non-covalent interactions. In such embodiments, the first binding moiety and/or the second binding moiety comprises a moiety that participates in a non-covalent interaction. For example, in some embodiments, the linker comprises a leucine zipper, wherein the first binding portion comprises a first portion of the leucine zipper, and wherein the second binding portion comprises a second portion of the leucine zipper. In some embodiments, the linker comprises a double stranded nucleic acid comprising two strands having complementary regions, wherein the first binding moiety comprises a nucleic acid strand, and wherein the second binding moiety comprises a second nucleic acid strand.

In some embodiments, the nucleic acid linker comprises a polynucleotide, such as an oligonucleotide, double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA. In some embodiments, the nucleic acid linker comprises any of 200 nucleotides or less, e.g., 180 nucleotides or less, 160 nucleotides or less, 140 nucleotides or less, 120 nucleotides or less, 100 nucleotides or less, 80 nucleotides or less, 60 nucleotides or less, 40 nucleotides or less, 20 nucleotides or less, or 10 nucleotides or less.

In some embodiments, the linker has a length, such as a linear chain based on the primary structure of the linker, e.g., amino acids. In some embodiments, the length of the linker is assessed based on a primary structure, such as a linear chain of amino acids. In some embodiments, the length of the linker is no more than 250 angstroms, for example no more than any of 240 angstroms, 230 angstroms, 220 angstroms, 210 angstroms, 200 angstroms, 190 angstroms, 180 angstroms, 170 angstroms, 160 angstroms, 150 angstroms, 140 angstroms, 130 angstroms, 120 angstroms, 110 angstroms, 100 angstroms, 90 angstroms, 80 angstroms, 70 angstroms, 60 angstroms, 50 angstroms, 40 angstroms, 30 angstroms, 20 angstroms, 15 angstroms, 10 angstroms, or 5 angstroms. In some embodiments, the length of the linker is any of about 250 angstroms, 240 angstroms, 230 angstroms, 220 angstroms, 210 angstroms, 200 angstroms, 190 angstroms, 180 angstroms, 170 angstroms, 160 angstroms, 150 angstroms, 140 angstroms, 130 angstroms, 120 angstroms, 110 angstroms, 100 angstroms, 90 angstroms, 80 angstroms, 70 angstroms, 60 angstroms, 50 angstroms, 40 angstroms, 30 angstroms, 20 angstroms, 15 angstroms, 10 angstroms, or 5 angstroms. In some embodiments, the length of the linker is reduced to a minimum length required to provide for the attachment of at least the first binding moiety and the second binding moiety without interfering with the binding of the respective binding molecule to the target molecule. In some embodiments, the length of the linker is configured to achieve minimal entropy loss and minimal interference with binding of the binding molecule to the target molecule.

In some embodiments, the linker is no more than 120 amino acids in length, such as any one of no more than 115 amino acids, 110 amino acids, 105 amino acids, 100 amino acids, 95 amino acids, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 25 amino acids, 20 amino acids, 15 amino acids, 10 amino acids, or 5 amino acids. In some embodiments, the linker is any of about 120 amino acids, 115 amino acids, 110 amino acids, 105 amino acids, 100 amino acids, 95 amino acids, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 25 amino acids, 20 amino acids, 15 amino acids, 10 amino acids, or 5 amino acids in length.

In some embodiments, the linker is a chemical linker, such as a synthetic chemical structure or a polymer. In some embodiments, the linker comprises a plurality of polyethylene glycol subunits. In some embodiments, the linker is a non-peptidyl polymer.

D. Other configurations, alternatives and variants of conjugate molecules

Various configurations of conjugate molecules are provided that include a second binding moiety that specifically recognizes a target site, wherein the second binding moiety is a second antibody moiety that includes an antibody variable domain having an N-terminal truncation ("N-terminal truncation"). In some embodiments, the conjugate molecule comprises a linker. In some embodiments, the conjugate molecule does not comprise a linker. In some embodiments, the conjugate molecule comprises a first moiety that is not a binding moiety, such as an enzyme, a drug, or a toxin. In some embodiments, the conjugate molecule is a multispecific conjugate molecule, such as a bispecific co-conjugate. In some embodiments, the conjugate molecule, such as a co-conjugate, is a multimeric conjugate molecule comprising at least a third binding moiety. In some embodiments, the conjugate molecule is a CAR, including a multi-specific CAR, e.g., a bispecific CAR. In some embodiments, the conjugate molecule is a conjugate, such as a co-conjugate conjugated to a drug or label, including bispecific conjugates.

In some embodiments, the conjugate molecule comprises a first moiety that is a non-immunoglobulin conjugate molecule that specifically binds to a target. These alternative conjugate molecules may include, for example, any engineered protein scaffold known in the art. Such scaffolds may comprise one or more CDRs of an antibody directed against a target. Such scaffolds include, for example, anticalin, which is based on lipocalin scaffolds, a protein structure characterized by a rigid β -barrel that supports four hypervariable loops forming ligand binding sites. The novel binding specificities can be engineered by directed random mutagenesis in the loop region with functional display and directed selection (see e.g., skerra,2008,FEBS J.275:2677-83). Other suitable scaffolds may include, for example, adnectin or monomers based on the tenth extracellular domain of human fibronectin III (see, e.g., koide and Koide,2007,Methods Mol.Biol.352:95-109); an affibody, based on the Z domain of staphylococcal protein A (see, e.g., nygren et al, 2008,FEBS J.275:2668-76)); DARPin, ankyrin-based repeat proteins (see, e.g., stumpp et al, 2008,Drug.Discov.Today 13:695-701); fynomer, SH3 domain based on human Fyn protein kinase (see e.g., grablovski et al, 2007, J.biol. Chem. 282:3196-204); affitin, based on Sac7d from sulfolobus acidophilus (see, e.g., krehhenbrink et al, 2008, J.mol. Biol.383: 1058-68); affilin, based on human y-B-crystallin (see, e.g., ebersbach et al, 2007, J.mol. Biol. 372:172-85); an affibody, based on the A domain of a membrane receptor protein (see, e.g., silverman et al 2005, biotechnol.23:1556-61); cysteine-rich knotting element peptides (see, e.g., kolmar,2008,FEBS J.275:2684-90); and engineered Kunitz-type inhibitors (see, e.g., nixon and Wood,2006, curr. Opin. Drug. Discovery. Dev. 9:261-68). For reviews, see, e.g., gebauer and Skerra,2009, curr. Opin. Chem. Biol.13:245-55.

In some embodiments, the disclosure includes amino acid sequence modifications of a conjugate molecule, such as a co-conjugate. In some embodiments, the antibody or antigen binding fragment thereof comprises amino acid sequence modifications. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity or solubility. Thus, it is contemplated that conjugate molecules, such as co-conjugates, variants, may be prepared. For example, co-conjugate variants may be prepared by introducing appropriate nucleotide changes into the encoding DNA and/or by synthesizing the desired antibody or polypeptide. Those skilled in the art understand that amino acid changes may alter post-translational processes of the co-conjugate or an antibody or antigen binding fragment thereof that is part of the co-conjugate, e.g., alter the number or position of glycosylation sites or alter membrane anchoring characteristics.

In some embodiments, a conjugate molecule provided herein, such as a co-conjugate, is chemically modified, for example, by covalently attaching any type of molecule to the conjugate molecule, such as a co-conjugate or an antibody or antigen binding fragment thereof. Such derivatives may include, for example, co-conjugates that have been chemically modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be made by known techniques including, but not limited to, specific chemical cleavage of tunicamycin, acetylation, formulation, metabolic synthesis, and the like. In addition, antibodies may contain one or more non-classical amino acids.

A variant may be a substitution, deletion or insertion of one or more codons encoding a polypeptide (co-conjugate or an antibody or antigen binding fragment thereof as part of a co-conjugate) that results in an amino acid sequence change as compared to the native sequence of the polypeptide. Amino acid substitutions may be the result of substitution of one amino acid with another amino acid having similar structure and/or chemical properties, e.g., substitution of leucine with serine, e.g., conservative amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions include no more than 25 amino acid substitutions relative to the original molecule, e.g., no more than any of 20 amino acid substitutions, 18 amino acid substitutions, 15 amino acid substitutions, 10 amino acid substitutions, 5 amino acid substitutions, 4 amino acid substitutions, 3 amino acid substitutions, or 2 amino acid substitutions. In a specific embodiment, the substitution is a conservative amino acid substitution at one or more predicted nonessential amino acid residues. The permissible variation can be determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting variants for activity exhibited by the full length or mature native sequence.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides comprising one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of an antibody molecule include fusion of the N-terminus or C-terminus of the antibody with an enzyme (e.g., an enzyme prodrug therapy for antibody targeting) or polypeptide that increases the serum half-life of the antibody.

Substantial modification of the biological properties of a conjugate molecule, such as a co-conjugate or an antibody or antigen binding fragment thereof, is achieved by selecting substitutions that have significantly different roles in the following cases: maintaining (a) the structure of the polypeptide backbone within the substituted region, e.g., in a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) a majority of the side chains. Alternatively, conservative substitutions (e.g., within amino acid groups having similar properties and/or side chains) may be made in order to maintain or not significantly alter the properties. Amino acids can be classified according to their similarity in side chain properties (see, e.g., lehninger, biochemistry 73-75 (2 d. 1975)): (1) nonpolar: ala (A), val (V), leu (L), ile (I), pro (P), phe (F), trp (W), met (M); (2) uncharged polarity: gly (G), ser (S), thr (T), cys (C), tyr (Y), asn (N), gln (Q); (3) acidity: asp (D), glu (E); and (4) alkaline: lys (K), arg (R), his (H).

Alternatively, naturally occurring residues can be grouped into several groups based on common side chain properties: (1) hydrophobicity: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr, asn, gln; (3) acidity: asp, glu; (4) alkaline: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe

Non-conservative substitutions require exchanging members of one of these classes with another class. Such substituted residues may also be introduced at conservative substitution sites or at the remaining (non-conservative) sites. Variations can be generated using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Cloned DNA may be subjected to site-directed mutagenesis (see, e.g., carter,1986,Biochem J.237:1-7; and Zoller et al, 1982,Nucl.Acids Res.10:6487-500), cassette mutagenesis (see, e.g., wells et al, 1985, gene 34:315-23), or other known techniques to produce co-conjugate variant DNA.

Any cysteine residue that does not participate in maintaining the correct conformation of the conjugate molecule, such as a co-conjugate or antibody or antigen binding fragment thereof, may also be substituted with, for example, another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, for example, cysteine bonds may be added to the co-conjugate or an antibody or antigen binding fragment thereof that is part of the co-conjugate to improve its stability (e.g., wherein the antibody is an antibody fragment such as an Fv fragment).

In some embodiments, the conjugate molecule, such as a co-conjugate or antibody or antigen binding fragment thereof, is "deimmunized". In some embodiments, a "deimmunized" conjugate molecule, such as a co-conjugate, comprises a humanized antibody or chimeric antibody, which has one or more changes in its amino acid sequence as compared to the corresponding original non-deimmunized antibody, resulting in reduced immunogenicity of the antibody. One of the procedures used to generate such antibody mutants involves the identification and removal of T cell epitopes of the antibody molecule. In a first step, the immunogenicity of the antibody molecule can be determined by several methods, for example by determining T cell epitopes in vitro or predicting such epitopes on a computer, as is known in the art. Once the critical residues for T cell epitope function are determined, mutations can be made to remove immunogenicity and preserve antibody activity. For reviews, see, for example, jones et al, 2009,Methods in Molecular Biology 525:405-23.

In certain aspects, covalent modification of a conjugate molecule, such as a co-conjugate, is included within the scope of the present disclosure. Covalent modification includes reacting a targeted amino acid residue of a conjugate molecule, such as a co-conjugate, with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues of the conjugate molecule. Other modifications include deamidation of the glutamine and asparagine residues to the corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; hydroxy phosphorylation of serine or threonyl residues; methylation of alpha-amino groups of lysine, arginine and histidine side chains (see, e.g., cright on, proteins: structure and Molecular Properties-86 (1983)); acetylation of the N-terminal amine; and amidation of any C-terminal carbonyl group.

Other types of covalent modifications of conjugate molecules, such as co-conjugates, included within the scope of the present disclosure include altering the native glycosylation pattern (see, e.g., beck et al, 2008, curr.pharm.biotechnol.9:482-501; and Walsh,2010,Drug Discov.Today15:773-80), and are described in, e.g., U.S. Pat. nos. 4,640,835;4,496,689;4,301,144;4,670,417;4,791,192; or 4,179,337 to various non-protein polymers such as polyethylene glycol, polypropylene glycol or polyoxyalkylene.

In some embodiments, a conjugate molecule of the present disclosure, such as a co-conjugate, may also be modified to form a chimeric molecule comprising a fusion of the co-conjugate with another heterologous polypeptide or amino acid sequence, such as an epitope tag (see, e.g., terpe,2003, appl. Microbiol. Biotechnol. 60:523-33), or the Fc region of an IgG molecule (see, e.g., aruffo, antibody Fusion Proteins 221-42 (chapow and Ashkenazi, 1999)).

In some embodiments, provided herein are also fusion proteins comprising a conjugate molecule provided herein, such as a co-conjugate and a heterologous polypeptide. In some embodiments, a heterologous polypeptide fused to a conjugate molecule, such as a co-conjugate, can be used to target the conjugate molecule to a particular cell.

The present disclosure also provides conjugates comprising a conjugate molecule of the present disclosure, such as a co-conjugate, covalently bound to one or more agents, such as through a linker (e.g., a synthetic linker).

In some embodiments, a conjugate molecule provided herein, such as a co-conjugate, is conjugated or recombinantly fused, for example, to a detectable molecule.

Such detection may be accomplished, for example, by coupling the co-conjugate to a detectable substance, including but not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, e.g. but not limited to streptavidin/biotin or avidinAnd biotin/biotin; fluorescent substances such as, but not limited to, umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin or aequorin; chemiluminescent materials such as, but not limited to, acridinium-based compounds or HALOTAG; radioactive materials such as, but not limited to, iodine @ ¹³¹ I、 ¹²⁵ I、 ¹²³ I and ¹²¹ I) The carbon is ¹⁴ C) Sulfur ³⁵ S, tritium ³ H) The indium is ¹¹⁵ In、 ¹¹³ In、 ¹¹² In and ¹¹¹ in, technetium ] ⁹⁹ Tc), thallium ²⁰¹ Ti, ga ] ⁶⁸ Ga and ⁶⁷ ga and Pd% ¹⁰³ Pd and molybdenum% ⁹⁹ Mo and xenon ¹³³ Xe and F ¹⁸ F)、 ¹⁵³ Sm、 ¹⁷⁷ Lu、 ¹⁵⁹ Gd、 ¹⁴⁹ Pm、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁶⁶ Ho、 ⁹⁰ Y、 ⁴⁷ Sc、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹⁴² Pr、 ¹⁰⁵ Rh、 ⁹⁷ Ru、 ⁶⁸ Ge、 ⁵⁷ Co、 ⁶⁵ Zn、 ⁸⁵ Sr、 ³² P、 ¹⁵³ Gd、 ¹⁶⁹ Yb、 ⁵¹ Cr、 ⁵⁴ Mn、 ⁷⁵ Se、 ¹¹³ Sn or ¹¹⁷ Sn; positron emitting metals using various positron emission tomography; and non-radioactive paramagnetic metal ions.

Also provided herein are conjugate molecules, such as co-conjugates, that are recombinantly fused or chemically conjugated (covalently or non-covalently conjugated) to a heterologous protein or polypeptide (or fragment thereof, e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids of the polypeptide) to produce fusion proteins, and uses thereof. In particular, provided herein are fusion proteins comprising a co-conjugate provided herein and a heterologous protein, polypeptide, or peptide. In one embodiment, a heterologous protein, polypeptide or peptide fused to an antibody can be used to target the co-conjugate to a particular cell type. For example, a co-conjugate that binds to a cell surface receptor expressed by a particular cell type may be fused or conjugated to a cytotoxic antibody or peptide.

Furthermore, conjugate molecules provided herein, such as co-conjugates, may be fused to a tag or "tag" sequence, e.g., a peptide, to facilitate purification. In particular embodiments, the tag or tag amino acid sequence is a hexahistidine peptide, such as the tag provided in a pQE vector (see e.g., QIAGEN, inc.), many of which are particularly commercially available. For example, hexahistidine provides for convenient purification of fusion proteins as described in Gentz et al 1989,Proc.Natl.Acad.Sci.USA 86:821-24. Other peptide tags that may be used for purification include, but are not limited to, hemagglutinin ("HA") tags, which correspond to epitopes derived from influenza hemagglutinin protein (Wilson et al, 1984, cell 37:767-78) and "FLAG" tags.

Methods for fusing or conjugating portions (including polypeptides) to conjugate molecules such as co-conjugates are known (see, e.g., arnon et al, drugs in Cancer Therapy, in Monoclonal Antibodies and Cancer Therapy 243-56 of Monoclonal Antibodies for Immunotargeting (Reisfeld et al, 1985), hellstrom et al, antibodies for Drug Delivery, in Controlled Drug Delivery 623-53 (Robinson et al, 2 nd edition 1987), thorpe, antibody Carriers, cytotoxic Agents in Cancer Therapy, A Review, in Monoclonal Antibodies: biological and Clinical Applications-506 (Pichera et al, 1985), analysis, results, and Future Prospective, therAN_SNeuses Radiolabeled Antibody in Cancer Therapy, in Monoclonal Antibodies for Cancer Detection and Therapy-16 (Baldwin et al, 1985), thorpe et al, 1982, immunol Rev.62:119-58, U.S. Pat. No. 5,336,603, 622,929, 5,359,046, 5,349,053, 5,851,723,125,125, 5,908, 181, 5,349, 97, and WO 35, 93/1981, and WO 97/1984, 1981, and WO 97/or WO 97, 1984, 19832, WO 97/1984, and WO 95/or WO 95).

For example, fusion proteins may be produced by gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling (collectively "DNA shuffling") techniques. DNA shuffling can be used to alter the activity of co-binders as provided herein, including, for example, co-binders with higher affinity and lower dissociation rates (see, for example, U.S. Pat. Nos. 5,605,793;5,811,238;5,830,721;5,834,252; and 5,837,458; patten et al, 1997,Curr.Opinion Biotechnol.8:724-33;Harayama,1998,Trends Biotechnol.16 (2): 76-82; hansson et al, 1999, J.mol. Biol.287:265-76; and Lorenzo and Blasco,1998,Biotechniques 24 (2): 308-13). The co-conjugate or antibodies provided herein for the co-conjugate can be altered by random mutagenesis via error-prone PCR, random nucleotide insertion, or other methods prior to recombination. Polynucleotides encoding antibodies provided herein may be recombined with one or more components, motifs, segments, parts, domains, fragments, etc., of one or more heterologous molecules.

The conjugate molecules provided herein, such as co-conjugates, can also be conjugated with a second antibody to form an antibody conjugate, as described, for example, in U.S. Pat. No. 4,676,980.

The conjugate molecules provided herein, such as co-conjugates, may also be attached to a solid support, which is particularly useful in immunoassays or may be useful for purifying target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

Conjugates of antibodies and agents may be prepared using a variety of bifunctional protein coupling agents, such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl- (4-vinyl sulfone) benzoate). The present disclosure further contemplates that conjugates of the co-conjugate and the agent may be prepared using any suitable method as disclosed in the art (see, e.g., bioconjugate Techniques (Hermanson, 2 nd ed. 2008)).

Conventional conjugation strategies for conjugate molecules such as co-conjugates and agents have been based on random conjugation chemistry involving either the epsilon amino group of a Lys residue or the thiol group of a Cys residue, which results in a heterologous conjugate. Recently developed techniques allow site-specific conjugation to polypeptides, resulting in homogeneous loading and avoiding antigen binding or altered pharmacokinetics of the conjugate subpopulation. These include engineering of "thiomab" which comprises cysteine substitutions at positions on the heavy and light chains that provide reactive sulfhydryl groups and do not disrupt the folding and assembly of immunoglobulins or alter antigen binding (see, e.g., junutula et al 2008, j. Immunol. Meth.332:41-52; and Junutula et al 2008,Nature Biotechnol.26:925-32). In another approach, selenocysteines are co-translationally inserted into antibody sequences by recording the stop codon UGA from termination to selenocysteine insertion, allowing site-specific covalent conjugation to occur at the nucleophilic selenol group of selenocysteine in the presence of other natural amino acids (see, e.g., hofer et al, 2008,Proc.Natl.Acad.Sci.USA 105:12451-56; and Hofer et al, 2009, biochemistry48 (50): 12047-57).

1. Chimeric Antigen Receptor (CAR)

In some aspects, the present disclosure provides Chimeric Antigen Receptors (CARs) comprising a conjugate molecule, such as a co-conjugate, provided herein. In some aspects, the present disclosure provides a cell that expresses a CAR provided herein, such as a CAR effector cell. In some embodiments, the cell is an immune cell, such as a T cell. The CARs provided herein comprise (a) an extracellular domain comprising a conjugate molecule described herein, and (b) an intracellular signaling domain. In some embodiments, the CAR comprises a transmembrane domain present between an extracellular domain and an intracellular domain.

In some embodiments, a spacer domain may be present between the extracellular domain and the transmembrane domain or between the intracellular domain and the transmembrane domain. The spacer domain may be any oligopeptide or polypeptide whose function is to link the transmembrane domain to an extracellular domain or an intracellular domain in the polypeptide chain. The spacer domain may comprise up to about 300 amino acids, including, for example, about 10 to about 100 or about 25 to about 50 amino acids.

The transmembrane domain may be from a natural or synthetic source. If the source is natural, the domain may be from any membrane-bound protein or transmembrane protein. Particularly useful transmembrane regions in the present invention may be derived from the α, β, δ or γ chain of a T cell receptor, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154 (i.e., at least the transmembrane region thereof). In some embodiments, the transmembrane domain may be synthetic, in which case it may comprise predominantly hydrophobic residues, such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine can be found at each end of the synthetic transmembrane domain. In some embodiments, a short oligopeptide or polypeptide linker of, for example, about 2 to about 10 amino acids in length (e.g., any of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) can form a linkage between the transmembrane domain and the intracellular signaling domain of the CAR. In some embodiments, the linker is a glycine-serine duplex.

In some embodiments, a transmembrane domain is used that naturally associates with one of the sequences in the CAR intracellular domain. In some embodiments, the transmembrane domains may be selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

The intracellular signaling domain of the CAR is responsible for activating at least one normal effector function of the immune cell in which the CAR has been placed. For example, the effector function of a T cell may be a cell lysis activity or a helper activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and directs a cell to perform a specific function. Although it is generally possible to use the entire intracellular signaling domain, in many cases it is not necessary to use the entire strand. In the case of using a truncated portion of the intracellular signaling domain, such a truncated portion can be used in place of the complete chain, provided that it transduces the effector function signal. Thus, the term "intracellular signaling sequence" is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

Examples of intracellular signaling domains for use in the CARs of the invention include cytoplasmic sequences of T Cell Receptors (TCRs) and co-receptors that cooperate to initiate signal transduction upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional ability.

It is known that the signal produced by TCR alone is not sufficient to fully activate T cells, and that a secondary signal or co-stimulatory signal is also required. Thus, T cell activation can be thought to be mediated by two different classes of intracellular signaling sequences: a sequence that initiates antigen-dependent primary activation by a TCR (primary signaling sequence) and a sequence that acts in an antigen-independent manner to provide a secondary signal or costimulatory signal (costimulatory signaling sequence).

The primary signaling sequence modulates primary activation of the TCR complex, either in a stimulatory manner or in an inhibitory manner. The primary signaling sequence acting in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. In some embodiments, the CAR construct comprises one or more ITAMs.

Examples of ITAM-containing primary signaling sequences particularly useful in the present invention include those derived from TCR ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b and CD66 d.

In some embodiments, the CAR comprises a primary signaling sequence derived from cd3ζ. For example, the intracellular signaling domain of the CAR may comprise the cd3ζ intracellular signaling sequence itself, or it may be combined with any other desired intracellular signaling sequence useful in the context of the CARs described herein. For example, the intracellular domain of the CAR can comprise a cd3ζ intracellular signaling sequence and a costimulatory signaling sequence. The costimulatory signaling sequence may be part of the intracellular domain of a costimulatory molecule, including, for example, CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B-H3, ligands that specifically bind to CD83, and the like.

In some embodiments, the intracellular signaling domain of the CAR comprises an intracellular signaling sequence of cd3ζ and an intracellular signaling sequence of CD 28. In some embodiments, the intracellular signaling domain of the CAR comprises an intracellular signaling sequence of cd3ζ and an intracellular signaling sequence of 4-1 BB. In some embodiments, the intracellular signaling domain of the CAR comprises an intracellular signaling sequence of cd3ζ and an intracellular signaling sequence of CD28 and 4-1 BB.

Also provided herein are effector cells (such as lymphocytes, e.g., T cells) that express the CARs described herein.

Also provided is a method of producing an effector cell expressing a CAR described herein, the method comprising introducing into the effector cell a vector comprising a nucleic acid encoding the CAR. In some embodiments, introducing the vector into the effector cell comprises transducing the effector cell with the vector. In some embodiments, introducing the vector into the effector cell comprises transfecting the effector cell with the vector. The vector may be transduced or transfected into effector cells using any method known in the art.

2. Immunoconjugates

In some embodiments, the conjugate molecule comprises an immunoconjugate comprising a conjugate molecule such as a co-conjugate (also referred to herein as an "immunoconjugate") attached to an effector molecule. In some embodiments, the effector molecule is a therapeutic agent, e.g., a cancer therapeutic agent, that is cytotoxic, cytostatic, or otherwise provides some therapeutic benefit. In some embodiments, the effector molecule is a label that can directly or indirectly generate a detectable signal.

In some embodiments, immunoconjugates (also referred to herein as "antibody-drug conjugates" or "ADCs") comprising a conjugate molecule and a therapeutic agent are provided. In some embodiments, the therapeutic agent is a toxin that is cytotoxic, cytostatic, or prevents or reduces the ability of a target cell to divide. The use of ADCs for the local delivery of cytotoxic agents or cytostatic agents, i.e. drugs that kill or inhibit tumor cells in cancer therapy (Syrigos and Epenetos, anticancer Research 19:605-614 (1999); niculascu-Duvaz and Springer, adv. Dr. Del. Rev. 26:151-172 (1997); U.S. Pat. No. 4,975,278) allows for the targeted delivery of drug moieties to target cells and intracellular accumulation therein, wherein systemic administration of these unconjugated therapeutic agents may result in unacceptable levels of toxicity to normal cells as well as target cells sought to be eliminated (Baldwin et al, lancet (3.15.1986): 603-605 (1986); thorpe, (1985), "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review," in Monoclonal Antibodies'84:Biological And Clinical Applications,A.Pinchera et al (code), pages 475-506). Maximum efficacy with minimal toxicity is thus sought.

Therapeutic agents used in immunoconjugates include, for example, daunorubicin, doxorubicin, methotrexate, and vindesine (Rowland et al, cancer immunol. Immunother.21:183-187 (1986)). Toxins used in immunoconjugates include bacterial toxins such as diphtheria toxins, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al, J.Nat. Cancer Inst.92 (19): 1573-1581 (2000); mandler et al, bioorganic & Med. Chem. Letters 10:1025-1028 (2000); mandler et al, bioconjugate chem.13:786-791 (2002)), maytansinoids (EP 91213; liu et al, proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode et al, caner Res.58:2928 (1998); hinman et al, cancer Res.53:3336-3342 (1993)). Toxins may exert their cytotoxic and cytostatic effects through mechanisms that include tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when bound to large antibodies or protein receptor ligands.

Toxins and fragments thereof having enzymatic activity that may be used include, for example, diphtheria chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from pseudomonas aeruginosa), ricin a chain, abrin a chain, morexicin (modeccin) a chain, alpha-sarcina, aleurone, caryophyllin protein, americans proteins (PAPI, PAPII and PAP-S), balsam pear inhibitors, curcin, crotin, soaping inhibitors, gelonin, mitomycin (mitogellin), restrictocin, phenol mycin, enomycin and trichothecene. See, for example, WO 93/21232 published on month 28 of 1993.

Immunoconjugates of conjugate molecules and one or more small molecule toxins, such as calicheamicin, maytansinoids, dolastatin, aurostatin, trichothecene, and CC1065, as well as derivatives of these toxins with toxin activity, are also contemplated herein.

In some embodiments, an immunoconjugate is provided comprising a therapeutic agent having intracellular activity. In some embodiments, the immunoconjugate is internalized and the therapeutic agent is a cytotoxin that blocks cellular protein synthesis, resulting in cell death. In some embodiments, the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome inactivating activity, including, for example, gelonin, bouganin, saporin, ricin a chain, bryodin, diphtheria toxin, restrictocin, pseudomonas exotoxin a, and variants thereof. In some embodiments, when the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome inactivating activity, the anti-AMC immunoconjugate must be internalized upon binding to the target cell to render the protein cytotoxic to the cell.

In some embodiments, an immunoconjugate is provided comprising a therapeutic agent for disrupting DNA. In some embodiments, the therapeutic agent for disrupting DNA is selected from the group consisting of: enediynes (e.g., calicheamicin and esperamicin) and non-enediynes small molecule agents (e.g., bleomycin, methionyl-EDTA-Fe (II)). Other cancer therapeutic agents useful according to the present application include, but are not limited to, daunorubicin, doxorubicin, distamycin A, cisplatin, mitomycin C, ecteinascidin, duocarmycin/CC-1065, and bleomycin/peleomycin.

The application further contemplates an immunoconjugate formed between a conjugate molecule and a compound having nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

In some embodiments, the immunoconjugate comprises an agent for disrupting tubulin. Such pharmacological agents may include, for example, rhizopus/maytansine, paclitaxel, vincristine and vinblastine, colchicine, reoxygenate (auristatin), dolastatin 10MMAE and petuloside (petuloside) a.

In some embodiments, the immunoconjugate comprises an alkylating agent comprising, for example, asaley NSC 167780, AZQ NSC 182986, BCNU NSC 409962, busulfan NSC 750, carboxyplatin NSC 271674, CBDCA NSC 241240, CCNU NSC 79037, CHIP NSC 256927, chlorambucil NSC 3088, chloromycetin NSC 178248, cisplatin NSC 119875, chloroethan NSC 338947, cyano morpholine doxorubicin NSC 357704, ethylene glycol methyldisulfonate (cyclist) NSC 348948, dianhydrogalactitol NSC 132313, fludopa NSC 73754, heloshan (hepsulfm) NSC 329680, sea anthracenone NSC 142982, melphalan NSC 8806, methyl CCNU NSC 37, mitomycin C142982, mitozolomide NSC 142982, nitrogen NSC 762, PCNU 142982, piperazine-containing, melbinin 142982, and other brassica 142982, and the spiroche 142982.

In some embodiments, the cancer therapeutic moiety of the immunoconjugate of the application may comprise an antimitotic agent, including, but not limited to, colchicine NSC 406042, halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10NSC 376128 (NG-rexoxetine derived), maytansine NSC 153858, rhizomycin NSC 332598, paclitaxel NSC 125973, paclitaxel derivative NSC 608832, thiocolchicine NSC 361792, trityl cysteine NSC 83265, vinblastine sulfate NSC 49842, and vincristine sulfate NSC 67574.

In some embodiments, the immunoconjugate comprises a topoisomerase I inhibitor, including, but not limited to, camptothecin NSC 94600, camptothecin, sodium salt NSC 100880, aminocamptothecin NSC 603071, camptothecin derivative NSC 95382, camptothecin derivative NSC 107124, camptothecin derivative NSC 643833, camptothecin derivative NSC 629971, camptothecin derivative NSC 295500, camptothecin derivative NSC 249910, camptothecin derivative NSC 606985, camptothecin derivative NSC 374028, camptothecin derivative NSC 176323, camptothecin derivative NSC 295501, camptothecin derivative NSC 606172, camptothecin derivative NSC 606173, camptothecin derivative NSC 610458, camptothecin derivative NSC 618939, camptothecin derivative NSC 610457, camptothecin derivative NSC 610459, camptothecin derivative NSC 606499, camptothecin derivative NSC 610456, camptothecin derivative NSC 364830, camptothecin derivative NSC 606497, and amycin 354646.

In some embodiments, the immunoconjugate comprises a topoisomerase II inhibitor, including, but not limited to, doxorubicin NSC 123127, amonafide NSC 308847, m-AMSA NSC 249992, anthracnose derivative NSC 355644, pyrazoloacridine NSC 366140, bisacodyl hydrochloride NSC 337766, daunomycin NSC 82151, deoxydoxorubicin NSC 267469, mitoxantrone NSC 301739, minoril NSC 269148, N-dibenzyl daunomycin NSC 268242, oxathiolane (oxanthrazole) NSC 349174, N-phenylhydrazide NSC 164011, VM-26NSC 122819, and VP-16NSC 141540.

In some embodiments, the immunoconjugate comprises an RNA or DNA antimetabolite, including but not limited to L-alanin NSC 153353, 5-azacytidine NSC 102816, 5-fluorouracil NSC 19893, avermectin NSC 163501, aminopterin derivative NSC 132483, aminopterin derivative NSC 184692, aminopterin derivative NSC 134033, folic acid antagonist NSC 633713, folic acid antagonist NSC 623017, baker's soluble folic acid antagonist NSC 139105, dichloroallyl lawsonin NSC 126771, bunana NSC 126771, tegafur (prodrug) NSC 126771, 5, 6-dihydro-5-azacytidine NSC 126771, methotrexate NSC 740, methotrexate derivative NSC 126771N- (phosphonoacetyl) -L-aspartic acid (PALA) NSC 126771, pyrazolofuranomycin NSC 126771, trimemesate NSC 126771, 3-HP NSC 126771, 2' -deoxy-5-fluorouridine NSC 126771, 5-HP NSC 126771, alpha-TGDR NSC 126771, an Afedrimycin glycinate NSC 126771, ara-C NSC 126771, 5-aza-2 ' -deoxycytidine NSC 126771, beta-TGDR NSC 126771, cyclosporin NSC 126771, guanazole NSC 1895, hydroxyurea NSC 126771, inosine hydroxyacetaldehyde NSC 126771, macbecin Il NSC 126771, pyrazoloimidazole NSC 126771, thioguanine NSC 752 and thiopurine NSC 755.

In some embodiments, the immunoconjugate comprises a highly radioactive atom. A variety of radioisotopes may be used to produce radioconjugated antibodies. Examples include ²¹¹ At、 ¹³¹ I、 ¹²⁵ I、 ⁹⁰ Y、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹⁵³ Sm、 ²¹² Bi、 ³² P、 ²¹² Radioisotopes of Pb and Lu.

In some embodiments, the conjugate molecule may be conjugated to a "receptor" (such as streptavidin) for tumor pretargeting, wherein the conjugate molecule-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a scavenger, and then administration of a "ligand" (such as avidin) conjugated to a cytotoxic agent (such as a radionucleotide).

In some embodiments, the immunoconjugate may comprise a conjugate molecule conjugated to a prodrug-activating enzyme. In some such embodiments, the prodrug activating enzyme converts a prodrug (e.g., a peptide-based chemotherapeutic agent, see WO 81/01145) to an active drug, such as an anticancer drug. Enzymes that can be conjugated to antibodies include, but are not limited to, alkaline phosphatase, which can be used to convert phosphate-containing prodrugs to free drugs; arylsulfatase, which can be used to convert sulfate-containing prodrugs into free drugs; cytosine deaminase, which can be used to convert non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; proteases, such as Serratia (serratia) protease, thermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsins B and L), which can be used to convert peptide-containing prodrugs into free drugs; d-alanylcarboxypeptidase which can be used to convert prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes, such as β -galactosidase and neuraminidase, which can be used to convert glycosylated prodrugs into free drugs; beta-lactamase useful for converting a drug derived from beta-lactam into a free drug; and penicillin amidases, such as penicillin V amidase and penicillin G amidase, which can be used to convert drugs derivatized with phenoxyacetyl or phenylacetyl groups, respectively, on their amine nitrogen into free drugs. In some embodiments, the enzyme may be covalently bound to the antibody moiety by recombinant DNA techniques well known in the art. See, e.g., neuberger et al, nature 312:604-608 (1984).

In some embodiments, the therapeutic moiety of the immunoconjugate may be a nucleic acid. Nucleic acids that may be used include, but are not limited to, antisense RNAs, genes, or other polynucleotides, including nucleic acid analogs such as thioguanine and thiopurine.

The application further provides immunoconjugates comprising a conjugate molecule attached to an effector molecule, wherein the effector molecule is a label that can indirectly or directly produce a detectable signal. These immunoconjugates can be used in research or diagnostic applications, for example for in vivo detection of cancer. The label is preferably capable of producing a detectable signal, either directly or indirectly. For example, the label may be radio-opaque or a radioisotope, such as ³ H、 ¹⁴ C、 ³² P、 ³⁵ S、 ¹²³ I、 ¹²⁵ I、 ¹³¹ I, a step of I; fluorescent (fluorophore) or chemiluminescent (chromophore) compounds, such as fluorescein isothiocyanate, rhodamine or fluorescein; enzymes such as alkaline phosphatase, beta-galactosidase, or horseradish peroxidase; an imaging agent; or metal ions. In some embodiments, the label is a radioactive atom for scintigraphy studies, e.g ⁹⁹ Tc or ¹²³ I, or spin labeling for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. Zirconium-89 can be used in combination with various The metal chelator complexes and is conjugated to an antibody, for example for PET imaging (WO 2011/056983).

3. Nucleic acid

Nucleic acid molecules encoding the conjugate molecules, including constructs thereof, are also contemplated. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a full-length binder molecule, such as a co-binder, is provided. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a multispecific binder molecule (e.g., a multispecific binder molecule, bispecific binder molecule, or bispecific T cell adapter) or polypeptide portion thereof is provided. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a CAR is provided. In some embodiments, a nucleic acid (or set of nucleic acids) encoding an immunoconjugate or polypeptide portion thereof is provided.

The application also includes variants of these nucleic acid sequences. For example, a variant comprises a nucleotide sequence that hybridizes under at least moderately stringent hybridization conditions to a nucleic acid sequence encoding a conjugate molecule of the application (including constructs thereof).

The application also provides vectors into which the nucleic acids of the application are inserted.

Briefly, expression of a conjugate molecule, including constructs thereof (e.g., CARs), or polypeptide portions thereof, by a nucleic acid encoding the conjugate molecule, or polypeptide portions thereof, can be accomplished by inserting the nucleic acid into a suitable expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, including, for example, promoters (e.g., lymphocyte-specific promoters) and 3' untranslated regions (UTRs). The vector may be suitable for replication and integration in a eukaryotic host cell. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of desired nucleic acid sequences.

The nucleic acids of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, the invention provides a gene therapy vector.

Nucleic acids can be cloned into many types of vectors. For example, the nucleic acid may be cloned into vectors (including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids). Vectors of particular interest include expression vectors, replication vectors, probe-generating vectors and sequencing vectors.

Alternatively, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York), among other virology and molecular biology manuals. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors include an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene may be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to cells of the subject in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from tumor retroviruses such as murine leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity.

Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these promoters are located in a region 30-110bp upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible, so that promoter function is preserved when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements may be increased to 50bp apart before the activity begins to decrease.

One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV 40) early promoter, mouse Mammary Tumor Virus (MMTV), human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, moMuLV promoter, avian leukemia virus promoter, epstein barr virus immediate early promoter, rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked thereto when such expression is desired and can turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

To assess the expression of the polypeptide or part thereof, the expression vector to be introduced into the cell may also comprise a selectable marker gene or a reporter gene or both, to facilitate identification and selection of the expressing cell from the population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on separate DNA fragments and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present or expressed in the recipient organism or tissue, and encodes a polypeptide whose expression is manifested as some readily detectable property (e.g., enzymatic activity). The expression of the reporter gene is detected at a suitable time after the DNA is introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., ui-Tel et al 2000FEBS Letters 479:79-82). Suitable expression systems are well known and may be prepared or commercially available using known techniques. Typically, constructs with minimal 5' flanking regions that show the highest expression levels of the reporter gene are identified as promoters. Such promoter regions may be linked to a reporter gene and used to evaluate the ability of an agent to regulate promoter-driven transcription.

Methods for introducing and expressing genes into cells are known in the art. In the case of expression vectors, the vector may be readily introduced into a host cell, such as a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York). In some embodiments, the polynucleotide is introduced into the host cell by calcium phosphate transfection.

Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus 1, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. The use of lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo or in vivo) is contemplated. In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated within the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome by a linker molecule associated with the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing the lipid, mixed with the lipid, combined with the lipid, contained in the lipid as a suspension, contained in or complexed with the micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector-related composition is not limited to any particular structure in solution. For example, they may exist in bilayer structures, micelles, or "folded" structures. They may also simply be dispersed in solution, possibly forming aggregates of inconsistent size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include naturally occurring fat droplets in the cytoplasm, as well as a class of compounds containing long chain aliphatic hydrocarbons and derivatives thereof, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Regardless of the method used to introduce exogenous nucleic acid into a host cell or to expose the cell to the inhibitors of the invention, a variety of assays can be performed in order to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those of skill in the art, such as southern and northern blotting, RT-PCR, and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, identify agents within the scope of the invention, for example, by immunological methods (ELISA and western blot) or by the assays described herein.

E. Additional features and details regarding conjugate molecules and portions thereof

In some embodiments, the second binding moiety and/or the first binding moiety of the conjugate molecule, such as a co-conjugate or a component thereof, is derived from a monoclonal antibody, an antibody fragment, a humanized antibody, or a human antibody. Monoclonal antibodies are well known in the art and methods of preparation and screening are described, for example, in Kohler et al, 1975,Nature 256:495-97; U.S. Pat. nos. 4,816,567; munson et al, 1980, anal biochem.107:220-39; skerra et al, 1993,Curr.Opinion in Immunol.5:256-62; pluckthun, 1992, immunol. Revs.130:151-88; bass et al, 1990,Proteins 8:309-14; WO 92/09690; and Bowers et al, 2011,Proc Natl Acad Sci USA.108:20455-60. Antibody fragments are well known in the art and methods of preparation and screening are described, for example, in Hudson et al, 2003,Nature Med.9:129-34; morimoto et al, 1992,J.Biochem.Biophys.Methods 24:107-17; brennan et al, 1985, science229:81-83; carter et al, 1992, bio/Technology 10:163-67; U.S. Pat. nos. 5,869,046; WO 93/16185; U.S. patent No. 5,571,894; U.S. Pat. nos. 5,587,458; woolven et al, 1999,Immunogenetics 50:98-101; and Streltsov et al, 2004,Proc Natl Acad Sci USA.101:12444-49. Humanized antibodies and human antibodies are well known in the art and methods of preparation and screening are described, for example, in Jones et al, 1986,Nature 321:522-25; riechmann et al 1988,Nature 332:323-27; verhoeyen et al, 1988,Science 239:1534-36; padlan et al, 1995,FASEB J.9:133-39; sims et al, 1993, J.Immunol.151:2296-308; chothia et al, 1987, J.mol. Biol.196:901-17; carter et al, 1992,Proc.Natl.Acad.Sci.USA 89:4285-89; presta et al, 1993, J.Immunol.151:2623-32; tan et al, 2002, J.Immunol.169:1119-25; lazar et al, 2007, mol. Immunol.44:1986-98; hoogenboom,2005, nat. Biotechnol.23:1105-16; dufner et al, 2006,Trends Biotechnol.24:523-29; feldhaus et al, 2003, nat. Biotechnol.21:163-70; schlabchy et al 2004,Protein Eng.Des.Sel.17:847-60; foote and Winter,1992, J.mol.biol.224:487-99); dall' acquat et al 2005,Methods 36:43-60; studnica et al 1994,Protein Engineering 7:805-14; U.S. Pat. nos. 5,766,886;5,770,196;5,821,123; and 5,869,619; PCT publication WO 93/11794; kozbor,1984, J.Immunol.133:3001-05; brodeur et al Monoclonal Antibody Production Techniques and Applications 51-63 (1987); and Boerner et al, 1991, J.Immunol.147:86-95; jakobovits, A.,1995, curr. Opin. Biotechnol.6 (5): 561-66; brUggemann and Taussing,1997, curr. Opin. Biotechnol.8 (4): 455-58; examples of such are U.S. Pat. nos. 6,075,181 and 6,150,584.

III. target

The conjugate molecules provided herein comprise an element, such as a first binding moiety and a second binding moiety, that specifically recognizes one or more targets comprising a target site (e.g., epitope). In some embodiments, the first binding moiety and the second binding moiety specifically recognize different epitopes on the same target, e.g., the first binding moiety recognizes a first epitope on a target such as a polypeptide, and the second binding moiety recognizes a second epitope on the target. In some embodiments, the first binding moiety and the second binding moiety specifically recognize the same epitope on the same target, e.g., the first binding moiety and the second binding moiety specifically recognize a homodimer. In some embodiments, the first binding moiety and the second binding moiety specifically recognize different epitopes on different targets, e.g., the first binding moiety recognizes a first epitope on a first target such as a polypeptide and the second binding moiety recognizes a second epitope on a second target such as a polypeptide. In such embodiments, the first target and the second target are adjacent to each other. In some embodiments, the first target and the second target form a single complex, such as a protein complex.

In some embodiments, the target is a polypeptide, a multiprotein complex, a nucleic acid, a carbohydrate, a glycan, a lipid molecule, a physiological metabolite, or a small molecule compound. In some embodiments, the target molecule is a polypeptide. In some embodiments, the target molecule is a protein. In some embodiments, the target molecule is a multiprotein complex. In some embodiments, the target molecule is a nucleic acid. In some embodiments, the target molecule is a DNA molecule. In some embodiments, the target molecule is an RNA molecule. In some embodiments, the target molecule is a lipid molecule. In some embodiments, the target molecule is a sugar. In some embodiments, the target molecule is a carbohydrate. In some embodiments, the target molecule is a glycan. In some embodiments, the target molecule is a physiological metabolite. In some embodiments, the target molecule is a small molecule compound.

In some embodiments, the target is a polypeptide, a multiprotein complex, a nucleic acid, a carbohydrate, a glycan, a lipid molecule, a physiological metabolite, or a small molecule compound. In some embodiments, the target is an intracellular molecule, a disease marker, a neoantigen, or a cell surface molecule. In some embodiments, the target molecule is a cancer antigen or a cancer marker. In some embodiments, the target is EGFR. In some embodiments, the expression level of the target is less than 1×10 ⁶ Less than 1X 10 ⁵ Less than 1X 10 ⁴ Less than 1X 10 ³ Or less than 1X 10 ² Cells.

Binding moieties (e.g., K) with high binding affinity to target molecules _D Is 1X 10 ^-12 M) binding moieties (e.g., K) with low binding affinity to target molecules _D Is 1X 10 ^-9 M) is more likely to have a high binding affinity (e.g., K) for non-specific targets _D Is 1X 10 ^-9 M). In some embodiments, binding moieties having low binding affinity for the target molecule are selected to make the co-conjugate. In some embodiments, a co-conjugate (e.g., K _D Is 1X 10 ^-12 M or less) comprises a first binding moiety and a second binding moiety, K of one or both _D Is at least 1X 10 ^-10 M, at least 1X 10 ^-9 At least 1X 10 ^-8 At least 1X 10 ^-7 Or at least 1X 10 ^-6 M, so that non-specific binding can be reduced or minimized.

In some embodiments, binding of the conjugate molecule relative to a control conjugate molecule, e.g., a control co-conjugate, is reported. As described herein, in some embodiments, a control conjugate molecule (e.g., a control co-conjugate molecule) comprises an antibody variable domain that does not have an N-terminal truncation in the second binding moiety. In some embodiments, a conjugate molecule, such as a co-conjugate, comprises a second antibody moiety comprising an N-terminally truncated antibody variable domain that binds to a second target site with an affinity that is at least about 3-fold, e.g., at least about any one of 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 250-fold, 500-fold, or 1000-fold, that of a control conjugate molecule, such as a control co-conjugate, comprising an N-terminally truncated antibody variable domain that does not have the second antibody moiety. In some embodiments, the conjugate molecule and the control conjugate molecule comprise the same linker, e.g., have the same amino acid sequence. In some embodiments, the conjugate molecule and the control conjugate molecule comprise the same first binding moiety. In some embodiments, provided herein is a linker control conjugate molecule, such as a co-conjugate, wherein the linker conjugate molecule is identical to the test conjugate molecule except that the last three C-terminal amino acids in the linker of the control linker conjugate molecule, e.g., the last three C-terminal amino acids in the linker of the control linker conjugate molecule, are GGG.

In some embodiments, the first binding moiety or the second binding moiety of the co-conjugate binds to the target with a KD of at least 1X 10 ^-10 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 Less than 1X 10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, the first binding moiety or the second binding moiety of the co-conjugate binds to the target with a KD of at least 1X 10 ^-9 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 Less than 1X 10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, the first binding moiety or the second binding moiety of the co-conjugate binds to the target with a KD of at least 1X 10 ^-8 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 Less than 1X 10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, the first binding moiety or the second binding moiety of the co-conjugate binds to the target with a KD of at least 1X 10 ^-7 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 Less than 1X 10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, the first binding moiety or the second binding moiety of the co-conjugate binds to the target with a KD of at least 1X 10 ^-6 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 Less than 1X 10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, the first binding moiety or the second binding moiety of the co-conjugate binds to the target with a KD of at least 1X 10 ^-5 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 Less than 1X 10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M。

In some embodiments, the first binding moiety of the co-conjugate has a relatively high affinity and binds to the target with a KD of less than 1X 10 ^-10 M. In some embodiments, the first binding moiety of the co-conjugate binds to the target with a KD of less than 1X 10 ^- ¹¹ M. In some embodiments, the first binding moiety of the co-conjugate binds to the target with a KD of less than 1X 10 ^-12 M. These co-conjugates may have a second binding moiety with a lower binding affinity. In some embodimentsIn this case, the second binding moiety of the co-conjugate binds to the target with a KD of at least 1X 10 ^-9 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 Less than 1X 10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, the second binding moiety of the co-conjugate binds to the target with a KD of at least 1X 10 ^-8 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 Less than 1X 10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, the second binding moiety of the co-conjugate binds to the target with a KD of at least 1X 10 ^-7 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 Less than 1X 10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, the second binding moiety of the co-conjugate binds to the target with a KD of at least 1X 10 ^-6 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 Less than 1X 10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, the second binding moiety of the co-conjugate binds to the target with a KD of at least 1X 10 ^-5 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 Less than 1X 10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^- ¹⁶ M。

In some embodiments, both the first binding moiety and the second binding moiety of the co-conjugate bind to the target with a KD of at least 1X 10 ^-10 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 M is less than 1×10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, both the first binding moiety and the second binding moiety of the co-conjugate bind to the target with a KD of at least 1X 10 ^-9 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 M is less than 1×10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, both the first binding moiety and the second binding moiety of the co-conjugate bind to the target with a KD of at least 1X 10 ^-8 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 M is less than 1×10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, both the first binding moiety and the second binding moiety of the co-conjugate bind to the target with a KD of at least 1X 10 ^-7 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 M is less than 1×10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, both the first binding moiety and the second binding moiety of the co-conjugate bind to the target with a KD of at least 1X 10 ^-6 M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-10 M is less than 1×10 ^- ¹¹ M is less than 1×10 ^-12 M is less than 1×10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M. In some embodiments, the first binding moiety and the second binding moiety of the co-conjugate bind to the target with a KD of at least 1X 10-5M; and the co-conjugate binds to the target with a KD of less than 1×10 ^-9 M is less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 M is less than 1×10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M or less than 1X 10 ^-16 M。

The first binding moiety or the second binding moiety may have a high binding affinity for non-specific molecules, which affinity may beTo be reduced or minimized in the co-conjugate. In some embodiments, the first binding moiety or the second binding moiety of the co-conjugate binds to a non-specific molecule with a KD of less than 1X 10 ^-10 M; and the co-conjugate binds to a non-specific molecule with a KD of at least 1X 10 ^-10 M, at least 1X 10 ^-9 M, at least 1X 10 ^-8 M, at least 1X 10 ^-7 M, at least 1X 10 ^-6 M, at least 1X 10 ^-5 M, at least 1X 10 ^-4 M or at least 1X 10 ^-3 M. In some embodiments, the first binding moiety or the second binding moiety of the co-conjugate binds to a non-specific molecule with a KD of less than 1X 10 ^-9 M; and the co-conjugate binds to a non-specific molecule with a KD of at least 1X 10 ^- ⁹ M, at least 1X 10 ^-8 M, at least 1X 10 ^-7 M, at least 1X 10 ^-6 M, at least 1X 10 ^-5 M, at least 1X 10 ^-4 M or at least 1X 10 ^- ³ M. In some embodiments, the first binding moiety or the second binding moiety of the co-conjugate binds to a non-specific molecule with a KD of less than 1X 10 ^-8 M; and the co-conjugate binds to a non-specific molecule with a KD of at least 1X 10 ^-8 M, at least 1X 10 ^-7 M, at least 1X 10 ^-6 M, at least 1X 10 ^-5 M, at least 1X 10 ^-4 M or at least 1X 10 ^-3 M。

IV composition and kit

In some aspects, the present disclosure provides a composition comprising a conjugate molecule, such as a co-conjugate, provided herein. In some aspects, the present disclosure provides a pharmaceutical composition comprising a conjugate molecule, such as a co-conjugate, provided herein, and a pharmaceutically acceptable carrier. In some aspects, the present disclosure provides a detection agent comprising a conjugate molecule, such as a co-conjugate, provided herein. In one aspect, the invention provides a diagnostic agent comprising a conjugate molecule, such as a co-conjugate, provided herein. In one aspect, the invention provides a therapeutic agent comprising a conjugate molecule, such as a co-conjugate, provided herein.

In some aspects, the present disclosure provides a cell that expresses a conjugate molecule, such as a co-conjugate, provided herein. In some embodiments, the cell is an immune cell.

In some embodiments, the present disclosure provides a composition comprising a conjugate molecule, such as a co-conjugate, provided herein. In some embodiments, the composition further comprises a second agent. In some embodiments, the second agent is a therapeutic agent. In some embodiments, the second agent is a therapeutic antibody. In some embodiments, the second agent is a therapeutic compound. In some embodiments, the second agent is a therapeutic small molecule compound.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising a conjugate molecule, such as a co-conjugate, provided herein. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a conjugate molecule provided herein, such as a co-conjugate, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions include any standard pharmaceutical carrier known in the art, such as phosphate buffered saline, water and emulsions, such as oil and water emulsions, and various types of wetting agents. These pharmaceutical compositions may be prepared in liquid unit dosage form or any other form of administration sufficient to deliver the co-conjugates of the present disclosure to a target area of a subject in need of treatment. For example, the pharmaceutical composition may be prepared in any manner suitable for the chosen mode of administration, such as intravascular, intramuscular, subcutaneous or intraperitoneal administration. Other optional components, such as pharmaceutical grade stabilizers, buffers, preservatives, excipients, and the like, can be readily selected by one of skill in the art. The preparation of pharmaceutical compositions with appropriate consideration of pH, isotonicity, stability, and the like is within the skill in the art.

Pharmaceutical compositions comprising the co-conjugates in aqueous solution or lyophilized or other dried form are prepared for storage by mixing the co-conjugates with the desired purity with optional physiologically acceptable carriers, excipients or stabilizers (see, e.g., remington' sPharmaceutical Sciences (18 th edition 1980)). Acceptable carriers, excipients or stabilizersIs non-toxic to recipients at the dosages and concentrations employed, and includes buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants, e.g. TWEEN ^TM 、PLURONICS ^TM Or polyethylene glycol.

The conjugate molecules of the present disclosure, such as co-conjugates, may be formulated in any suitable form for delivery to target cells/tissues, for example as microcapsules or macroemulsions (Remington, supra; park et al 2005,Molecules 10:146-61; malik et al 2007, curr. Drug. Deliv. 4:141-51), as sustained release formulations (Putney and Burke,1998,Nature Biotechnol.16:153-57) or in liposome form (Maclean et al 1997, int. J. Oncol.11:325-32; kontermann,2006, curr. Opin. Mol. Ther. 8:39-45).

The conjugate molecules provided herein, such as co-conjugates, may also be embedded in microcapsules, e.g., prepared by coacervation techniques or by interfacial polymerization, e.g., hydroxymethyl cellulose or gelatin-microcapsules and poly (methyl methacrylate) microcapsules, respectively; in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or in a coarse emulsion. Such techniques are disclosed, for example, in Remington, supra.

Various compositions and delivery systems are known and may be used with the conjugate molecules described herein, such as co-conjugates, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies; receptor-mediated endocytosis (see, e.g., wu and Wu,1987, J.biol. Chem. 262:4429-32); nucleic acids constructed as part of a retrovirus or other vector, and the like. In another embodiment, the composition may be provided as a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see, e.g., langer, supra; sefton,1987, crit. Ref. Biomed. Eng.14:201-40; buchwald et al 1980,Surgery 88:507-16; and Saudek et al, 1989, N.Engl. J. Med. 321:569-74). In another embodiment, the polymeric material may be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., a co-conjugate as described herein) or a composition of the disclosure (see, e.g., medical Applications of Controlled Release (Langer and Wise, inc.), controlled Drug Bioavailability, drug Product Design and Performance (Smolen and Ball, inc., 1984), ranger and Peppas,1983, J.macromol. Sci. Rev. Macromol. Chem.23:61-126; levy et al, 1985,Science 228:190-92; during et al, 1989, ann. Neurol.25:351-56; howard et al, 1989, J. Neurosurg.71:105-12; U.S. Pat. Nos. 5,679,377, 5,916,597;5,912,015;5,989,463, and 5,128,326; PCT publications WO 99/15154 and WO 99/20253). Examples of polymers for sustained release formulations include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-vinyl acetate), poly (methacrylic acid), polyethylene glycol (PLG), polyanhydrides, poly (N-vinylpyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactide (PLA), poly (lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable.

In another embodiment, a controlled or sustained release system may be placed in proximity to a specific target tissue, such as the nasal cavity or lungs, thus requiring only a portion of the systemic dose (see, e.g., goodson, volume Medical Applications of Controlled Release, volume 2, 115-38 (1984)). For example, langer,1990,Science 249:1527-33 discusses a controlled release system. Any technique known to those skilled in the art may be used to produce a sustained release formulation comprising one or more co-conjugates as described herein (see, e.g., U.S. Pat. No. 4,526,938; PCT publication Nos. WO 91/05548 and WO 96/20698; ning et al, 1996,Radiotherapy&Oncology 39:179-89; song et al, 1995,PDA J.of Pharma.Sci. & Tech.50:372-97; cleek et al, 1997, pro.int 'l.Symp.control. Rel.Bioact. Mater.24:853-54; and Lam et al, 1997,Proc.Int'l.Symp.Control Rel.Bioact.Mater.24:759-60).

In some embodiments, the present disclosure provides a detection agent comprising a conjugate molecule, such as a co-conjugate, provided herein.

In some embodiments, the present disclosure provides a diagnostic agent comprising a conjugate molecule, such as a co-conjugate, provided herein.

In some embodiments, the present disclosure provides a diagnostic agent comprising a therapeutic agent comprising a conjugate molecule, such as a co-conjugate, provided herein.

A conjugate molecule, such as a co-conjugate, provided herein may form a functional domain of the molecule. For example, in some embodiments, antibodies having a conjugate molecule, such as a co-conjugate, such as an antigen recognition domain, of the present disclosure are also provided herein. In some embodiments, provided herein are multispecific antibodies having a conjugate molecule of the present disclosure, such as a co-conjugate, e.g., one of its antigen recognition domains. In some embodiments, provided herein are also bispecific antibodies having a co-conjugate of the disclosure, e.g., one of its antigen recognition domains. In some embodiments, provided herein are chimeric antigen receptors having a conjugate molecule of the disclosure, such as a co-conjugate, e.g., an antigen recognition domain thereof.

In some embodiments, the present disclosure provides a Chimeric Antigen Receptor (CAR) comprising a conjugate molecule, such as a co-conjugate, provided herein. In some embodiments, the CAR is expressed in a cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a T cell, a T cell precursor, a Natural Killer (NK) cell, or an Antigen Presenting Cell (APC).

In some embodiments, the conjugate molecule, such as a co-conjugate, is a peptide or protein. Also provided herein are nucleic acid molecules encoding peptide or protein conjugate molecules, such as co-conjugates, and vectors comprising nucleic acids encoding the peptide or protein. Thus, "nucleic acid" includes nucleic acids encoding the conjugate molecules disclosed herein, as well as nucleic acids encoding their functional subsequences, sequence variants, and modified forms, so long as the foregoing nucleic acids retain at least detectable or measurable activity or function. Nucleic acid, which may also be referred to herein as a gene, polynucleotide, nucleotide sequence, primer, oligonucleotide or probe, refers to a natural or modified purine and pyrimidine containing polymer of any length, i.e., polyribonucleotides or polydeoxyribonucleotides or mixed polyribonucleotides and alpha-anomeric forms thereof. Two or more purine and pyrimidine containing polymers are typically linked by a phosphate linkage or an analogue thereof. The terms are used interchangeably to refer to all forms of nucleic acid, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleic acid may be single-stranded, double-stranded or triple-stranded, linear or circular. Nucleic acids include genomic DNA and cDNA. The RNA nucleic acid may be a spliced or non-spliced mRNA, rRNA, tRNA or antisense nucleic acid. Nucleic acids include naturally occurring nucleotides, synthetic nucleotides, and nucleotide analogs and derivatives.

Because of the degeneracy of the genetic code, nucleic acid molecules include sequences that are degenerate relative to nucleic acid molecules encoding the conjugate molecules of the present disclosure. When used in reference to nucleic acid sequences, the term "complementary" means that the regions referred to are 100% complementary, i.e., exhibit 100% base pairing, with no mismatches. Nucleic acids can be produced using any of a variety of known standard cloning and chemical synthesis methods, and can be intentionally altered by site-directed mutagenesis or other recombinant techniques known to those of skill in the art. The purity of the polynucleotide can be determined by sequencing, gel electrophoresis, UV spectroscopy.

Nucleic acids may be inserted into nucleic acid constructs in which expression of the nucleic acid is affected or regulated by an "expression control element" referred to herein as an "expression cassette". The term "expression control element" refers to one or more nucleic acid sequence elements that regulate or affect the expression of a nucleic acid sequence to which it is operably linked. Expression control elements may suitably include promoters, enhancers, transcription terminators, gene silencers, initiation codons (e.g., ATG) prior to the gene encoding the protein, and the like.

An expression control element operably linked to the nucleic acid sequence controls transcription of the nucleic acid sequence and, where appropriate, translation thereof. The term "operatively connected" refers to a juxtaposed relationship wherein the components mentioned are in a relationship that allows them to function in their intended manner. Typically, the expression control elements are juxtaposed at the 5 'or 3' end of the gene, but may also be introns.

Expression control elements include elements that constitutively activate transcription, either inducible (i.e., require an external signal or stimulus to activate) or non-repressible (i.e., require a signal to turn off transcription; when the signal is no longer present, transcription is activated or "de-repressed"). Also included in the expression cassettes of the present disclosure are control elements (i.e., tissue-specific control elements) sufficient to allow for control of gene expression for a particular cell type or tissue. Typically, such elements are located upstream or downstream (i.e., 5 'and 3') of the coding sequence. Promoters are usually located 5' to the coding sequence. Promoters produced by recombinant DNA or synthetic techniques may be used to provide transcription of the polynucleotides of the present disclosure. "promoter" generally refers to the smallest sequence element that is sufficient to direct transcription.

The nucleic acid may be inserted into a plasmid for transformation into a host cell and for subsequent expression and/or genetic manipulation. Plasmids are nucleic acids that can stably proliferate in a host cell; the plasmid may optionally contain expression control elements to drive expression of the nucleic acid. As used herein, a vector is synonymous with a plasmid. Plasmids and vectors typically contain at least an origin of replication and a promoter for propagation in a cell. Plasmids and vectors may also comprise expression control elements for expression in a host cell, and thus may be used, for example, to express and/or genetically manipulate nucleic acids encoding peptide sequences, to express peptide sequences in host cells and organisms (e.g., subjects in need of treatment), or to produce peptide sequences.

As used herein, the term "transgene" refers to a polynucleotide that is introduced into a cell or organism by artificial means. For example, cells having a transgene that has been introduced by genetic manipulation or "transformation" of the cell. Cells into which the transgene is introduced or their progeny are referred to as "transformed cells" or "transformants". Typically, the transgene is contained in the progeny of the transformant, or is part of an organism that develops from the cell. The transgene may be inserted into chromosomal DNA or maintained as a self-replicating plasmid, YAC, minichromosome, or the like.

Bacterial system promoters include T7 and inducible promoters such as the pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) and tetracycline responsive promoters of phage lambda. Insect cell system promoters include constitutive or inducible promoters (e.g., ecdysone). Mammalian cell constitutive promoters include SV40, RSV, bovine Papilloma Virus (BPV) and other viral promoters, or inducible promoters derived from the mammalian cell genome (e.g., metallothionein IIA promoter; heat shock promoter) or from mammalian viruses (e.g., adenovirus late promoter; inducible mouse mammary tumor virus long terminal repeat). Alternatively, the retroviral genome may be genetically modified to introduce and direct expression of peptide sequences in an appropriate host cell.

Also provided herein are vectors designed for in vivo applications, including in vivo delivery and expression systems thereof. Specific non-limiting examples include adenovirus vectors (U.S. Pat. nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S. Pat. No. 5,604,090), herpes simplex virus vectors (U.S. Pat. No. 5,501,979), retrovirus vectors (U.S. Pat. nos. 5,624,820, 5,693,508 and 5,674,703), BPV vectors (U.S. Pat. No. 5,719,054), CMV vectors (U.S. Pat. No. 5,561,063), parvoviruses, rotaviruses, norwalk viruses, and lentiviral vectors (see, e.g., U.S. Pat. No. 6,013,516). Vectors include those that deliver genes to intestinal cells, including stem cells (Croyle et al, gene Ther.5:645 (1998); S.J.Henning, adv.Drug Deliv. Rev.17:341 (1997); U.S. Pat. Nos. 5,821,235 and 6,110,456). Many of these vectors have been approved for human research.

Yeast vectors include constitutive and inducible promoters (see, e.g., ausubel et al, in: current Protocols In Molecular Biology, volume 2, chapter 13, editors Greene publication. Assoc. & Wiley Interscience,1988; grant et al Methods In Enzymology,153:516 (1987), editors Wu & Grossman; bitterMethods In Enzymology,152:673 (1987), editors Berger & Kimmel, acad.Press, N.Y.; and Strater et al, the Molecular Biology of the Yeast Saccharomyces (1982) eds. Cold Spring Harbor Press, volumes I and II). Constitutive yeast promoters such as ADH or LEU2 or inducible promoters such as GAL may be used (R.Rothstein In: DNA Cloning, A Practical Approach, vol.11, chapter 3, editions D.M.Glover, IRL Press, wash., D.C., 1986). Vectors that facilitate integration of exogenous nucleic acid sequences into yeast chromosomes are known in the art, for example, via homologous recombination. Yeast Artificial Chromosomes (YACs) are typically used when the inserted polynucleotide is too large (e.g., greater than about 12 Kb) for more conventional vectors.

The expression vector may also contain a selectable or identifiable marker (e.g., β -galactosidase) that confers resistance to selection pressure, thereby allowing for the selection, growth, and expansion of cells with the vector. Alternatively, the selectable marker may be on a second vector that is co-transfected into the host cell with a first vector that contains a nucleic acid encoding a peptide sequence. Selection systems include, but are not limited to, the herpes simplex virus thymidine kinase gene (Wigler et al, cell 11:223 (1977)), the hypoxanthine-guanine phosphoribosyl transferase gene (Szybalska et al, proc. Natl. Acad. Sci. USA 48:2026 (1962)), and the adenine phosphoribosyl transferase gene (Lowy et al, cell 22:817 (1980)) which can be used in tk-, hgprt-, or aprt-cells, respectively. Furthermore, antimetabolite resistance can be used as a selection basis for: dhfr, which confers resistance to methotrexate (O' Hare et al, proc. Natl. Acad. Sci. USA 78:1527 (1981)); the gpt gene, which confers resistance to mycophenolic acid (Mulligan et al, proc. Natl. Acad. Sci. USA 78:2072 (1981)); neomycin gene, which confers resistance to aminoglycoside G-418 (Colberre-Garapin et al, J.mol. Biol.150:1 (1981)); puromycin; and a hygromycin Gene which confers hygromycin resistance (Santerre et al, gene 30:147 (1984)). Additional selection genes include trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells to replace histidine with histidine (Hartman et al, proc. Natl. Acad. Sci. USA 85:8047 (1988)); and ODC (ornithine decarboxylase) conferring resistance to the ornithine decarboxylase inhibitor 2- (difluoromethyl) -DL-ornithine DFMO (McConlovue (1987) In: current Communications In Molecular Biology, cold Spring Harbor Laboratory).

Thus, provided herein are also transformed cells or host cells (in vitro, ex vivo, and in vivo) that produce the conjugate molecules disclosed herein, such as co-conjugates, wherein expression of the conjugate molecules is conferred by a nucleic acid encoding the co-conjugates. Transformed cells and host cells expressing a conjugate molecule, such as a co-conjugate, typically comprise a nucleic acid encoding the conjugate molecule. In some embodiments, the transformed cell or host cell is a prokaryotic cell. In another embodiment, the transformed cell or host cell is a eukaryotic cell. In various aspects, the eukaryotic cell is a yeast or mammalian (e.g., human, primate, etc.) cell.

As used herein, a "transformed" cell or "host" cell is a cell into which nucleic acid has been introduced, which can be propagated and/or transcribed to express the encoded peptide sequence. The term also includes any progeny or subclones of the host cell. Transformed cells and host cells include, but are not limited to, microorganisms such as bacteria and yeast; and plant, insect and mammalian cells. For example, bacteria transformed with recombinant phage nucleic acid, plasmid nucleic acid, or cosmid nucleic acid expression vectors; yeast transformed with a recombinant yeast expression vector; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, caMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., ti plasmid); insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus); and animal cell systems infected with recombinant viral expression vectors (e.g., retrovirus, adenovirus, vaccinia virus) or transformed animal cell systems engineered for transient or stable proliferation or expression.

In some embodiments, the present disclosure provides a cell that expresses a conjugate molecule, such as a co-conjugate, provided herein. In some embodiments, the cell expressing the conjugate molecule is an immune cell. In some embodiments, the cell expressing the conjugate molecule is a T cell, a T cell precursor, a Natural Killer (NK) cell, or an Antigen Presenting Cell (APC). In some embodiments, the present disclosure provides a host cell that expresses a conjugate molecule, such as a co-conjugate, provided herein. In some embodiments, the host cell expressing the conjugate molecule is an immune cell. In some embodiments, the host cell expressing the conjugate molecule is a T cell, a T cell precursor, a Natural Killer (NK) cell, or an Antigen Presenting Cell (APC).

In some embodiments, the present disclosure provides a complex comprising a conjugate molecule provided herein, such as a co-conjugate and a target.

Also provided herein are kits comprising conjugate molecules provided herein, such as co-conjugates or compositions thereof (e.g., pharmaceutical compositions), packaged in suitable packaging materials. The kit optionally includes a label or package insert that includes a description of the components or instructions for use of the components therein, either in vitro, in vivo, or ex vivo.

The term "packaging material" refers to the physical structure that contains the components of the kit. The packaging material may keep the components sterile and may be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoule, vial, tube, etc.).

Kits provided herein may include a label or insert. The label or insert includes "print" such as paper or cardboard, a kit or packaging material (e.g., a box) alone or adhered to the components, or attached to, for example, an ampoule, tube or vial containing the kit components. The label or insert may additionally include a computer-readable medium, such as a magnetic disk (e.g., hard disk, card, storage disk); optical discs such as CD-ROM or DVD-ROM/RAM, DVD, MP3, magnetic tape; or an electronic storage medium such as RAM and ROM or hybrids of these media, e.g., magnetic/optical storage media; a flash memory medium; or a memory type card. The label or insert may include information identifying manufacturer information, lot number, manufacturer location, and date.

Kits provided herein can additionally include other components. Each component of the kit may be enclosed in a separate container, and all of the different containers may be enclosed in a single package. The kit may also be designed for refrigeration. The kit may further be designed to contain a conjugate molecule provided herein, such as a co-conjugate, or a cell containing a nucleic acid encoding a conjugate molecule provided herein, such as a co-conjugate. The cells in the kit may be stored under appropriate storage conditions until ready for use.

V. preparation method, library and screening technique

The conjugate molecules described herein, such as co-conjugates, may be produced by any method known in the art for the synthesis of peptides, nucleic acids or other molecules, in particular by chemical synthesis or recombinant expression techniques. The practice of the present disclosure employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g., maniatis et al (1982) Molecular Cloning: ALaboratory Manual, cold Spring Harbor Laboratory Press; sambrook et al (1989), molecular Cloning: A Laboratory Manual, second edition, cold Spring Harbor Laboratory Press; sambrook et al (2001) Molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, NY; ausubel et al Current Protocols in Molecular Biology, john Wiley & Sons (1987 and yearly updates); current Protocols in Immunology, john Wiley & Sons (1987 and annual update) Gait (eds.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; eckstein (eds.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; birren et al (eds.) (1999) Genome Analysis, A Laboratory Manual, cold Spring Harbor Laboratory Press; borrebaeck (eds.) (1995) Antibody Engineering, second edition, oxford University Press; lo (braid) (2006) Antibody Engineering: methods and Protocols (Methods in Molecular Biology); volume 248, humana Press, inc; each of which is incorporated by reference in its entirety.

Peptides and peptidomimetics can be produced and isolated using methods known in the art. Peptides can be synthesized in whole or in part using chemical methods (see, e.g., caruthers (1980), nucleic Acids Res. Symp. Ser.215; horn (1980); and Banga, A.K., therapeutic Peptides and Proteins, formulation, processing and Delivery Systems (1995) Technomic Publishing Co., lancaster, pa.). Peptide synthesis can be performed using a variety of solid phase techniques (see, e.g., roberge Science 269:202 (1995); merrifield, methods enzymes 289:3 (1997)) and automated synthesis can be accomplished according to manufacturer's instructions, e.g., using an ABI 431A peptide synthesizer (Perkin Elmer). Peptides and peptidomimetics can also be synthesized using combinatorial methods. Synthetic residues and polypeptides incorporating the mimetics can be synthesized using a variety of procedures and methods known in the art (see, e.g., organic Syntheses Collective Volumes, gilman et al, john Wiley & Sons, inc., NY). The modified peptides can be produced by chemical modification (see, e.g., belosus, nucleic Acids Res.25:3440 (1997); frenkel, free radio. Biol. Med.19:373 (1995); and Blommers Biochemistry 33:7886 (1994)). Peptide sequence variation, derivatization, substitution, and modification can also be performed using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR-based mutagenesis. Site-directed mutagenesis (Carter et al, nucleic acids Res.,13:4331 (1986); zoller et al, nucleic acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al, gene 34:315 (1985)), restriction-selective mutagenesis (Wells et al, philos. Trans. R. Soc. London Sera 317:415 (1986)) and other techniques can be performed on cloned DNA to produce peptide sequences, variants, fusions and chimeras, as well as variations, derivatives, substitutions and modifications thereof.

Conjugate molecules, such as co-conjugates, described herein comprising antigen binding fragments of antibodies can be prepared using a variety of techniques known in the art, including using hybridomas and recombinant techniques, or a combination thereof. For example, monoclonal Antibodies can be produced using hybridoma techniques, including those known in the art and described in, for example, harlow et al Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd edition 1988); hammerling et al, in Monoclonal Antibodies and T-Cell hybrid 563 681 (Elsevier, N.Y., 1981), each of which is incorporated herein by reference in its entirety. Other methods of producing co-conjugates are also known in the art.

In some embodiments, the co-conjugates and antibodies provided herein for the co-conjugates can be produced by culturing cells transformed or transfected with a vector containing a nucleic acid encoding the co-conjugate or encoding the antibody. Polynucleotide sequences encoding the polypeptide components of the co-conjugates or antibodies of the present disclosure can be obtained using standard recombinant techniques. The desired polynucleotide sequence may be isolated and sequenced from a co-conjugate or antibody producing cell such as a hybridoma cell. Alternatively, polynucleotides may be synthesized using nucleotide synthesizers or PCR techniques. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotide in a host cell. Many vectors available and known in the art may be used for the purposes of this disclosure. The choice of the appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Host cells suitable for expressing the antibodies of the present disclosure include prokaryotes such as archaebacteria and eubacteria, including gram negative organisms or gram positive organisms, eukaryotic microorganisms such as filamentous fungi or yeast, invertebrate cells such as insect or plant cells, and vertebrate cells such as mammalian host cell lines. The host cells are transformed with the above-described expression vectors and cultured in a suitably modified conventional nutrient medium for inducing promoters, selecting transformants or amplifying the genes encoding the desired sequences. The co-conjugate produced by the host cell is purified using standard protein purification methods as known in the art.

Methods for co-conjugate generation, including vector construction, expression and purification, are further described in Pluckthun et al, antibody Engineering: producing antibodies in Escherichia coli: from PCR to fermentation 203-52 (McCafferty et al, 1996); kwong and Rader, e.colli Expression and Purification of Fab Antibody Fragments, in Current Protocols in Protein Science (2009); tachibana and Takekoshi, production of Antibody Fab Fragments in Escherischia coli, in Antibody Expression and Production (Al-Rubai, eds.); and Therapeutic Monoclonal Antibodies: from Bench to Clinic (An, 2009).

Of course, alternative methods known in the art are contemplated for preparing the co-conjugates. For example, suitable amino acid sequences or portions thereof may be produced by direct peptide synthesis using Solid phase techniques (see, e.g., stewart et al, solid-Phase Peptide Synthesis (1969); and Merrifield,1963, J.Am. Chem. Soc. 85:2149-54). In vitro protein synthesis may be performed using manual techniques or automation. The different moieties of the co-conjugate may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired co-conjugate.

In certain aspects, provided herein is a combinatorial library (e.g., a co-conjugate library) that can be used to develop a conjugate molecule, such as a co-conjugate, that specifically recognizes a target as described herein.

In some embodiments, a library is provided comprising a plurality of co-binders or a plurality of polynucleotides encoding a plurality of co-binders, each co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, wherein the first binding moiety is linked to the second binding moiety via a peptide linker through the N-terminus of the antibody variable domain, wherein at least two co-binders in the library differ from each other in peptide linker sequence. In some embodiments, the first target site and the second target site are non-overlapping binding sites on the target molecule. In some embodiments, the antibody variable domain has an N-terminal truncation ("N-terminally truncated antibody variable domain"). In some embodiments, at least two co-binders in the library differ from each other in terms of N-terminal truncation of the antibody variable domain of the second antibody moiety.

In some embodiments, the diversity of the library is at least about 5000, e.g., the library contains at least about 5000 unique co-binder sequences.

In some embodiments, substantially all of the plurality of co-conjugates comprise the same first binding moiety and second binding moiety. In such embodiments, the library comprises co-binders comprising unique linker sequences.

In some embodiments, at least two of the plurality of co-conjugates, e.g., any of at least about 10, 25, 50, 100, 250, 500, and 1,000, comprise different first and/or second binding moieties.

In some embodiments, methods of screening for co-binders that specifically bind to a second target site with a desired affinity are provided, the methods comprising: (1) Contacting the library described herein with a target molecule comprising a second target site to form a complex between a co-conjugate that specifically binds the target molecule and the target molecule, and (2) identifying the co-conjugate that binds the second target site with a desired affinity.

In some embodiments, methods of screening for co-conjugates that specifically bind to a target molecule with a desired affinity are provided, the methods comprising: (1) Contacting the library described herein with a target molecule to form a complex between a co-conjugate that specifically binds the target molecule and the target molecule, and (2) identifying the co-conjugate that binds the target molecule with a desired affinity.

In some embodiments, the combinatorial library comprises a collection of any one or more of: (a) a second binding moiety; (b) a first binding moiety; (c) a linker; and/or (d) another feature described herein, such as a label.

In some embodiments, the library comprises antibodies from a plurality of antibodiesAt least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 x 10 of the second binding moiety described herein ³ At least 1X 10 ⁴ At least 1X 10 ⁵ At least 1X 10 ⁶ At least 1X 10 ⁷ At least 1X 10 ⁸ At least 1X 10 ⁹ At least 1X 10 ¹⁰ Or at least 1X 10 ¹¹ And a variable region, wherein each variable region comprises a truncation of 1 to 18 amino acids. In some embodiments, the library comprises about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1 x 10 of the second binding moiety described herein from the plurality of antibodies ³ About 1X 10 ⁴ About 1X 10 ⁵ About 1X 10 ⁶ About 1X 10 ⁷ About 1X 10 ⁸ About 1X 10 ⁹ About 1X 10 ¹⁰ Or about 1X 10 ¹¹ And a variable region, wherein each variable region comprises an N-terminal truncation of 1 to 18 amino acids. In some embodiments, the truncation is in the FR1 region of the variable region.

In some embodiments, the library comprises at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 x 10 of the first binding moiety described herein from a plurality of antibodies ³ At least 1X 10 ⁴ At least 1X 10 ⁵ At least 1X 10 ⁶ At least 1X 10 ⁷ At least 1X 10 ⁸ At least 1X 10 ⁹ At least 1X 10 ¹⁰ Or at least 1X 10 ¹¹ A variable region. In some embodiments, the library comprises about 2, about 5, about 10, about 15, about 20, about 25, a first binding moiety described herein from a plurality of antibodies,About 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1 x 10 ³ About 1X 10 ⁴ About 1X 10 ⁵ About 1X 10 ⁶ About 1X 10 ⁷ About 1X 10 ⁸ About 1X 10 ⁹ About 1X 10 ¹⁰ Or about 1X 10 ¹¹ A variable region.

In some embodiments, the library comprises at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 x 10 ³ At least 1X 10 ⁴ At least 1X 10 ⁵ At least 1X 10 ⁶ At least 1X 10 ⁷ At least 1X 10 ⁸ At least 1X 10 ⁹ At least 1X 10 ¹⁰ Or at least 1X 10 ¹¹ And a plurality of connectors. In some embodiments, the library comprises about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1 x 10 ³ About 1X 10 ⁴ About 1X 10 ⁵ About 1X 10 ⁶ About 1X 10 ⁷ About 1X 10 ⁸ About 1X 10 ⁹ About 1X 10 ¹⁰ Or about 1X 10 ¹¹ The linkers described herein.

In some embodiments, the library comprises a partial form of a conjugate molecule described herein, e.g., a second binding moiety covalently attached to a linker. The plurality of members in the library may each comprise a different second binding moiety (e.g., a second binding moiety having a different CDR sequence or a different N-terminal truncation) and/or a different linker sequence. In such embodiments, such library features may be used to effectively identify binder molecules, such as co-binders. In some embodiments, the N-terminal amino acid of the linker in each member of the library is further linked to the C-terminal amino acid of the first binding moiety.

In some embodiments, a single second binding moiety covalently attached to a linker may be fused to a different first binding moiety to form a library for the purpose of identifying a suitable co-conjugate.

In some embodiments, the library comprises a first variable region of the first binding moiety, wherein the N-terminal amino acid of the linker is linked to the C-terminal amino acid of the first variable region. In some embodiments, the library comprises a plurality of first variable regions of the first binding moiety, wherein the N-terminal amino acid of the linker is linked to the C-terminal amino acid of the first variable region. In some embodiments, the library comprises a plurality of first variable regions of a plurality of first binding moieties, wherein the N-terminal amino acid of the linker is linked to the C-terminal amino acid of the first variable region.

Accordingly, as will be appreciated by those of ordinary skill in the art based on the above description, the present disclosure provides libraries of binder molecules, such as co-binders, comprising: (i) Any first subsection of the library selected from the group consisting of: a second heavy chain variable region comprising an N-terminally truncated second antibody portion of 1 to 18 amino acids; a plurality of second heavy chain variable regions of a second antibody portion, wherein each second heavy chain variable region comprises an N-terminal truncation of 1 to 18 amino acids; a plurality of second heavy chain variable regions of a plurality of second antibody portions, wherein each second heavy chain variable region comprises an N-terminal truncation of 1 to 18 amino acids; a second light chain variable region of a second antibody portion comprising an N-terminal truncation of 1 to 18 amino acids; a plurality of second light chain variable regions of a second antibody portion, wherein each second light chain variable region comprises an N-terminal truncation of 1 to 18 amino acids; and a plurality of second light chain variable regions of a plurality of second antibody portions, wherein each second light chain variable region comprises an N-terminal truncation of 1 to 18 amino acids; (ii) Any linker moiety of a library selected from the group consisting of: a polypeptide linker; and a plurality of polypeptide linkers; and (iii) any second subsection of the library selected from the group consisting of: a first heavy chain variable region of a first antibody portion; a plurality of first heavy chain variable regions of a first antibody portion; a plurality of first heavy chain variable regions of a plurality of first antibody portions; a first light chain variable region of a first antibody portion; a plurality of first light chain variable regions of a first antibody portion; and a plurality of first light chain variable regions of the plurality of first antibody portions, wherein the N-terminal amino acid of the linker is linked to the C-terminal amino acid of the first light chain variable region, and wherein the C-terminal amino acid of the linker is linked to the N-terminal amino acid of the truncated first light chain variable region. Thus, one of ordinary skill in the art will understand that the library of co-conjugates provided herein includes any and all combinations or permutations of the present disclosure and, in particular, the subfractions of the libraries provided in this paragraph for (i), (ii), and (iii).

In some embodiments, the library comprises (i) a first variable region and a second variable region that bind non-overlapping epitopes on the same target, (ii) a first variable region and a second variable region that do not bind the same target, (iii) a first variable region and a second variable region that bind non-overlapping epitopes on the same target, or (iv) a first variable region and a second variable region that do not bind the same target.

In some embodiments, a library of first binding moieties (paratope P1) and a library of second binding moieties (paratope P2) that bind to an epitope of a target antigen can be independently constructed. The library of first binding moieties (paratope P1) or the library of second binding moieties (paratope P2) can be constructed in a gene expression vector that allows transcription and translation of the cloned gene into a recombinant protein.

The library of first binding moieties (paratope P1) may be sequences encoding camelids VHH, scFv, fab, affibodies, affilin, affimer, affitin, alpha bodies, anticalin, aptamers, affibodies, DARPin, fynomer, kunitz domain peptides, monomers, or nanocamp, etc. Similarly, the library of second binding moieties (P2) may be sequences encoding camelids VHH, scFv, fab, affibodies, affilin, affimer, affitin, alpha bodies, anticalin, aptamers, affibodies, DARPin, fynomer, kunitz domain peptides, monomers, or nanocamp, etc. Any combination of different libraries of first binding moieties (paratope P1) and second binding moieties (paratope P2) is contemplated herein.

In some embodiments, the library of first binding moieties (paratope P1) and the library of second binding moieties (paratope P2) in the expression vector may both be sequences encoding camelid VHHs. In some embodiments, the library of first binding moieties (paratope P1) and the library of second binding moieties (paratope P2) in the expression vector may both be sequences encoding scFv. In another preferred embodiment, the library of first binding moieties (paratope P1) and the library of second binding moieties (paratope P2) in the expression vector may be one sequence encoding a camelid VHH and the other sequence encoding a scFv.

For example, to construct a library of first binding moieties (paratope P1) or a library of second binding moieties (paratope P2), mRNA encoding the heavy chain antibody variable region (VHH) can be isolated from alpaca immunized against the target molecule, transcribed into cDNA, and cloned into phagemid vectors for phage display library construction. Similarly, to construct a library of first binding moieties (P1 paratope) or a library of second binding moieties (P2 paratope), mRNA encoding the heavy and light chain antibody variable domains can be isolated from animals immunized against the target antigen, transcribed into cDNA, and cloned into phagemid vectors as scFv for phage display library construction. While the expression vector may be part of a phage display library construction, it may also be part of a yeast display, bacterial display, mammalian cell display, ribosome display or mRNA display library construction.

The library of first binding moieties (P1 paratope) or the library of second binding moieties (P2 paratope) may be the original library, and they may also be primary response or secondary response immune libraries. The library of first binding moieties (P1 paratope) or the library of second binding moieties (P2 paratope) may be a synthetic library. The library of first binding moieties (P1 paratope) or the library of second binding moieties (P2 paratope) may be an affinity enriched original library, an immune library or a synthetic library for binding to a target antigen of interest. For example, a library of first binding moieties (P1 paratope) or a library of second binding moieties (P2 paratope) can be cloned into a phagemid of a phage display library construct. These phage display libraries are then bound to immobilized target antigens. Phage display proteins that interact with the target antigen will remain attached while all other proteins are washed away. The attached phage may then be eluted and used to produce more phage by infecting the appropriate bacterial host. The new phage constitute an enriched mixture that contains significantly less unbound phage than was present in the initial mixture. By this procedure, a library of first binding moieties (paratope P1) or a library of second binding moieties (paratope P2) can be enriched for sequences encoding those paratopes that bind the target antigen. An affinity enriched library of the first binding moiety (paratope P1) or an affinity enriched library of the second binding moiety (paratope P2) may be more suitable for screening.

In certain aspects, provided herein is a method of screening for a binder molecule, such as a co-binder, that binds to a target, comprising (i) obtaining a library provided herein; and (ii) contacting the library of candidates from step (i) with a target to identify a binder molecule, such as a co-binder, that specifically binds the target.

In certain aspects, provided herein is a method of screening for a binder molecule, such as a co-binder, that binds to a target, comprising (i) expressing a library of expression vectors encoding a library provided herein; (ii) obtaining a library provided herein; and (iii) contacting the library of candidates from step (ii) with a target to identify a binder molecule, such as a co-binder, that specifically binds the target.

In another aspect, provided herein is a method of screening for a binder molecule, such as a co-binder, that binds to a target, comprising (i) expressing a library of expression vectors encoding a library of co-binders provided herein; (ii) obtaining a library provided herein; (iii) Contacting the library of candidates from step (ii) with a target to form a complex between binder molecules, such as co-binders, that specifically bind the target; (iv) Enriching complexes between binding molecules, such as co-binders, that specifically bind to the target; and (v) identifying a conjugate molecule, such as a co-conjugate, that specifically binds to the target.

In some embodiments, the screening methods provided herein identify binder molecules, such as co-binders, wherein the affinity of the conjugate molecule for the target is no less than 50-fold, no less than 60-fold, no less than 70-fold, no less than 80-fold, no less than 90-fold, no less than 100-fold, no less than 110-fold, no less than 120-fold, no less than 130-fold, no less than 140-fold, no less than 150-fold, no less than 160-fold, no less than 170-fold, no less than 180-fold, no less than 190-fold, no less than 200-fold, no less than 250-fold, no less than 300-fold, no less than 350-fold, no less than 400-fold, no less than 450-fold, no less than 500-fold, no less than 600-fold, no less than 700-fold, no less than 800-fold, no less than 900-fold, no less than 1000-fold, no less than 1100-fold, no less than 1200-fold, no less than 1300-fold, no less than 1400-fold, no less than 1500-fold, no less than 1600-fold, no less than 1700-fold, no less than 300-fold, no less than 350-fold, no less than 400-fold, no less than 4000-fold, no less than 5000-fold, or no less than 5000-fold.

In some embodiments of the screening methods provided herein, a conjugate molecule, such as a co-conjugate, identified by the methods binds to K of the target _D Less than 1X 10 ^-8 M is less than 1×10 ^-9 M is less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 M is less than 1×10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M is less than 1×10 ^-16 M is less than 1×10 ^-17 M or less than 1X 10 ^-18 M。

In some embodiments, provided herein are methods of screening for co-binders, wherein an expression vector can be constructed that contains a first coding region for a subpart of a library of a first binding moiety (paratope P1), a second coding region for a subpart of a library of a second binding moiety (paratope P2), and a third coding region for a subpart of a linker L library that connects the first binding moiety (paratope P1) and the second binding moiety (paratope P2). In phage display systems, expression vectors are expressed as fusions with phage coat proteins (e.g., pIII) such that they are displayed on the surface of the virion. The displayed fusion protein corresponds to the genetic sequence within the phage. By this means, those displayed proteins can be identified which contain a first binding moiety (paratope P1) and a second binding moiety (paratope P2) linked by a linker, which have a high affinity binding to the target antigen, and which are candidates for co-binders. Similarly, candidate co-binders may be screened using yeast display, bacterial display, mammalian cell display, ribosome display or mRNA display library constructs.

The present disclosure demonstrates that identification of co-conjugates that specifically bind to a target can be accomplished by a variety of methods available to one of ordinary skill in the art. For example, polynucleotides encoding co-conjugates that specifically bind to a target can be sequenced from sorted host cells or panning phage as described above. The corresponding polypeptide sequences of the co-conjugates can be identified by translating the polynucleotide sequences encoding the co-conjugates using genetic code tables well known in the art. Alternatively, the co-conjugate can be identified by amino acid sequencing and/or mass spectrometry of the co-conjugate and/or the protein complex between the co-conjugate and the target.

In some embodiments, the first binding moiety (paratope P1) in the expression vector contains sequences encoding more than one different binding moiety. In some embodiments, the first binding moiety (paratope P1) in the expression vector comprises a sequence encoding more than 2, more than 5, more than 10, more than 20, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 1X 10 ⁴ More than 1X 10 ⁵ Or more than 1X 10 ⁶ Sequences of the different binding moieties. In some embodiments, the second binding moiety (paratope P2) in the expression vector contains sequences encoding more than one different paratope. In some embodiments, the second binding moiety (paratope P2) in the expression vector comprises a sequence encoding at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 x 10 ³ At least 1X 10 ⁴ At least 1X 10 ⁵ At least 1X 10 ⁶ At least 1X 10 ⁷ At least 1X 10 ⁸ At least 1X 10 ⁹ At least 1X 10 ¹⁰ Or at least 1X 10 ¹¹ Sequences of the different binding moieties.

The linker L in the expression vector contains sequences encoding more than one linker. In some embodiments, the linker L in the expression vector contains a sequence encoding at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 x 10 ³ At least 1X 10 ⁴ At least 1X 10 ⁵ At least 1X 10 ⁶ At least 1X 10 ⁷ At least 1X 10 ⁸ At least 1X 10 ⁹ At least 1X 10 ¹⁰ Or at least 1X 10 ¹¹ Sequences of the different linkers. In some embodiments, the linker L in the expression vector contains a sequence encoding at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 x 10 ³ At least 1X 10 ⁴ At least 1X 10 ⁵ At least 1X 10 ⁶ At least 1X 10 ⁷ At least 1X 10 ⁸ At least 1X 10 ⁹ At least 1X 10 ¹⁰ Or at least 1X 10 ¹¹ Sequences of different recombinant proteins.

In some embodiments, a library of co-conjugates is provided, each co-conjugate comprising a first binding moiety and a second binding moiety that bind to the same target molecule, wherein the library comprises at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1X 10 ³ At least 1X 10 ⁴ At least 1X 10 ⁵ At least 1X 10 ⁶ At least 1X 10 ⁷ At least 1X 10 ⁸ At least 1X 10 ⁹ At least 1X 10 ¹⁰ Or at least 1X 10 ¹¹ A plurality of different joints, said joints being connected in pairsAnd a second binding portion.

The present disclosure shows that enrichment of a conjugate molecule, such as a co-conjugate, that specifically binds to a target with a complex between the target can be achieved by a variety of methods available to one of ordinary skill in the art. For example, host cells expressing the library of co-conjugates can be sorted by suitable sorting means (e.g., fluorescence activated cell sorting, "FACS" or magnetic bead-based sorting) to positively select cells expressing co-conjugates with high affinity, thereby obtaining a cell population enriched for pluripotent cells. In a specific embodiment, host cells expressing the library of co-binders can be sorted based on the amount of staining using the labeled target, wherein enrichment of the high affinity co-binders can be fine-tuned by adjusting the concentration of the labeled target. In this fine tuning of high affinity co-conjugate enrichment, only K when low concentrations of labeled target are used _D Co-binders above a determinable level can be stably stained and sorted. The lower the concentration, the higher the binding stringency. High affinity binders can maintain good target binding at high stringency, but weaker binders cannot. In certain embodiments, the concentration is less than 1X 10 ^-8 M is less than 1×10 ^-9 M is less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 M is less than 1×10 ^-13 M is less than 1×10 ^-14 M or less than 1X 10 ^-15 M labeled targets stain host cells expressing the library of co-binders. In certain embodiments, a concentration of about 1X 10 is used ^-8 M, about 1X 10 ^-9 M, about 1X 10 ^-10 M, about 1X 10 ^-11 M, about 1X 10 ^-12 M, about 1X 10 ^-13 M, about 1X 10 ^-14 M or about 1X 10 ^-15 M labeled targets stain host cells expressing the library of co-binders.

Alternatively, host cells expressing the library of co-binders may be sorted based on the amount of staining using a labeled target, wherein the labeled target bound to host cells expressing the low affinity co-binders has been washed away by washing under various stringent conditions, thereby enriching for host cells expressing the high affinity co-binders. In such a caseIn embodiments, the stringency of the wash may be fine tuned and controlled by various methods known to those of ordinary skill in the art. For example, host cells expressing the library of co-binders may be washed with unlabeled target molecules that compete with the labeled targets. The stringency of the wash can be controlled by adjusting the ratio between unlabeled and labeled targets such that host cells expressing only high affinity co-binders will remain stained by the labeled targets and positively sorted, enriching for host cells expressing high affinity co-binders. The stringency of the wash can be controlled by adjusting the intensity of the wash buffer used, e.g., by washing with different detergent solutions. In certain embodiments, the concentration is greater than 1X 10 ^-3 M is greater than 1×10 ^-4 M, or greater than 1X 10 ^-5 M、1×10 ^-6 M is greater than 1×10 ^-6 M, or greater than 1X 10 ^-7 M is greater than 1×10 ^-8 M is greater than 1×10 ^-9 M is greater than 1×10 ^-10 M is greater than 1×10 ^-11 M, or greater than 1X 10 ^-12 The unlabeled target of M washes host cells expressing the library of co-binders. In certain embodiments, a concentration of about 1X 10 is used ^-3 M, about 1X 10 ^-4 M, or about 1X 10 ^-5 M、1×10 ^-6 M, about 1X 10 ^-6 M or about 1X 10 ^-7 M, about 1X 10 ^-8 M, about 1X 10 ^-9 M, about 1X 10 ^- ¹⁰ M, about 1X 10 ^-11 M or about 1X 10 ^-12 The unlabeled target of M washes host cells expressing the library of co-binders.

In addition, the washing time may vary. After incubating the library members with the target protein, unbound library members or proteins need to be washed away. The longer the washing time, the higher the stringency. Weaker binders may dissociate from the target protein during the washing step, but high affinity binders do not. Similarly, the time of incubation of the target protein may also vary. Strong binders tend to bind targets faster than weak binders. By limiting the incubation time, high affinity binders are better enriched than low affinity binders.

One or more rounds of enrichment can be performed to enrich for co-binders with high affinity for the target. In some embodiments, after 3-4 rounds of increased stringency selection, the resulting library of co-binders will be enriched in high affinity co-binders. In some embodiments, the library of co-binders is enriched for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 rounds to obtain a library of co-binders having a high affinity for the co-binders.

In some embodiments described herein, host cells expressing the high affinity co-conjugate may also be enriched by negative sorting (removing) cells that are not stained by the labeled target.

The strategies described above for enriching complexes between co-binders specifically binding to targets and targets may also be used and may be combined with other techniques described herein and known to one of ordinary skill in the art. For example, the binding measured in SPR, in ELISA, in phage display panning, in yeast display, in mammalian cell display can be fine tuned by reducing the concentration of labeled target for binding or by adjusting the stringency of the wash as described above, so that only high affinity co-conjugates will stably bind to the labeled target, enriching for complexes between co-conjugates specifically binding to the target and the target.

In some embodiments, the conjugate molecules provided herein, such as co-conjugates, variants, and/or antibody variants, are prepared by in vitro affinity maturation, with improved properties, such as affinity, stability, or expression levels, as compared to the parental construct. In vitro affinity maturation, as with the natural prototype, is based on the principle of mutation and selection. Libraries of conjugate molecules, such as co-conjugates, variants, and/or antibody variants, provided herein are displayed on the surface of an organism (e.g., phage, bacterial, yeast, or mammalian cells), or associated (e.g., covalently or non-covalently) with their encoding mRNA or DNA. Affinity selection of displayed conjugate molecules such as co-conjugates, variants, and/or antibody variants allows for isolation of organisms or complexes carrying genetic information encoding antibodies. Two or three rounds of mutation and selection using a display method such as phage display typically produce antibody fragments with affinities in the low nanomolar range. Affinity matured binder molecules such as co-binders, variants and/or antibody variants may have nanomolar or even picomolar affinity for the target antigen.

Phage display is a widely used method for displaying and selecting conjugate molecules provided herein, such as co-conjugates, variants, and/or antibody variants. Conjugate molecules such as co-conjugates, variants and/or antibody variants are displayed as fusions with phage coat proteins on Fd or M13 phage surfaces. Selection involves exposure to an antigen to allow phage-displayed conjugate molecules, such as co-conjugates, variants, and/or antibody variants, to bind to their targets, a process known as "panning" to recover antigen-bound phage, and used to infect bacteria to produce phage for further rounds of selection. For reviews, see, e.g., hoogenboom,2002, methods. Mol. Biol.178:1-37; and Bradbury and Marks,2004,J.Immunol.Methods 290:29-49.

In yeast display systems (see, e.g., boder et al, 1997, nat. Biotech.15:553-57; and Chao et al, 2006,Nat.Protocols 1:755-68), antibody variants of a conjugate molecule, such as a co-conjugate, provided herein may be displayed as a single chain variable fusion (scFv), in which the heavy and light chains are linked by a flexible linker. The scFv is fused to an adhesion subunit of the yeast lectin protein Aga2p, which is attached to the yeast cell wall by disulfide bonds with Aga1 p. Displaying the protein by Aga2p projects the protein from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen libraries to select antibodies with improved affinity or stability. Binding to the soluble antigen of interest is determined by labeling the yeast with a biotinylated antigen and a secondary reagent such as streptavidin conjugated to a fluorophore. Changes in antibody surface expression can be measured by immunofluorescence labeling of hemagglutinin or c-Myc epitope tags flanking the scFv. Expression has been shown to correlate with the stability of the displayed protein, and thus antibodies with improved stability and affinity can be selected (see, e.g., shusta et al 1999, J.mol. Biol.2 92:949-56). Another advantage of yeast display is that the displayed protein folds in the endoplasmic reticulum of eukaryotic yeast cells, utilizing endoplasmic reticulum partners and quality control mechanisms. Once maturation is complete, the antibody affinity can be conveniently "titrated" while displayed on the yeast surface, thereby eliminating the need to express and purify each clone. The theoretical limit of yeast surface display is that functional library sizes may be smaller than other display methods; however, the most recent method uses a mating system of yeast cells to produce a size estimate of 10 ¹⁴ For example, see U.S. patent publication 2003/0186374; and Blaise et al 2004,Gene 342:211-18).

In ribosome display, antibody-ribosome-mRNA (ARM) complexes are generated in cell-free systems for selection. DNA libraries encoding specific libraries of binder molecules, such as co-binders, variants or antibody variants, are genetically fused to spacer sequences lacking stop codons. The spacer sequence remains attached to the peptidyl tRNA post-translationally and occupies the ribosomal channel, allowing the protein of interest to extend out of the ribosome and fold. The resulting complex of mRNA, ribosome and protein can bind to surface-bound ligands, allowing simultaneous isolation of antibodies and their encoding mRNA by affinity capture with the ligand. The ribosome-bound mRNA is then reverse transcribed back into cDNA, which can then be subjected to mutagenesis and used for the next round of selection (see, e.g., fukuda et al, 2006,Nucleic Acids Res.34:e127). In mRNA display, puromycin was used as an adapter molecule to establish covalent bonds between the antibody and the mRNA (Wilson et al, 2001,Proc.Natl.Acad.Sci.USA 98:3750-55).

Since these methods are performed entirely in vitro, they offer two major advantages over other selection techniques. First, the diversity of the library is not limited by the transformation efficiency of the bacterial cells, but only by the number of ribosomes present in the tube and the different mRNA molecules. Second, random mutations can be easily introduced after each round of selection, for example by non-proofreading polymerase, since no library has to be transformed after any diversification step.

In mammalian cell display systems (see, e.g., bowers et al, 2011,Proc Natl Acad Sci USA.108:20455-60), a complete human IgG library was constructed based on germline sequence V gene segments linked to pre-recombinant D (J) regions. The full length V regions of the heavy and light chains are assembled with human heavy and light chain constant regions and transferred into mammalian cell lines (e.g., HEK 293). The transfected library was amplified and several rounds of negative selection were performed on Streptavidin (SA) coupled magnetic beads followed by one round of positive selection on SA coupled magnetic beads coated with biotinylated target proteins, peptide fragments or epitopes. Positively selected cells are expanded and then sorted by multiple rounds of FACS to isolate single cell clones displaying antibodies that specifically bind to the target protein, peptide fragment or epitope. Heavy and light chain pairs from these single cell clones were re-transfected with AID for further maturation. Several rounds of mammalian cell display plus AID-triggered somatic hypermutation produced high-specificity, high-affinity antibodies.

Diversity can also be introduced into CDRs of an antibody library or the entire V gene in a targeted manner or by random introduction. The former approach involves targeting all CDRs of an antibody in turn by high or low level mutagenesis, or targeting isolated hot spots of somatic hypermutation (see, e.g., ho et al 2005, j. Biol. Chem. 280:607-17) or residues suspected to affect affinity based on experimental basis or structural reasons. In a specific embodiment, the somatic hypermutation is triggered by AID, e.g., using SHM-XEL ^TM The platform (AnaptysBio, san Diego, calif.) was used for somatic hypermutation. Using E.coli mutants, random mutations can be introduced into the entire V gene and error-prone replication can be performed with DNA polymerase (see, e.g., hawkins et al, 1992, J.mol. Biol. 226:889-96) or RNA replicase. Diversity can also be introduced by replacing naturally diverse regions via DNA shuffling or similar techniques (see, e.g., lu et al, 2003, J. Biol. Chem.278:43496-507; U.S. Pat. Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops extending to framework region residues (see, e.g., bond et al, 2005, J.mol. Biol. 348:699-709), use loop deletions and insertions in the CDRs, or use hybridization-based diversification (see, e.g., U.S. patent publication No. 2004/0005709). Additional methods for generating diversity in CDRs are proprietary in, for example, the united states U.S. Pat. No. 7,985,840. Other methods that may be used to generate antibody libraries and/or antibody affinity maturation are disclosed, for example, in U.S. patent nos. 8,685,897 and 8,603,930, and U.S. publication nos. 2014/0170705, 2014/0094392, 2012/0028301, 2011/0183855 and 2009/007538, each of which is incorporated herein by reference.

Screening of the library may be accomplished by a variety of techniques known in the art. For example, the targets may be immobilized on a solid support, column, needle or cellulose/poly (vinylidene fluoride) membrane/other filter, expressed on host cells adhered to an adsorption plate or used for cell sorting, or conjugated with biotin for capture with streptavidin coated beads, or used in any other method for panning a display library.

For a review of in vitro affinity maturation methods, see, e.g., hoogenboom,2005,Nature Biotechnology 23:1105-16; quiroz and Sinclair,2010,Revista Ingeneria Biomedia 4:39-51; and references therein.

VI methods of use

A. Detection method

The conjugate molecules, such as co-conjugates, described herein, having high affinity and/or high specificity can be used as detection agents for detecting targets. In some embodiments, the target is a disease marker, and the conjugate molecules described herein, such as co-conjugates, can be used as diagnostic agents for diagnosing a disease by detecting the disease marker as a target molecule. In some aspects, provided herein is a method for detecting a label, such as a target, in a sample comprising (i) contacting the sample with a conjugate molecule, such as a co-conjugate, provided herein under conditions sufficient to form a complex of the conjugate molecule and the label, and (ii) detecting the presence of the complex in the sample. In some aspects, provided herein is a method of diagnosing a disease in a subject comprising (i) contacting a sample from an individual with a conjugate molecule provided herein, such as a co-conjugate, under conditions sufficient to form a complex of the conjugate molecule and a disease marker, wherein the conjugate molecule specifically binds to the marker, and (ii) detecting the presence of the complex in the sample.

Early detection of disease is important for a variety of diseases including cancer, infectious diseases, cardiovascular diseases, brain damage and alzheimer's disease. In general, the earlier a disease is diagnosed, the greater the likelihood of cure or successful control. Cancer screening tests include mammography of breast cancer, pap smear of cervical cancer or high risk HPV detection, colonoscopy of colon cancer, and the like. Early diagnosis of cancer and other diseases may increase survival chances and ensure that patients receive the most effective treatment as early as possible.

Detecting disease markers in blood or other body fluids for early diagnosis is an attractive method because it is non-invasive, easy to perform testing and cost-effective. For the example of cancer, the disease marker may be protein-based (e.g., detection via ELISA) or DNA-based (e.g., detection via PCR or NGS), as in "liquid biopsies". The main problem in the identification of disease markers is that the concentration of disease markers is very low at the earliest stages of disease progression. For example, HER2 amplified tumor cells are known to express about 2×10 ⁶ HER2 protein molecules/cell, and HER2 gene amplified by about 25 copies/cell (kallinonimi et al PNAS, 6 months 1992, 89 (12) 5321-5325). Thus, one tumor cell equivalent of HER2 tumor markers circulating in the blood is only about 4×10 ² Protein molecules per ml of blood and about 5X 10 ^-3 Individual copies of DNA per ml of blood. This calculation shows that detecting DNA markers is much more difficult than detecting protein markers for early diagnostic purposes.

It is estimated that K is typically a strong antibody-antigen interaction _D Is about 1X 10 ^-10 M (Foote and Eisen, proc Natl Acad Sci U S A.1995Feb 28;92 (5): 1254-1256). This means that when protein diseases are marked with less than 1X 10 ^-12 M (i.e. less than 6X 10) ⁸ Individual molecules/ml blood), the binding of very high affinity antibodies to disease markers is thermodynamically unfavorable. Thus, this calculation suggests that antibodies typically do not have sufficient binding affinity for early detection of very low concentrations of disease markers, such as single tumor cell equivalent HER2 markers. Biotin-streptavidin binding is known to be one of the strongest non-covalent binding interactions,and report K _D Is about 1X 10 ^-15 M (Foote and Eisen, proc Natl Acad Sci U S A.1995Feb 28;92 (5): 1254-1256). The binding molecules, such as co-conjugates, having high binding affinity described herein are particularly useful for disease marker detection.

In addition to the problem of insufficient binding affinity, antibodies also lack specificity/selectivity for detecting very low levels of disease markers. The concentration range of plasma proteins is known to cover at least 10 log orders, from about 6 x 10 ⁷ IL-6 basal level of individual molecules/ml to about 3X 10 ¹⁷ Human serum albumin (e.g., geyer et al Mol System biol.2017, month 9; 13 (9): 942doi: 10.15252/msb.20151297) at a concentration of one molecule per ml. To be about 4×10 ² Individual tumor cell equivalent levels of individual molecules/ml to detect HER2 protein, anti-HER 2 antibodies need not only have high binding affinity, but also need to have at least 1 x 10 for unintended plasma proteins ⁵ Specificity/selectivity of fold. For example, an anti-HER 2 antibody should preferably have a KD for HER2 that is 1X 10 lower than its KD for unintended, non-specific plasma proteins ⁵ Fold to minimize non-specific background. Such high levels of antibody specificity are difficult to achieve because antibodies are known to have relatively limited sequence and structural diversity at the antigen binding site (e.g., peng et al Proc Natl Acad Sci U S A.2014, 7, 1; 111 (26): E2656-E2665.Doi:10.1073/pnas. 1401131111).

As described above, it is estimated that K is a typical strong antibody-antigen interaction _D Is about 1X 10 ^-10 M (Foote and Eisen, proc Natl Acad Sci U S A.1995Feb 28;92 (5): 1254-1256). It is therefore concluded that even high affinity antibodies typically do not have sufficient binding affinity and/or sufficient specificity to detect disease markers in the blood as early as possible. It is known that most antibodies do not bind unique epitopes and that they cross-react with unintended proteins, despite the lower binding affinity. Furthermore, non-specific proteins that are not associated with the intended target may have epitopes that are similar to but not identical to the specific epitope. Then, the binding affinity of the antibody to a similar epitope is lower than to a specific epitope, which results in a corresponding comparison with what is called a non-specific background Low signal cross-reactivity. It is this non-specific background that interferes with specific antibody-antigen binding interactions, particularly when the target is expected to be present at very low concentrations and the non-specific protein target is not expected to be present at relatively high concentrations, such as in the case of early detection of disease markers in the blood. Thus, in order to detect a disease marker as early as possible, the conjugate of the disease marker needs to have a very high binding affinity. The conjugates also need to be able to minimize non-specific binding to unintended targets even if these unintended targets are present in relatively high concentrations. In some embodiments, the conjugate molecules described herein, such as co-conjugates, having significantly better binding affinity and better specificity are particularly useful for disease marker detection and early disease diagnosis.

In one aspect, provided herein is a method of detecting a label in a sample, comprising (i) contacting the sample with a conjugate molecule, such as a co-conjugate, provided herein under conditions sufficient to form a complex of the conjugate molecule and the label, and (ii) detecting the complex in the sample.

In some embodiments of the methods provided herein, the complex is detected by measuring a labeled reagent conjugated to the complex. In some embodiments of the methods provided herein, the complex is detected by measuring a labeled reagent conjugated to a conjugate molecule, such as a co-conjugate.

In some embodiments of the method of detecting a marker, the sample is a body fluid, tissue, or cell. In some embodiments of the method of detecting a marker, the sample is blood, bone marrow, plasma, serum, urine, or cerebrospinal fluid. In some embodiments, the complex is formed in vitro. In some embodiments, the complex is formed in vivo. In some embodiments, the complex is detected in vitro. In some embodiments, the complex is detected in vivo. In some embodiments, the complex is formed in vitro and the complex is detected in vitro. In some embodiments, the complex is formed in vivo and the complex is detected in vivo. In some embodiments, the complex is formed under physiological conditions. In some embodiments, at 37deg.CAnd forming a complex. In some embodiments, shear stress in physiological vessels, e.g., 10-70 dynes/cm ² (shear stress range in artery) or 1-6 dynes/cm ² (shear stress range in the vein) to form a complex. In some embodiments, the physiological vascular shear stress is between about 10 and 70 dynes/cm at 37 degrees celsius ² (shear stress range in artery) or 1-6 dynes/cm ² (shear stress range in the vein) to form a complex. In some embodiments, the complex is detected under physiological conditions. In some embodiments, the complex is detected at 37 ℃. In some embodiments, shear stress in physiological vessels, e.g., 10-70 dynes/cm ² (range of shear stress in arteries) or 1-6 dynes/cm ² The complex was detected (range of shear stress in the vein). In some embodiments, the physiological vascular shear stress is between about 10 and 70 dynes/cm at 37 degrees celsius ² (shear stress range in artery) or 1-6 dynes/cm ² The complex was detected (range of shear stress in the vein). In some embodiments, the complex is formed under normal laboratory conditions (e.g., at room temperature or 25 ℃). In some embodiments, the complex is detected under normal laboratory conditions (e.g., at room temperature or 25 ℃). In some embodiments, the complex is formed under normal laboratory conditions (e.g., room temperature or 25 ℃) and the complex is detected under normal laboratory conditions (e.g., room temperature or 25 ℃).

In some embodiments of the methods provided herein, the complex is detected by measuring a labeled reagent conjugated to the complex. In some embodiments, the labeling reagent may be a colorimetric reagent, a fluorescent reagent, a chemiluminescent reagent, a radioisotope, a metal ion, an enzyme, a polymer, or an affinity tag. The colorimetric reagent may be, for example, PNPP (p-nitrophenylphosphate), ABTS (2, 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) or OPD (o-phenylenediamine). The fluorescent agent may be, for example, quantaBlu or QuantaRedTM (Thermo Scientific, waltham, mass.). The luminescent agent may be, for example, luminol or fluorescein.

In some embodiments of the methods provided herein, the labeling agent is a fluorescent molecule, radioisotope, metal ion, enzyme, biotin, polymer, or antibody.

In some embodiments, a conjugate molecule, such as a co-conjugate, may be conjugated to an affinity tag for detection. In some embodiments, the affinity tag may be a glutathione-S-transferase, HA-tag, his-tag, FLAG-tag, or biotin.

In some embodiments, complexes with a conjugate molecule, such as a co-conjugate and a target molecule, can be detected by a second antibody that recognizes the conjugate molecule. The second antibody may be, for example, an anti-human IgA, anti-human IgD, anti-human IgE, anti-human IgG or anti-human IgM antibody. The second antibody may be a monoclonal antibody or a polyclonal antibody. The secondary antibody may be derived from any mammalian organism, including mice, rats, hamsters, goats, camels, chickens, rabbits, and the like. The secondary antibody may also be recombinant. The secondary antibody may be conjugated to an enzyme (e.g., horseradish peroxidase (HRP), alkaline Phosphatase (AP), luciferase, etc.) or a dye (e.g., a colorimetric dye, a fluorescent dye, a Fluorescence Resonance Energy Transfer (FRET) -dye, a Time Resolved (TR) -FRET dye, etc.). In some embodiments, the secondary antibody may be conjugated with a Fluorescein (FITC) -based dye, such as fluorescein isothiocyanate. In some embodiments, the second antibody may be conjugated to Alexa 488 (Life technologies) conjugation.

The presence or absence of the complex may also be detected by enzyme-linked immunosorbent assay (ELISA), including multiplex ELISA, immunohistochemical assay (IHC), immunofluorescent assay (IF), western Blot (WB), flow cytometry, fluorescent Immunosorbent Assay (FIA), chemiluminescent Immunoassay (CIA), radioimmunoassay (RIA), enzyme-multiplied immunoassay, solid Phase Radioimmunoassay (SPROA), fluorescence Polarization (FP) assay, fluorescence Resonance Energy Transfer (FRET) assay, time resolved fluorescence resonance energy transfer (TR-FRET) assay, surface plasmon resonance assay (SPR) or dot blot assay. Methods and protocols for performing immunoassays and biophysical protein interaction assays are well known in the art. See, e.g., wild d., the Immunoassay Handbook, elsevier Science, 4 th edition (2013); fu H., protein-Protein Interactions, humana Press, 4 th edition (2004).

As will be appreciated by those of ordinary skill in the art, a higher level of a disease marker in a human sample may indicate a higher likelihood that a human subject has the disease. Schouwers et al Clin Chem Lab Med (2014) 52 (4): 547-51. In some embodiments, the detecting step may further comprise determining the level of the target molecule in the sample. Determining the level of the target molecule may comprise comparing the level of the target molecule in a sample from the subject to a control level of the target molecule in a sample from a healthy individual, wherein an increase in the level of the target molecule in the sample from the subject compared to the control level indicates that the subject has the disease. Determining the level of the target molecule may comprise correlating the level with a likelihood that the subject is suffering from the disease, wherein a higher level indicates a higher likelihood of suffering from the disease.

The level of a target molecule in a human sample over a period of time may be indicative of the progression of a disease associated with the target molecule over the period of time. The time period may be a treatment time course, wherein a change in the level of the target molecule may be indicative of the efficacy of the treatment. In some embodiments, the present disclosure provides a method of monitoring target molecule levels in a patient at different time points, comprising determining target molecule levels in two or more samples taken from the patient at different time points and comparing the target molecule levels in the two or more samples. A decrease in the level of the target molecule in the sample obtained at a subsequent time point relative to the level of the target molecule in the sample obtained at the first time point may indicate that the patient's condition is improving or that the treatment the patient receives is effective. An increase in the level of the target molecule in the sample obtained at a subsequent time point relative to the level of the target molecule in the sample obtained at the first time point may indicate that the condition of the human subject is deteriorating. In some embodiments, one or more samples are obtained at the beginning of a particular treatment session and one or more samples are obtained at a later point in time throughout the treatment session.

In some embodiments, detection and diagnosis may be accomplished, for example, by conjugating a conjugate molecule, such as a co-conjugate, disclosed herein to a detectable substance, including but not limited to a radioactive substance, such as but not limited to zirconium # ⁸⁹ Zr, iodine ¹³¹ I、 ¹²⁵ I、 ¹²⁴ I、 ¹²³ I、and ¹²¹ I, C% ¹⁴ C、 ¹¹ C) Sulfur ³⁵ S, tritium ³ H) The indium is ¹¹⁵ In、 ¹¹³ In、 ¹¹² In and ¹¹¹ in, technetium ] ⁹⁹ Tc), thallium ²⁰¹ Ti, ga ] ⁶⁸ Ga、 ⁶⁷ Ga and Pd% ¹⁰³ Pd and molybdenum% ⁹⁹ Mo and xenon ¹³³ Xe and F ¹⁸ F)、 ¹⁵ O、 ¹³ N、 ⁶⁴ Cu、 ⁹⁴ mTc、 ¹⁵³ Sm、 ¹⁷⁷ Lu、 ¹⁵⁹ Gd、 ¹⁴⁹ Pm、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁶⁶ Ho、 ⁸⁶ Y、 ⁹⁰ Y、 ⁴⁷ Sc、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹⁴² Pr、 ¹⁰⁵ Rh、 ⁹⁷ Ru、 ⁶⁸ Ge、 ⁵⁷ Co、 ⁶⁵ Zn、 ⁸⁵ Sr、 ³² P、 ¹⁵³ Gd、 ¹⁶⁹ Yb、 ⁵¹ Cr、 ⁵⁴ Mn、 ⁷⁵ Se、 ¹¹³ Sn and Sn ¹¹⁷ Sn; and positron emitting metals using various positron emission tomography, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent substances such as, but not limited to, umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin and aequorin, and non-radioactive paramagnetic metal ions.

Conjugates disclosed herein having a conjugate molecule, such as a co-conjugate, which is detectably labeled as provided herein, are useful for diagnostic purposes to detect, diagnose, or monitor diseases, such as cancer, infectious diseases, cardiovascular diseases, brain injury, and alzheimer's disease.

Accordingly, in one aspect, provided herein is a method of diagnosing a disease in a subject comprising (i) contacting a sample with a conjugate molecule, such as a co-conjugate, provided herein under conditions sufficient to form a complex of the conjugate molecule and a disease marker, wherein the conjugate molecule specifically binds the marker, and (ii) detecting the complex in the sample.

In certain embodiments of the methods provided herein, the complex is detected by measuring a labeled reagent conjugated to the complex, as further described above in this section.

In certain embodiments of the methods provided herein, the labeling agent is a labeling agent as further described in this section above.

In some embodiments of the method, the sample is a body fluid, tissue, or cell. In some embodiments of the method, the sample is blood, bone marrow, plasma, serum, urine, or cerebrospinal fluid. In some embodiments, the complex is formed in vitro. In some embodiments, the complex is formed in vivo. In some embodiments, the complex is detected in vitro. In some embodiments, the complex is detected in vivo. In some embodiments, the complex is formed in vitro and the complex is detected in vitro. In some embodiments, the complex is formed in vivo and the complex is detected in vivo. In some embodiments, the complex is formed under physiological conditions. In some embodiments, the complex is formed at 37 ℃. In some embodiments, shear stress in physiological vessels, e.g., 10-70 dynes/cm ² (shear stress range in artery) or 1-6 dynes/cm ² (shear stress range in the vein) to form a complex. In some embodiments, the physiological vascular shear stress is between about 10 and 70 dynes/cm at 37 degrees celsius ² (shear stress range in artery) or 1-6 dynes/cm ² (shear stress range in the vein) to form a complex. In some embodiments, the complex is detected under physiological conditions. In some embodiments, the complex is detected at 37 ℃. In some embodiments, shear stress in physiological vessels, e.g., 10-70 dynes/cm ² (range of shear stress in arteries) or 1-6 dynes/cm ² The complex was detected (range of shear stress in the vein). In some embodiments, the physiological vascular shear stress is between about 10 and 70 dynes/cm at 37 degrees celsius ² (shear stress range in artery) or 1-6 dynes/cm ² The complex was detected (range of shear stress in the vein). In some embodiments, the complex is formed under normal laboratory conditions (e.g., at room temperature or 25 ℃). In some embodiments, the complex is detected under normal laboratory conditions (e.g., at room temperature or 25 ℃). In some embodiments, the complex is formed under normal laboratory conditions (e.g., room temperature or 25 ℃) and the complex is detected under normal laboratory conditions (e.g., room temperature or 25 ℃).

In some embodiments of the methods provided herein, the concentration of the label present in the sample does not exceed 1 x 10 ^-8 M is not more than 0.5X10 ^-8 M is not more than 1×10 ^-9 M is not more than 0.5X10 ^-9 M is not more than 1×10 ^-10 M is not more than 0.5X10 ^- ¹⁰ M is not more than 1×10 ^-11 M is not more than 0.5X10 ^-11 M is not more than 1×10 ^-12 M is not more than 0.5X10 ^-12 M is not more than 1×10 ^-13 M is not more than 0.5X10 ^-13 M is not more than 1×10 ^-14 M is not more than 0.5X10 ^-14 M is not more than 1×10 ^-15 M is not more than 0.5X10 ^-15 M is not more than 1×10 ^-16 M is not more than 0.5X10 ^-16 M is not more than 1×10 ^-17 M is not more than 0.5X10 ^-17 M is not more than 1×10 ^-18 M is not more than 0.5X10 ^-18 M is not more than 1×10 ^-19 M is not more than 0.5X10 ^-19 M is not more than 1×10 ^-20 M is not more than 0.5X10 ^-20 M is not more than 1×10 ^-21 M or not more than 0.5X10 ^-21 M。

In some embodiments of the methods provided herein, the concentration of label present in the sample is less than 1 x 10 ^-8 M is less than 0.5X10 ^-8 M is less than 1×10 ^-9 M is less than 0.5X10 ^-9 M is less than 1×10 ^-10 M is less than 0.5X10 ^-10 M is less than 1×10 ^-11 M is less than 0.5X10 ^-11 M is less than 1×10 ^-12 M is less than 0.5X10 ^-12 M is less than 1×10 ^-13 M is less than 0.5X10 ^-13 M is less than 1×10 ^-14 M is less than 0.5X10 ^-14 M is less than 1×10 ^-15 M is less than 0.5X10 ^-15 M is less than 1×10 ^-16 M is less than 0.5X10 ^-16 M is less than 1×10 ^-17 M is less than 0.5X10 ^-17 M is less than 1×10 ^-18 M is less than 0.5X10 ^-18 M is less than 1×10 ^-19 M is less than 0.5X10 ^-19 M is less than 1×10 ^-20 M is less than 0.5X10 ^-20 M is less than 1×10 ^-21 M or less than 0.5X10 ^-21 M。

In some embodiments of the methods provided herein, the concentration of the label present in the sample is about 1 x 10 ^-8 M, about 0.5X10) ^-8 M, about 1X 10 ^-9 M, about 0.5X10) ^-9 M, about 1X 10 ^-10 M, about 0.5X10) ^-10 M, about 1X 10 ^-11 M, about 0.5X10) ^-11 M, about 1X 10 ^-12 M, about 0.5X10) ^-12 M, about 1X 10 ^-13 M, about 0.5X10) ^-13 M, about 1X 10 ^-14 M, about 0.5X10) ^- ¹⁴ M, about 1X 10 ^-15 M, about 0.5X10) ^-15 M, about 1X 10 ^-16 M, about 0.5X10) ^-16 M, about 1X 10 ^-17 M, about 0.5X10) ^-17 M, about 1X 10 ^-18 M, about 0.5X10) ^-18 M, about 1X 10 ^-19 M, about 0.5X10) ^-19 M, about 1X 10 ^-20 M, about 0.5X10) ^-20 M, about 1X 10 ^-21 M or about 0.5X10 ^-21 M。

In some embodiments, the detection method may further comprise determining expression of a disease marker on a cell or tissue sample of the subject using the co-conjugates disclosed herein; and comparing the level of the disease marker to a control level, e.g., a level in a normal tissue sample (e.g., from a non-diseased subject or from the same subject prior to onset of the disease), whereby an increase in the measured level of the disease marker compared to the control level is indicative of the disease. Such diagnostic methods may allow health professionals to take preventive measures or aggressive treatments earlier than other methods, thereby preventing the development or further exacerbation of the disease.

The conjugate molecules disclosed herein, such as co-conjugates, may also be used to determine target molecule levels in biological samples using classical immunohistological methods as provided herein or well known to those skilled in the art (see, e.g., jalkanen et al, 1985, J.cell. Biol.101:976-985; and Jalkanen et al, 1987, J.cell. Biol. 105:3087-3096), such as enzyme-linked immunosorbent assays (ELISA) and Radioimmunoassays (RIA). Suitable analytical labels are known in the art and include enzymatic labels, such as glucose oxidase; radioisotopes, e.g. such as iodine @, for example ¹²⁵ I、 ¹²¹ I) The carbon is ¹⁴ C) Sulfur ³⁵ S, tritium ³ H) The indium is ¹²¹ In, technetium ] ⁹⁹ Tc); luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin.

In one aspect, the present disclosure provides detection and diagnosis of human diseases. In some embodiments, the diagnosing comprises: a) Administering (e.g., parenterally, subcutaneously, or intraperitoneally) an effective amount of a conjugate having a conjugate molecule, such as a co-conjugate, disclosed herein to a subject; b) Waiting a time interval after administration to allow preferential concentration of the conjugate at the site of expression of the disease marker in the subject (and in some aspects, clearance of unbound conjugate or fusion protein to background levels); c) Determining a background level; and d) detecting the conjugate in the subject such that detection of a conjugate above the background level indicates that the subject has the disease. Background levels can be determined by a variety of methods, including comparing the amount of conjugate detected to standard values previously determined for a particular system.

It should be understood that the number of the devices,the size of the subject and the imaging system used will determine the amount of imaging portion required to produce the diagnostic image and can be readily determined by one skilled in the art. For example, in the case of radioisotopes conjugated to conjugate molecules described herein, the injected radiation dose is typically about 5 to 20 millicuries for human subjects ⁹⁹ Tc is in the range of Tc. The conjugate will then preferentially accumulate at the cell site expressing the target molecule. In vivo tumor imaging is described in s.w. burchiel et al, "Immunopharmacokinetics of Radiolabeled Antibodies and Their fragments" (Chapter 13in Tumor Imaging:The Radiochemical Detection of Cancer,S.W.Burchiel and b.a. rhodes, eds., masson Publishing inc. (1982).

Depending on several variables, including the type of detectable agent used and the mode of administration, the time interval following administration that allows the conjugate to preferentially concentrate at the site of the subject and allows unbound conjugate to be cleared to background levels is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment, the time interval after administration is 5 to 20 days or 5 to 10 days. In some embodiments, disease monitoring is performed by repeating the diagnostic methods provided herein, e.g., one month after initial diagnosis, six months after initial diagnosis, one year or more after initial diagnosis.

The presence of the conjugate or fusion protein in the subject can be detected using methods known in the art for in vivo scanning. These methods depend on the type of detectable agent used. The skilled person will be able to determine the appropriate method for detecting a particular detectable agent. Methods and apparatus useful in the diagnostic methods of the present disclosure include, but are not limited to, computed Tomography (CT), whole-body scanning such as Position Emission Tomography (PET), magnetic Resonance Imaging (MRI), and ultrasound scanning. In some embodiments, a conjugate molecule, such as a co-conjugate, disclosed herein is conjugated to a radioisotope and detected in a subject using a radiation-responsive surgical instrument. In another embodiment, a conjugate molecule, such as a co-conjugate, disclosed herein is conjugated to a fluorescent compound and detected in a subject using a fluorescent response scanning instrument. In another embodiment, disclosed hereinBinding molecules such as co-binders with positron emitting metals such as zirconium # ⁸⁹ Zr) or any other positron emitting metal conjugate provided herein or well known in the art that can be detected by positron emission tomography, and detected in a subject using positron emission tomography. In yet another embodiment, a conjugate molecule disclosed herein, such as a co-conjugate, is conjugated to a paramagnetic label and detected in a subject using Magnetic Resonance Imaging (MRI).

Also contemplated herein is the use of a conjugate molecule disclosed herein, such as a co-conjugate, in place of an antibody in an application such as ELISA, IHC, IF, IP, WB, flow cytometry, flow cell sorting, imaging, multiplex ELISA, or multiplex antibody array. The use of the conjugate molecules disclosed herein, such as co-conjugates, in the detection of markers associated with food and environmental safety detection and monitoring is also contemplated herein.

In certain embodiments of the various methods provided herein, the subject is a mammal. In some embodiments, the subject is a mammal selected from the group consisting of: guinea pigs (cavnnae/guinea pig), pigs (Sus/pig), cynomolgus monkeys (Macaca Fascicularis) (monkeys, e.g., cynomolgus monkey (cynomolgus monkey)), apes (gibbons, gorillas, chimpanzees, and humans), dogs (dogs), rodents (rats), and mice (mice). In a specific embodiment, the subject is a human.

B. Therapeutic method

For therapeutic applications, the conjugate molecules described herein, such as co-conjugates, also have significant advantages due to typical bivalent antibodies. Many drug targets such as GPCRs, ion channels, tyrosine kinase receptors, cytokine receptors are expressed at much lower levels on the cell surface than HER2, and they require high affinity binders to block their binding to their natural ligand or to act as antagonists or agonists.

Thus, in one aspect, provided herein is a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a conjugate molecule provided herein, such as a co-conjugate, wherein the disease can be treated by activating or inhibiting a target that specifically binds to the conjugate molecule, such as the co-conjugate. As described herein, a conjugate molecule, such as a co-conjugate, provided herein has increased binding affinity and/or specificity relative to a single binding moiety in a co-conjugate or a control conjugate molecule, such as a control co-conjugate described herein. Thus, in one aspect, provided herein is a method of increasing the binding affinity of a first binding moiety to a target or its use, comprising constructing a co-conjugate of the first binding moiety and a second binding moiety according to any one of the configurations or embodiments provided herein or any combination thereof. In some aspects, provided herein is a method of increasing the therapeutic or diagnostic efficacy of a binding moiety or use thereof by linking the binding moiety to another binding moiety such that the resulting binding molecule binds to a target with sufficient affinity and specificity for therapeutic or diagnostic use and produces the desired biological efficacy.

In some embodiments, a conjugate molecule, such as a co-conjugate, disclosed herein is an agonist of a target, and provided herein are methods of treating a disease treatable by activating a biological function of the target in a subject, the method comprising administering to the subject a therapeutically effective amount of the conjugate molecule that specifically binds to the target. In some embodiments, a conjugate molecule, such as a co-conjugate, disclosed herein is an antagonist of a target, and provided herein are methods of treating a disease treatable by inhibiting a biological function of the target in a subject, the method comprising administering to the subject a therapeutically effective amount of the conjugate molecule that specifically binds to the target.

In some embodiments of the methods provided herein, the expression level of the target is less than 1 x 10 ⁷ Below 0.5X10) ⁷ Less than 1X 10 ⁶ Below 0.5X10) ⁶ Less than 1X 10 ⁵ Below 0.5X10) ⁵ Less than 1X 10 ⁴ Below 0.5X10) ⁴ Less than 1X 10 ³ Below 0.5X10) ³ Less than 1X 10 ² Or less than 0.5X10 ² Cells. In some embodiments of the methods provided herein, the expression level of the target does not exceed 1 x 10 ⁷ No more than 0.5X10 ⁷ No more than 1X 10 ⁶ Not only is not provided withExceeding 0.5X10 ⁶ No more than 1X 10 ⁵ No more than 0.5X10 ⁵ No more than 1X 10 ⁴ No more than 0.5X10 ⁴ No more than 1X 10 ³ No more than 0.5X10 ³ No more than 1X 10 ² Or not more than 0.5X10 ² Cells. In some embodiments of the methods provided herein, the expression level of the target is about 1 x 10 ⁷ About 0.5×10 ⁷ About 1X 10 ⁶ About 0.5×10 ⁶ About 1X 10 ⁵ About 0.5×10 ⁵ About 1X 10 ⁴ About 0.5×10 ⁴ About 1X 10 ³ About 0.5×10 ³ About 1X 10 ² Or about 0.5X10 ² Cells.

One class of drug targets are secreted molecules in bodily fluids (e.g., blood), such as growth factors, cytokines, chemokines, which may also benefit from the high affinity conjugate molecules disclosed herein, such as co-conjugates, that inhibit or activate their biological functions. In some embodiments of the methods provided herein, less than 1×10 protein targets are secreted in a body fluid sample, such as blood, serum, plasma, bone marrow, or cerebrospinal fluid ¹⁰ Less than 0.5X10 ¹⁰ Less than 1X 10 ⁹ Less than 0.5X10 ⁹ Less than 1X 10 ⁸ Less than 0.5X10 ⁸ Less than 1X 10 ⁷ Less than 0.5X10 ⁷ Less than 1X 10 ⁶ Less than 0.5X10 ⁶ Less than 1X 10 ⁵ Less than 0.5X10 ⁵ Less than 1X 10 ⁴ Less than 0.5X10 ⁴ Less than 1X 10 ³ Less than 0.5X10 ³ Less than 1X 10 ² Or less than 0.5X10 ² Each molecule/ml. In some embodiments of the methods provided herein, the secreted protein target is about 1 x 10 in a body fluid sample, such as blood, serum, plasma, bone marrow, or cerebrospinal fluid ¹⁰ About 0.5×10 ¹⁰ About 1X 10 ⁹ About 0.5×10 ⁹ About 1X 10 ⁸ About 0.5×10 ⁸ About 1X 10 ⁷ About 0.5×10 ⁷ About 1X 10 ⁶ About 0.5×10 ⁶ About 1X 10 ⁵ About 0.5×10 ⁵ About 1X 10 ⁴ About 0.5×10 ⁴ About 1X 10 ³ About 0.5×10 ³ About 1X 10 ² Or about 0.5X10 ² Each molecule/ml.

Many drug targets belong to the family of proteins whose members share nearly identical sequences in functionally conserved regions (e.g., protein binding interaction regions or regions responsible for enzymatic activity), and it is therefore difficult to generate therapeutic antibodies that can specifically target functionally conserved regions of specific family members without affecting the function of other members of the family of proteins. When the second binding moiety in the co-conjugate enhances binding affinity and stabilizes binding of the first binding moiety to the functionally conserved region, a conjugate molecule, such as a co-conjugate, having a first binding moiety that binds to the functionally conserved region with relatively low affinity may still inhibit or activate the function of the protein target. Selective inhibition or activation of the function of a particular target molecule can be achieved if the second binding moiety binds to a unique sequence in the target that is not shared with other members of the protein family.

Thus, in some embodiments, provided herein is a conjugate molecule, such as a co-conjugate, having a first binding moiety that binds to a functionally conserved region or binding site of a protein family with low affinity such that it does not stably bind to a protein target or family member thereof alone; and a second binding moiety that binds to a unique sequence or binding site in the target molecule that is different from the other members of the family. The resulting conjugate molecules, such as co-conjugates, selectively inhibit or activate the function of the target molecule without affecting other members of the family. In some embodiments, provided herein are co-conjugates having a first binding moiety, a second binding moiety, and a linker linking the first binding moiety and the second binding moiety, wherein the first binding moiety and the second binding moiety bind to non-overlapping epitopes on a target molecule, and wherein the first binding moiety preferentially binds to a functionally conserved region or binding site of the target molecule with low binding affinity (e.g., KD of at least 1 x 10 ^-9 M). In some embodiments, provided herein are conjugate molecules, such as co-conjugates, having a first binding moiety, a second binding moiety, and a linker connecting the first binding moiety and the second binding moiety, wherein the first binding moiety and the second binding moiety bind simultaneouslyNon-overlapping epitopes on the target molecule, and wherein the first binding moiety preferably binds to a functionally conserved region or binding site of the target molecule with low binding affinity (e.g., a KD of at least 1x 10 ^-9 M). In some embodiments, the first binding moiety of the co-conjugate binds to the target molecule with a KD of at least 1X 10 ^-9 M, at least 1X 10 ^-8 M, at least 1X 10 ^-7 M, at least 1X 10 ^-6 。

The primary B cell library or primary immune response library contains a greater variety of low affinity binding moieties than the secondary immune response library, which binding moieties in the secondary immune response are more selective but have a higher affinity due to affinity maturation. However, low affinity binding moieties with rapid dissociation rates generally do not work as well as those with higher affinity binding moieties with slower dissociation rates. The binding affinity of a desired binding moiety capable of inhibiting or activating the function of a drug target can be greatly improved with a co-conjugate comprising a second binding moiety that cooperates and acts synergistically with the desired first binding moiety. In some embodiments, provided herein are co-conjugates having a first binding moiety and a second binding moiety that bind simultaneously to non-overlapping epitopes on a drug target, wherein the first binding moiety has the ability to inhibit or activate the function of the drug target upon binding, and a relatively low binding affinity (e.g., KD of at least 1x 10 ^-9 M), and wherein the binding affinity is improved by at least more than 50-fold due to the presence of a second binding moiety that simultaneously and synergistically binds to different non-overlapping epitopes on the drug target. In some embodiments, provided herein are co-conjugates having a first binding moiety and a second binding moiety that bind to a non-overlapping epitope on a drug target, wherein the first binding moiety has the ability to inhibit or activate the function of the drug target upon binding, and a relatively low binding affinity (e.g., KD of at least 1x 10 ^-9 M), and wherein the binding affinity is improved by at least more than 50-fold due to the presence of a second binding moiety that binds a different non-overlapping epitope on the drug target. In some embodiments, the work of a drug target may be inhibited or activatedThe first binding moiety is capable of binding to the drug target with a KD of at least 1x 10 ^-9 M. In some embodiments, the first binding moiety that inhibits or activates a function of the drug target has a KD of at least 1x 10 when bound to the drug target ^-8 M. In some embodiments, the first binding moiety that inhibits or activates a function of the drug target has a KD of at least 1x 10 when bound to the drug target ^-7 M. In some embodiments, the first binding moiety that inhibits or activates a function of the drug target has a KD of at least 1x10 when bound to the drug target ^-6 M. In some embodiments, the binding affinity is improved by more than 100-fold. In some embodiments, the binding affinity is improved by more than 200-fold. In some embodiments, the binding affinity is improved by more than 500-fold. In some embodiments, the binding affinity is improved by more than 1000-fold. In some embodiments, the binding affinity is improved by more than 2000-fold. In some embodiments, the binding affinity is improved by more than 5000-fold. In some embodiments, the binding affinity is improved by more than 10,000-fold.

In addition, the B cell library of the original or primary immune response contains a greater diversity of low affinity binding moieties, and their binding affinity can be greatly improved by the synergistic second binding moiety in the co-conjugate. Thus, the co-conjugates provided herein can bind any region of interest in a target molecule with high affinity and/or broad affinity profile for different purposes (affinity tuning). In some embodiments, the first binding moiety of the co-conjugate binds to the target molecule with a KD of at least 1X10 ^-9 M, at least 1X 10 ^-8 M, at least 1X 10 ^-7 M or at least 1X 10 ^-6 M, and the binding affinity of the co-conjugate is improved by more than 10-fold, more than 20-fold, more than 50-fold, more than 100-fold, more than 200-fold, more than 500-fold, more than 1000-fold, more than 2000-fold, more than 5000-fold, or more than 10,000-fold due to the presence of a second binding moiety that binds to a different non-overlapping epitope in the same target molecule.

In some embodiments, provided herein is a method for treating a disease in a subject in need thereof. The method may comprise administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein. For example, a pharmaceutical composition may comprise a conjugate molecule provided herein, such as a co-conjugate. Diseases that may be treated or prevented using the methods of the present disclosure include cancer, infectious diseases, cardiovascular diseases, brain injuries, autoimmune diseases, and neurodegenerative diseases such as, for example, alzheimer's disease. In some embodiments, provided herein is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition having a conjugate molecule, such as a co-conjugate, provided herein. In some embodiments of the present disclosure, a conjugate molecule, such as a co-conjugate, is used as part of a CAR-T construct that binds to a target molecule, such as a tumor antigen or a neoantigen, with higher affinity and specificity.

The conjugate molecules provided herein, such as co-conjugates, may also be used to specifically target a therapeutic agent to a diseased cell, tissue or organ. In some embodiments, provided herein are therapeutic uses of conjugate molecules, such as co-conjugates, conjugated (covalently or non-covalently) or recombinantly fused to one or more therapeutic agents. In this context, for example, a conjugate molecule such as a co-conjugate may be conjugated or recombinantly fused with a therapeutic agent such as a cytotoxin, e.g., a cytostatic or cytocidal agent, or a radiometal ion, e.g., an alpha-emitter. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. The therapeutic agent may be a chemotherapeutic agent such as, but not limited to, anthracyclines (e.g., doxorubicin and daunorubicin (formerly daunorubicin)); taxanes (e.g., paclitaxel/Taxol) and docetaxel; antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and dacarbazine); or alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin (II) (DDP), and cisplatin); antibiotics (e.g., actinomycin D, bleomycin, mithramycin and Anthramycin (AMC)), ryoxetine molecules (e.g., ryoxetine PHE, bryostatin 1, sorastatin (solastatin) 10, monomethyl ryoxetine E (MMAE) and monomethyl ryoxetine F (MMAF)), hormones (e.g., glucocorticoids, progestins, androgens and estrogens), nucleoside analogs (e.g., gemcitabine), DNA repair enzyme inhibitors (e.g., etoposide and topotecan), kinase inhibitors (e.g., compound ST1571, also known as glifeverc (Gleevec) or imatinib mesylate), cytotoxic agents (e.g., maytansine, paclitaxel, cytochalasin B, ponin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, tolithromycin, photorhaponin, mitomycin 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof; farnesyl transferase inhibitors (e.g., R115777, BMS-214662, such as those disclosed in U.S. patent No. 6,458,935); topoisomerase inhibitors (e.g., camptothecin, irinotecan, SN-38, topotecan, 9-aminocamptothecin, GG-211 (GI 147211), DX-8951f, IST-622, lubitecan, pyrazoloacridine, XR-5000, sainttopin (saintpin), UCE6, UCE1022, TAN-1518A, TAN 1518B, KT6006, KT6528, ED-110, NB-506, fagaronine (Fagaronine), methoberlin (coralyne), beta-lapaquinone, and butterfly mycin); DNA minor groove binders (e.g., hoescht dye 33342 and Hoechst dye 33258); adenosine deaminase inhibitors (e.g., fludarabine phosphate and 2-chlorodeoxyadenosine); or a pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof. The therapeutic agent may be an immunotherapeutic agent such as, but not limited to, cetuximab, bevacizumab, herceptin, rituximab.

Furthermore, conjugate molecules provided herein, such as co-conjugates, may be conjugated to a therapeutic agent, such as a radioactive metal ion, for example, an alpha emitter, such as ²¹³ Bi or macrocyclic chelators useful for conjugation of radiometal ions, including but not limited to ¹³¹ In、 ¹³¹ LU、 ¹³¹ Y、 ¹³¹ Ho、 ¹³¹ Sm; or macrocyclic chelating agents such as 1,4,7, 10-tetraazacyclododecane-N, N' -tetraacetic acid (DOTA)。

Furthermore, conjugate molecules provided herein, such as co-conjugates, can be conjugated (covalently or non-covalently) or recombinantly fused to a therapeutic agent that alters a given biological response. Thus, therapeutic agents should not be construed as limited to classical chemotherapeutic agents. For example, the therapeutic agent may be a protein, peptide or polypeptide having a desired biological activity. Such proteins may include, for example, toxins (e.g., abrin, ricin a, pseudomonas exotoxin, cholera toxin, and diphtheria toxin); proteins such as tumor necrosis factor, gamma-interferon, alpha-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, apoptosis agents (e.g., TNF-gamma, AIM I, AIMII, fas ligand, and VEGF), anti-angiogenic agents (e.g., angiostatin, endostatin, and components of the coagulation pathway such as tissue factor); biological response modifiers (e.g., cytokines such as interferon gamma, interleukin-1, interleukin-2, interleukin-5, interleukin-6, interleukin-7, interleukin-9, interleukin-10, interleukin-12, interleukin-15, interleukin-23, granulocyte-macrophage-colony stimulating factor, and granulocyte-colony stimulating factor); growth factors (e.g., growth hormone) or coagulants (e.g., calcium, vitamin K, tissue factors such as, but not limited to, hageman factor (factor XII), high Molecular Weight Kininogens (HMWK), prekallikrein (PK), thrombospondin-factor II (prothrombin), factor V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipids, and fibrin monomers).

Therapeutic agents conjugated or recombinantly fused to the co-conjugates provided herein can be selected to achieve a desired prophylactic or therapeutic effect. It will be appreciated that the skill level of the clinician or other medical personnel can determine which therapeutic agent to conjugate or recombinantly fuse with the co-conjugates provided herein in view of the following factors: the nature of the disease, the severity of the disease, and the condition of the subject.

The co-conjugates or pharmaceutical compositions described herein may be administered once or may be divided into a plurality of smaller doses for administration over a time interval. It will be appreciated that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known test protocols or by inference from in vivo or in vitro test data. It is noted that the concentration and dosage values may also vary with the severity of the condition to be alleviated. It will be further understood that the specific dosage regimen may be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

In some embodiments, one or more of the co-conjugates described herein is in a liquid pharmaceutical formulation described in section 4.4 above.

Methods for administering pharmaceutical compositions having the co-conjugates described herein are well known in the art. It will be appreciated that the appropriate route of administration of the pharmaceutical composition can be readily determined by the skilled clinician. Exemplary routes of administration include intravenous injection, intramuscular injection, intradermal injection, or subcutaneous injection. Furthermore, it should be understood that the formulation of the pharmaceutical composition can be easily adapted to the route of administration.

The methods provided herein for treating a disease are intended to include (1) preventing the disease even if a subject who may be susceptible to the disease but has not experienced or exhibited symptoms of the disease does not develop clinical symptoms of the disease; (2) Inhibiting the disease, i.e., preventing or reducing the progression of the disease or its clinical symptoms; or (3) alleviating the disease, i.e., causing regression of the disease or its clinical symptoms. Also provided herein are methods of preventing a disease comprising pre-arresting a clinical symptom indicative of the disease. The therapeutically effective amount of the pharmaceutical composition used in the methods of the present disclosure will vary depending on the pharmaceutical composition used, the disease and its severity, and the age, weight, etc., of the subject to be treated, all of which are within the skill of the attending clinician.

Exemplary embodiments

Embodiment 1. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) a heavy chain variable region of a first antibody (VHAb 1); (ii) Heavy chain variable region (VHA) of the second antibodyb2 Comprising an N-terminal truncation of 1 to 18 amino acids; and (iii) a polypeptide linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VHAb2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein VHAb1 and VHAb2 bind non-overlapping epitopes on the target.

Embodiment 2. The co-conjugate of embodiment 1, wherein the N-terminal truncation of VHAb2 is 1 to 10 amino acids.

Embodiment 3. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VHAb1; (ii) A VHAb2 comprising a truncation or deletion in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the VHAb2 comprising the truncation or deletion; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein X of said polypeptide linker ₃ Amino acids and the VHAb2 complementarity determining region 1 (CDR 1) are 8 to 25 amino acids apart; and wherein VHAb1 and VHAb2 bind non-overlapping epitopes on the target.

Embodiment 4. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VHAb1; (ii) A VHAb2 comprising a truncation or deletion in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the VHAb2 comprising the truncation or deletion; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein X of said polypeptide linker ₃ Amino acids and the VHAb2 complementarity determining region 1 (CDR 1) are not more separatedOver 25 amino acids; and wherein VHAb1 and VHAb2 bind non-overlapping epitopes on the target.

Embodiment 5. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VHAb1; (ii) An N-terminally truncated VHAb2 comprising 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VHAb2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein VHAb1 and VHAb2 bind non-overlapping epitopes on the target.

Embodiment 6. The co-conjugate of embodiment 5, wherein the N-terminal truncation of the FR1 is 1 to 10 amino acids.

Embodiment 7. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) a heavy chain variable region of a first antibody (VHAb 1); (ii) A light chain variable region (VLAb 2) of a second antibody comprising an N-terminal truncation of 1 to 18 amino acids; and (iii) a polypeptide linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VLAb2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein VHAb1 and VLAb2 bind non-overlapping epitopes on the target.

Embodiment 8. The co-conjugate of embodiment 7 wherein the N-terminus of the VLAb2 is truncated to 1 to 10 amino acids.

Embodiment 9. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VHAb1; (ii) A VLAb2 comprising a truncation or deletion in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the VLAb2 comprising the truncation or deletion; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein X of said polypeptide linker ₃ Amino acids and the VLAb2 complementarity determining region 1 (CDR 1) are 5 to 22 amino acids apart; and wherein VHAb1 and VLAb2 bind non-overlapping epitopes on the target.

Embodiment 10. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VHAb1; (ii) A VLAb2 comprising a truncation or deletion in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the VLAb2 comprising the truncation or deletion; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein X of said polypeptide linker ₃ Amino acids and the VLAb2 complementarity determining region 1 (CDR 1) are no more than 22 amino acids apart; and wherein VHAb1 and VLAb2 bind non-overlapping epitopes on the target.

Embodiment 11. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VHAb1; (ii) An N-terminally truncated VLAb2 comprising 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VLAb2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein VHAb1 and VLAb2 bind non-overlapping epitopes on the target.

Embodiment 12. The co-conjugate of embodiment 11, wherein the N-terminal truncation of the FR1 is 1 to 10 amino acids.

Embodiment 13. Co-conjugate specifically binding to target wherein the co-conjugate comprises: (i) the light chain variable region of the first antibody (VLAb 1); (ii) A light chain variable region (VLAb 2) of a second antibody comprising an N-terminal truncation of 1 to 18 amino acids; and (iii) a polypeptide linker linking the C-terminal amino acid of VLAb1 to the N-terminal amino acid of truncated VLAb2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein VLAb1 and VLAb2 bind to non-overlapping epitopes on the target.

Embodiment 14. The co-conjugate of embodiment 13 wherein the N-terminus of the VLAb2 is truncated to 1 to 10 amino acids.

Embodiment 15. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VLAb1; (ii) A VLAb2 comprising a truncation or deletion in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VLAb 1C-terminal amino acid to the N-terminal amino acid of the VLAb2 comprising the truncation or deletion; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein X of said polypeptide linker ₃ Amino acids and the VLAb2 complementarity determining region 1 (CDR 1) are 5 to 22 amino acids apart; and wherein VLAb1 and VLAb2 bind to non-overlapping epitopes on the target.

Embodiment 16. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VLAb1; (ii) A VLAb2 comprising a truncation or deletion in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VLAb 1C-terminal amino acid to the N-terminal amino acid of the VLAb2 comprising the truncation or deletion; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and is also provided withWherein X of the polypeptide linker ₃ Amino acids and the VLAb2 complementarity determining region 1 (CDR 1) are no more than 22 amino acids apart; and wherein VLAb1 and VLAb2 bind to non-overlapping epitopes on the target.

Embodiment 17. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VLAb1; (ii) An N-terminally truncated VLAb2 comprising 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the C-terminal amino acid of VLAb1 to the N-terminal amino acid of truncated VLAb2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein VLAb1 and VLAb2 bind to non-overlapping epitopes on the target.

Embodiment 18. The co-conjugate of embodiment 17, wherein the N-terminal truncation of the FR1 is 1 to 10 amino acids.

Embodiment 19. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) the light chain variable region of the first antibody (VLAb 1); (ii) A heavy chain variable region (VHAb 2) of a second antibody comprising an N-terminal truncation of 1 to 18 amino acids; and (iii) a polypeptide linker linking the C-terminal amino acid of VLAb1 to the N-terminal amino acid of truncated VHAb2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein VLAb1 and VHAb2 bind non-overlapping epitopes on the target.

Embodiment 20. The co-conjugate of embodiment 19, wherein the N-terminus of the VHAb2 is truncated to 1 to 10 amino acids.

Embodiment 21. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VLAb1; (ii) A VHAb2 comprising a truncation or deletion in the framework 1 (FR 1) region; and (iii) combining said VLAb 1C-terminal amino acid with said polypeptide comprising said truncation or deletion A polypeptide linker linked to the N-terminal amino acid of VHAb2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein X of said polypeptide linker ₃ Amino acids and the VHAb2 complementarity determining region 1 (CDR 1) are 8 to 25 amino acids apart; and wherein VLAb1 and VHAb2 bind non-overlapping epitopes on the target.

Embodiment 22. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VLAb1; (ii) A VHAb2 comprising a truncation or deletion in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the VLAb 1C-terminal amino acid to the N-terminal amino acid of the VHAb2 comprising the truncation or deletion; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein X of said polypeptide linker ₃ Amino acids and the VHAb2 complementarity determining region 1 (CDR 1) are no more than 25 amino acids apart; and wherein VLAb1 and VHAb2 bind non-overlapping epitopes on the target.

Embodiment 23. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VLAb1; (ii) An N-terminally truncated VHAb2 comprising 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a polypeptide linker linking the C-terminal amino acid of VLAb1 to the N-terminal amino acid of truncated VHAb2; wherein the three amino acids at the C-terminal end of the polypeptide linker are X ₁ -X ₂ -X ₃ Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R; x is X ₂ V, A, L, S, G, R, K, M, C, F, T, P or E; and X is ₃ Is G; and wherein VLAb1 and VHAb2 bind non-overlapping epitopes on the target.

Embodiment 24. The co-conjugate of embodiment 23, wherein the N-terminal truncation of FR1 is 1 to 10 amino acids.

Embodiment 25. The co-conjugate of any one of embodiments 1 to 24, wherein the three amino acids at the C-terminus of the polypeptide linker are VVG, VAG, VLG, VSG, VGG, VRG, VKG, VMG, VCG, VFG, VTG, VPG or VEG.

Embodiment 26. The co-conjugate according to any one of embodiments 1 to 24, wherein the three amino acids at the C-terminal end of the polypeptide linker is LVG, LAG, LLG, LSG, LGG, LRG, LKG, LMG, LCG, LFG, LTG, LPG or LEG.

Embodiment 27. The co-conjugate according to any one of embodiments 1 to 24, wherein the three amino acids at the C-terminal end of the polypeptide linker is WVG, WAG, WLG, WSG, WGG, WRG, WKG, WMG, WCG, WFG, WTG, WPG or WEG.

Embodiment 28. The co-conjugate according to any one of embodiments 1 to 24, wherein the three amino acids at the C-terminal end of the polypeptide linker is PVG, PAG, PLG, PSG, PGG, PRG, PKG, PMG, PCG, PFG, PTG, PPG or PEG.

Embodiment 29. The co-conjugate according to any one of embodiments 1 to 24, wherein the three amino acids at the C-terminal end of the polypeptide linker is SVG, SAG, SLG, SSG, SGG, SRG, SKG, SMG, SCG, SFG, STG, SPG or SEG.

Embodiment 30. The co-conjugate according to any one of embodiments 1 to 24, wherein the three amino acids at the C-terminal end of the polypeptide linker is GVG, GAG, GLG, GSG, GGG, GRG, GKG, GMG, GCG, GFG, GTG, GPG or GEG.

Embodiment 31. The co-conjugate according to any one of embodiments 1 to 24, wherein the three amino acids at the C-terminal end of the polypeptide linker is KVG, KAG, KLG, KSG, KGG, KRG, KKG, KMG, KCG, KFG, KTG, KPG or KEG.

Embodiment 32. The co-conjugate according to any one of embodiments 1 to 24, wherein the three amino acids at the C-terminal end of the polypeptide linker is DVG, DAG, DLG, DSG, DGG, DRG, DKG, DMG, DCG, DFG, DTG, DPG or DEG.

Embodiment 33. The co-conjugate according to any one of embodiments 1 to 24, wherein the three amino acids at the C-terminal end of the polypeptide linker is FVG, FAG, FLG, FSG, FGG, FRG, FKG, FMG, FCG, FFG, FTG, FPG or FEG.

Embodiment 34. The co-conjugate according to any one of embodiments 1 to 24, wherein the three amino acids at the C-terminal end of the polypeptide linker is MVG, MAG, MLG, MSG, MGG, MRG, MKG, MMG, MCG, MFG, MTG, MPG or MEG.

Embodiment 35. The co-conjugate according to any one of embodiments 1 to 24, wherein the three amino acids at the C-terminal end of the polypeptide linker is TVG, TAG, TLG, TSG, TGG, TRG, TKG, TMG, TCG, TFG, TTG, TPG or TEG.

Embodiment 36. The co-conjugate according to any one of embodiments 1 to 24, wherein the three amino acids at the C-terminal end of the polypeptide linker is NVG, NAG, NLG, NSG, NGG, NRG, NKG, NMG, NCG, NFG, NTG, NPG or NEG.

Embodiment 37. The co-conjugate of any one of embodiments 1 to 24, wherein the three amino acids at the C-terminus of the polypeptide linker is RVG, RAG, RLG, RSG, RGG, RRG, RKG, RMG, RCG, RFG, RTG, RPG or REG.

Embodiment 38. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) a heavy chain variable region of a first antibody (VHAb 1); (ii) A heavy chain variable region (VHAb 2) of a second antibody comprising an N-terminal truncation of 1 to 18 amino acids; and (iii) a linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VHAb 2; wherein VHAb1 and VHAb2 bind non-overlapping epitopes on the target.

Embodiment 39. The co-conjugate of embodiment 38, wherein the N-terminus of the VHAb2 is truncated to 1 to 10 amino acids.

Embodiment 40. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VHAb1; (ii) VHAb2 comprising a truncation or deletion of 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a linker linking the VHAb 1C-terminal amino acid to the VHAb 2N-terminal amino acid comprising the truncation or deletion; wherein VHAb1 and VHAb2 bind non-overlapping epitopes on the target.

Embodiment 41. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VHAb1; (ii) An N-terminally truncated VHAb2 comprising 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VHAb2; wherein VHAb1 and VHAb2 bind non-overlapping epitopes on the target.

Embodiment 42. The co-conjugate of embodiment 41, wherein the N-terminal truncation of FR1 is 1 to 10 amino acids.

Embodiment 43. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) a heavy chain variable region of a first antibody (VHAb 1); (ii) A light chain variable region (VLAb 2) of a second antibody comprising an N-terminal truncation of 1 to 18 amino acids; and (iii) a linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VLAb 2; wherein VHAb1 and VLAb2 bind non-overlapping epitopes on the target.

Embodiment 44. The co-conjugate of embodiment 43, wherein the N-terminus of VLAb2 is truncated to 1 to 10 amino acids.

Embodiment 45. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VHAb1; (ii) VLAb2 comprising a truncation or deletion of 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the VLAb2 comprising the truncation or deletion; wherein VHAb1 and VLAb2 bind non-overlapping epitopes on the target.

Embodiment 46. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VHAb1; (ii) An N-terminally truncated VLAb2 comprising 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a linker linking the VHAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VLAb2; wherein VHAb1 and VLAb2 bind non-overlapping epitopes on the target.

Embodiment 47. The co-conjugate of embodiment 46, wherein the N-terminal truncation of FR1 is 1 to 10 amino acids.

Embodiment 48. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) the light chain variable region of the first antibody (VLAb 1); (ii) A light chain variable region (VLAb 2) of a second antibody comprising an N-terminal truncation of 1 to 18 amino acids; and (iii) a linker linking the VLAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VLAb2; wherein VLAb1 and VLAb2 bind to non-overlapping epitopes on the target.

Embodiment 49. The co-conjugate of embodiment 48 wherein the N-terminus of the VLAb2 is truncated to 1 to 10 amino acids.

Embodiment 50. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VLAb1; (ii) VLAb2 comprising a truncation or deletion of 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a linker linking the VLAb 1C-terminal amino acid to the N-terminal amino acid of the VLAb2 comprising the truncation or deletion; wherein VLAb1 and VLAb2 bind to non-overlapping epitopes on the target.

Embodiment 51. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VLAb1; (ii) An N-terminally truncated VLAb2 comprising 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a linker linking the VLAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VLAb2; wherein VLAb1 and VLAb2 bind to non-overlapping epitopes on the target.

Embodiment 52. The co-conjugate of embodiment 51, wherein the N-terminal truncation of the FR1 is 1 to 10 amino acids.

Embodiment 53. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) the light chain variable region of the first antibody (VLAb 1); (ii) A heavy chain variable region (VHAb 2) of a second antibody comprising an N-terminal truncation of 1 to 18 amino acids; and (iii) a linker linking the VLAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VHAb 2; wherein VLAb1 and VHAb2 bind to non-overlapping epitopes on the target.

Embodiment 54. The co-conjugate of embodiment 53, wherein the N-terminus of the VHAb2 is truncated to 1 to 10 amino acids.

Embodiment 55. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VLAb1; (ii) VHAb2 comprising a truncation or deletion of 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a linker linking the VLAb 1C-terminal amino acid to the N-terminal amino acid of the VHAb2 comprising the truncation or deletion; wherein VLAb1 and VHAb2 bind to non-overlapping epitopes on the target.

Embodiment 56. A co-conjugate that specifically binds to a target, wherein the co-conjugate comprises: (i) VLAb1; (ii) An N-terminally truncated VHAb2 comprising 1 to 18 amino acids in the framework 1 (FR 1) region; and (iii) a linker linking the VLAb 1C-terminal amino acid to the N-terminal amino acid of the truncated VHAb2; wherein VLAb1 and VHAb2 bind to non-overlapping epitopes on the target.

Embodiment 57. The co-conjugate of embodiment 56, wherein the N-terminal truncation of the FR1 is 1 to 10 amino acids.

Embodiment 58 the co-conjugate of any one of embodiments 1 to 12 and 25 to 47, wherein the VHAb1 is further linked to a light chain variable region (VL).

Embodiment 59. The co-conjugate of any one of embodiments 1 to 6, 19 to 42 and 53 to 57, wherein the VHAb2 is further linked to VL.

Embodiment 60. The co-conjugate of any one of embodiments 13 to 37, 48 to 57 and 59, wherein the VLAb1 is further linked to a heavy chain variable region (VH).

Embodiment 61. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58 and 60, wherein the VLAb2 is further linked to VH.

Embodiment 62. The co-conjugate of any one of embodiments 1 to 6 and 25 to 42, wherein the VHAb1 is further linked to a first VL and the VHAb2 is further linked to a second VL.

Embodiment 63. The co-conjugate of any one of embodiments 7 to 12, 25 to 37, and 43 to 47, wherein said VHAb1 is further linked to VL and said VLAb2 is further linked to VH.

Embodiment 64. The co-conjugate of any one of embodiments 13 to 18, 25 to 37, and 48 to 52, wherein the VLAb1 is further linked to a first VH and the VLAb2 is further linked to a second VH.

Embodiment 65 the co-conjugate of any one of embodiments 19 to 37 and 53 to 57, wherein the VLAb1 is further linked to VH and the VHAb2 is further linked to VL.

Embodiment 66. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65, wherein said truncated VHAb2 further comprises 1 to 18 additional amino acids substituted for the amino acids truncated from said VHAb 2.

Embodiment 67. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65, wherein the VHAb2 comprising a deletion further comprises 1 to 18 amino acids that replace the amino acid deleted from the VHAb 2.

Embodiment 68. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62, and 65 to 67, wherein the N-terminal amino acid 1 of the truncated VHAb2 is not E or Q.

Embodiment 69. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62, and 65 to 68, wherein the N-terminal amino acid 1 of the truncated VHAb2 is not E, Q or R.

Embodiment 70. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 69, wherein the N-terminal amino acid 2 of the truncated VHAb2 is not I, L, M or V.

Embodiment 71. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 70, wherein the N-terminal amino acid 3 of the truncated VHAb2 is not Q or T.

Embodiment 72. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 70, wherein the N-terminal amino acid 3 of the truncated VHAb2 is not Q, T, H or R.

Embodiment 73. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 72, wherein the N-terminal amino acid 4 of the truncated VHAb2 is not L or V.

Embodiment 74. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 72, wherein the N-terminal amino acid 4 of the truncated VHAb2 is not L, V or R.

Embodiment 75. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 74, wherein the N-terminal 5 th amino acid of the truncated VHAb2 is not K, L, Q, R or V.

Embodiment 76. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62, and 65 to 75, wherein the N-terminal amino acid 6 of the truncated VHAb2 is not E or Q.

Embodiment 77. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 75, wherein the N-terminal amino acid 6 of the truncated VHAb2 is not E, K, Q or D.

Embodiment 78. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 77, wherein the N-terminal amino acid 7 of the truncated VHAb2 is not P, S or W.

Embodiment 79. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 77, wherein the N-terminal amino acid 7 of the truncated VHAb2 is not P, S, W, L or T.

Embodiment 80. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 79, wherein the N-terminal amino acid 8 of the truncated VHAb2 is not G.

Embodiment 81. The co-conjugate of any of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 79, wherein the N-terminal amino acid 8 of the truncated VHAb2 is not G, A or V.

Embodiment 82. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 81, wherein the N-terminal amino acid 9 of the truncated VHAb2 is not A, E, G, P or S.

Embodiment 83. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 82, wherein the N-terminal 11 th amino acid of the truncated VHAb2 is not A, E, G, T or V.

Embodiment 84. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 83, wherein the N-terminal amino acid 12 of the truncated VHAb2 is not L or V.

Embodiment 85 the co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 84, wherein the N-terminal 13 th amino acid of the truncated VHAb2 is not I, K, L, R or V.

Embodiment 86. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 85, wherein the N-terminal amino acid 14 of the truncated VHAb2 is not K, Q or R.

Embodiment 87. The co-conjugate of any of embodiments 1 to 6, 19 to 42, 53 to 60, 62, and 65 to 85, wherein the N-terminal amino acid 14 of the truncated VHAb2 is not K, Q, R or N.

Embodiment 88. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62, and 65 to 87, wherein the N-terminal 15 th amino acid of the truncated VHAb2 is not a or P.

Embodiment 89. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62, and 65 to 87, wherein the N-terminal 15 th amino acid of the truncated VHAb2 is not A, P, D, L or T.

Embodiment 90. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 89, wherein the N-terminal amino acid 16 of the truncated VHAb2 is not G, P, S or T.

Embodiment 91. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 90, wherein the N-terminal amino acid 17 of the truncated VHAb2 is not A, D, E, G, Q, R or S.

Embodiment 92. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 90, wherein the N-terminal amino acid 17 of the truncated VHAb2 is not A, D, E, G, Q, R, S, P, T or V.

Embodiment 93. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62, and 65 to 92, wherein the N-terminal 18 th amino acid of the truncated VHAb2 is not S or T.

Embodiment 94. The co-conjugate of any one of embodiments 1 to 6, 19 to 42, 53 to 60, 62 and 65 to 92, wherein the N-terminal 18 th amino acid of the truncated VHAb2 is not S, T, A, L or M.

Embodiment 95. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, and 63 to 64, wherein the truncated VLAb2 further comprises 1 to 18 additional amino acids substituted for the amino acids truncated from the VLAb 2.

Embodiment 96 the co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, and 63 to 64, wherein the VLAb2 comprising a deletion further comprises 1 to 18 amino acids substituted for the amino acid deleted from the VLAb 2.

Embodiment 97. The co-conjugate of any of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 96, wherein the VLAb2 is a light chain variable region of a human lambda (lambda) light chain (lambda VLAb 2).

Embodiment 98. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 97 wherein the N-terminal amino acid 1 of the truncated lambda VLAb2 is not N, Q, R or S.

Embodiment 99. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 97 wherein the N-terminal amino acid 1 of the truncated lambda VLAb2 is not N, Q, R, S or L.

Embodiment 100. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 99 wherein the N-terminal amino acid 2 of the truncated lambda VLAb2 is not A, F, L, P, S, T or Y.

Embodiment 101. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 100 wherein the N-terminal amino acid 3 of the truncated lambda VLAb2 is not A, E, G, M or V.

Embodiment 102. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64 and 95 to 101 wherein the N-terminal amino acid 4 of the truncated lambda VLAb2 is not L or V.

Embodiment 103. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 101 wherein the N-terminal amino acid 4 of the truncated lambda VLAb2 is not L, V or P.

Embodiment 104. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 103 wherein the N-terminal 5 th amino acid of the truncated lambda VLAb2 is not T.

Embodiment 105. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 103 wherein the N-terminal 5 th amino acid of the truncated lambda VLAb2 is not T or M.

Embodiment 106. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 105 wherein the N-terminal amino acid 6 of the truncated lambda VLAb2 is not Q.

Embodiment 107. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 106 wherein the N-terminal 7 th amino acid of the truncated lambda VLAb2 is not E, P or S.

Embodiment 108. The co-conjugate according to any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64 and 95 to 106 wherein the N-terminal amino acid 7 of the truncated lambda VLAb2 is not E, P, S, D, L or V.

Embodiment 109. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 108, wherein the N-terminal 8 th amino acid of the truncated lambda VLAb2 is not A, H, L, P, R, S or T.

Embodiment 110. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 109 wherein the N-terminal 9 th amino acid of the truncated lambda VLAb2 is not A, F or S.

Embodiment 111. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 110 wherein the N-terminal 11 th amino acid of the truncated lambda VLAb2 is not A, F, L or V.

Embodiment 112. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 110 wherein the N-terminal 11 th amino acid of the truncated lambda VLAb2 is not A, F, L, V, H or S.

Embodiment 113. The co-conjugate according to any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64 and 95 to 112, wherein the N-terminal amino acid 12 of the truncated lambda VLAb2 is not S or T.

Embodiment 114. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64 and 95 to 113 wherein the N-terminal 13 th amino acid of the truncated lambda VLAb2 is not A, E, G, K or V.

Embodiment 115. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 113 wherein the N-terminal 13 th amino acid of the truncated lambda VLAb2 is not A, E, G, K, V or R.

Embodiment 116. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 115 wherein the N-terminal 14 th amino acid of the truncated lambda VLAb2 is not A, G, S or T.

Embodiment 117. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 115 wherein the N-terminal 14 th amino acid of the truncated lambda VLAb2 is not A, G, S, T or L.

Embodiment 118. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 117 wherein the N-terminal 15 th amino acid of the truncated lambda VLAb2 is not L, P or T.

Embodiment 119. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 117 wherein the N-terminal 15 th amino acid of the truncated lambda VLAb2 is not L, P, T or S.

Embodiment 120. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 119, wherein the N-terminal 16 th amino acid of the truncated lambda VLAb2 is not A, G or R.

Embodiment 121. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 120 wherein the N-terminal 17 th amino acid of the truncated lambda VLAb2 is not A, G, K, Q or S.

Embodiment 122. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 120 wherein the N-terminal 17 th amino acid of the truncated lambda VLAb2 is not A, G, K, Q, S or E.

Embodiment 123. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 122 wherein the N-terminal 18 th amino acid of the truncated lambda VLAb2 is not K, M, R, S or T.

Embodiment 124. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95 to 122, wherein the N-terminal 18 th amino acid of the truncated lambda VLAb2 is not K, M, R, S, T or a.

Embodiment 125. The co-conjugate of any of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, and 95, wherein the VLAb2 is a light chain variable region of a human kappa (kappa) light chain (kappa VLAb 2).

Embodiment 126. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 wherein the N-terminal amino acid 1 of the truncated kappa VLAb2 is not A, D, E or V.

Embodiment 127. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95 and 125 wherein the N-terminal amino acid 1 of the truncated kappa VLAb2 is not A, D, E or N.

Embodiment 128 the co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 127 wherein the N-terminal amino acid 2 of the truncated kappa VLAb2 is not I or V.

Embodiment 129 the co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 127 wherein the N-terminal amino acid 2 of the truncated kappa VLAb2 is not I, V or T.

Embodiment 130. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 129 wherein the N-terminal amino acid 3 of the truncated kappa VLAb2 is not Q, R, V or W.

Embodiment 131. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 129 wherein the N-terminal amino acid 3 of the truncated kappa VLAb2 is not Q, R, V, W or T.

Embodiment 132. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 129 wherein the N-terminal amino acid 4 of the truncated kappa VLAb2 is not L or M.

Embodiment 133. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 132 wherein the N-terminal 5 th amino acid of the truncated kappa VLAb2 is not T.

Embodiment 134. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95 and 125 to 133 wherein the N-terminal amino acid 6 of the truncated kappa VLAb2 is not Q.

Embodiment 135. The co-conjugate according to any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95 and 125 to 134 wherein the N-terminal 7 th amino acid of the truncated kappa VLAb2 is not S or T.

Embodiment 136. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 135 wherein the N-terminal 8 th amino acid of the truncated kappa VLAb2 is not P.

Embodiment 137. The co-conjugate according to any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95 and 125 to 136 wherein the N-terminal amino acid 9 of the truncated kappa VLAb2 is not A, D, L or S.

Embodiment 138 the co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95 and 125 to 136 wherein the N-terminal amino acid 9 of the truncated kappa VLAb2 is not A, D, L, S, F or G.

Embodiment 139. The co-conjugate of any of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 138 wherein the N-terminal 10 th amino acid of the truncated kappa VLAb2 is not A, F, L, S or T.

Embodiment 140. The co-conjugate of any of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 139 wherein the N-terminal 11 th amino acid of the truncated kappa VLAb2 is not L, M, Q or V.

Embodiment 141. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 139 wherein the N-terminal 11 th amino acid of the truncated kappa VLAb2 is not L, M, Q, V, F or S.

Embodiment 142. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 141 wherein the N-terminal amino acid 12 of the truncated kappa VLAb2 is not P or S.

Embodiment 143. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 141 wherein the N-terminal amino acid 12 of the truncated kappa VLAb2 is not P, S or a.

Embodiment 144. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95 and 125 to 143 wherein the N-terminal 13 th amino acid of the truncated kappa VLAb2 is not A, I, L or V.

Embodiment 145. The co-conjugate of any of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 144 wherein the N-terminal 14 th amino acid of said truncated kappa VLAb2 is not S or T.

Embodiment 146. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 145, wherein the N-terminal 15 th amino acid of the truncated kappa VLAb2 is not L, P, T or V.

Embodiment 147. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95, and 125 to 146, wherein the N-terminal 16 th amino acid of the truncated kappa VLAb2 is not G or K.

Embodiment 148 the co-conjugate of any of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95 and 125 to 147 wherein the N-terminal 17 th amino acid of the truncated kappa VLAb2 is not D, E or Q.

Embodiment 149. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60 to 61, 63 to 64, 95 and 125 to 148 wherein the N-terminal 18 th amino acid of the truncated kappa VLAb2 is not K, P, Q or R.

Embodiment 150. An antigen binding protein comprising a heavy chain variable region (VHAb) of an antibody, wherein the VHAb comprises an N-terminal truncation of 1 to 18 amino acids, and wherein the VHAb is linked to a second molecule via a linker.

Embodiment 151 an antigen binding protein comprising a light chain variable region (VLAb) of an antibody, wherein the VLAb comprises an N-terminal truncation of 1 to 18 amino acids, and wherein the VLAb is linked to a second molecule via a linker.

The antigen binding protein of embodiment 150, wherein the VHAb is further linked to a light chain variable region.

Embodiment 153 the antigen binding protein of embodiment 151, wherein the VLAb is further linked to a heavy chain variable region.

The antigen binding protein of any one of embodiments 150 to 153, wherein the second molecule is a DNA molecule or a protein molecule.

Embodiment 155. The antigen binding protein of any one of embodiments 150 to 154, wherein said second molecule is an antibody or antigen binding fragment thereof.

Embodiment 156. The co-conjugate of any one of embodiments 1 to 149, wherein the FR1, CDR1, or both FR1 and CDR1 are determined according to the IMGT numbering scheme.

Embodiment 157 the co-conjugate of any of embodiments 1 to 149, wherein the FR1, CDR1, or both FR1 and CDR1 are determined according to the Kabat numbering scheme.

Embodiment 158. The co-conjugate of any one of embodiments 1 to 157, wherein the VH, VL, or both VH and VL are determined according to IMGT numbering schemes.

Embodiment 159 the co-conjugate of any one of embodiments 1 to 157, wherein the VH, VL, or both VH and VL are determined according to the Kabat numbering scheme.

Embodiment 160. The co-conjugate according to any one of embodiments 68 to 159, wherein amino acid positions are determined according to IMGT numbering scheme.

Embodiment 161. The co-conjugate of any of embodiments 68 to 159, wherein the amino acid position is determined according to the Kabat numbering scheme.

Embodiment 162. The co-conjugate of any one of embodiments 1 to 161, wherein said VHAb1, VHAb2 or both VHAb1 and VHAb2 are from a camelidae single chain VHH.

Embodiment 163. The co-conjugate of any one of embodiments 1 to 161, wherein said VHAb1, VHAb2, or both VHAb1 and VHAb2 are from IgG, igA, igE, igM or IgD.

Embodiment 164. The co-conjugate of any one of embodiments 1 to 161, wherein the VHAb1, VHAb2, or both VHAb1 and VHAb2 are camelidae single chain VHH.

Embodiment 165. The co-conjugate of any one of embodiments 1 to 161, wherein the VHAb1, VHAb2, or both VHAb1 and VHAb2 are part of a scFv.

Embodiment 166. The co-conjugate of any one of embodiments 1 to 161, wherein the VHAb1, VHAb2, or both VHAb1 and VHAb2 are part of a Fab.

Embodiment 167. The co-conjugate of any of embodiments 7 to 166, wherein the VLAb1, VLAb2, or both VLAb1 and VLAb2 are from IgG, igA, igE, igM or IgD.

Embodiment 168 the co-conjugate of any one of embodiments 7 to 166 wherein the VLAb1, VLAb2, or both VLAb1 and VLAb2 are part of a scFv.

Embodiment 169. The co-conjugate of any one of embodiments 7 to 166, wherein the VLAb1, VLAb2, or both VLAb1 and VLAb2 are part of a Fab.

Embodiment 170. The co-conjugate of any one of embodiments 1 to 169, wherein the linker is a rigid linker.

Embodiment 171. The co-conjugate of any one of embodiments 1 to 169, wherein the linker is a flexible linker.

Embodiment 172. The co-conjugate according to any one of embodiments 1 to 169, wherein the linker is a cleavable linker.

Embodiment 173. The co-conjugate of any one of embodiments 1-169, wherein the linker is a non-cleavable linker.

Embodiment 174. The co-conjugate of any of embodiments 38 to 173, wherein the linker is a peptide, a nucleic acid, or a chemical linker.

Embodiment 175. The co-conjugate of any of embodiments 1 to 174, wherein the linker connects the variable region of the first antibody and the variable region of the second antibody by a covalent bond.

Embodiment 176. The co-conjugate of any one of embodiments 1 to 175, wherein the linker is a peptide linker.

Embodiment 177. The co-conjugate of embodiment 176, wherein the peptide linker comprises a peptide sequence (EAAAK) _n Wherein n is 1 to 10An integer.

Embodiment 178 the co-conjugate of embodiment 176, wherein the peptide linker comprises the peptide sequence (XP) n, (XPP) n or (XPPP) n, wherein X is any amino acid, and wherein n is an integer from 1 to 20.

Embodiment 179 the co-conjugate according to embodiment 176, wherein said peptide linker comprises a peptide sequence (AP) _n Wherein n is an integer from 1 to 20.

Embodiment 180. The co-conjugate according to embodiment 176, wherein the peptide linker comprises the peptide sequence (EEEEKKK) _n Wherein n is an integer from 1 to 10.

Embodiment 181. The co-conjugate according to embodiment 176, wherein the peptide linker comprises a peptide sequence (G _x S _y ) _n Wherein x is 1 to 5, y is 1 to 5, and n is an integer of 1 to 10.

Embodiment 182 the co-conjugate of any one of embodiments 38 to 175, wherein the linker is a chemical linker.

Embodiment 183 the co-conjugate of embodiment 182, wherein the chemical linker comprises polyethylene glycol ("PEG").

Embodiment 184. The co-conjugate of any of embodiments 1 to 174, wherein the linker connects the variable region of the first antibody and the variable region of the second antibody by non-covalent binding.

Embodiment 185. The co-conjugate according to embodiment 184, wherein the linker comprises a leucine zipper.

The co-conjugate according to embodiment 186, wherein the linker comprises a double stranded nucleic acid having two complementary strands, each complementary strand being covalently linked to the variable region of the first antibody and the variable region of the second antibody, respectively.

Embodiment 187 the co-conjugate of any of embodiments 1 to 186, wherein the linker has a length of no more than 150 angstroms, no more than 140 angstroms, no more than 130 angstroms, no more than 120 angstroms, no more than 110 angstroms, no more than 100 angstroms, no more than 90 angstroms, no more than 80 angstroms, no more than 70 angstroms, no more than 60 angstroms, no more than 50 angstroms, no more than 40 angstroms, no more than 30 angstroms, no more than 20 angstroms, no more than 15 angstroms, no more than 10 angstroms, or no more than 5 angstroms.

Embodiment 188. The co-conjugate of any of embodiments 1 to 186, wherein the linker has a length of about 150 angstroms, about 140 angstroms, about 130 angstroms, about 120 angstroms, about 110 angstroms, about 100 angstroms, about 90 angstroms, about 80 angstroms, about 70 angstroms, about 60 angstroms, about 50 angstroms, about 40 angstroms, about 30 angstroms, about 20 angstroms, about 15 angstroms, about 10 angstroms, or about 5 angstroms.

The co-conjugate according to any one of embodiments 1 to 181, wherein the linker is no more than 60 amino acids, no more than 55 amino acids, no more than 50 amino acids, no more than 45 amino acids, no more than 40 amino acids, no more than 35 amino acids, no more than 30 amino acids, no more than 25 amino acids, no more than 20 amino acids, no more than 15 amino acids, no more than 10 amino acids, or no more than 5 amino acids in length.

Embodiment 190. The co-conjugate of any one of embodiments 1 to 181, wherein the linker is about 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 15 amino acids, about 10 amino acids, or about 5 amino acids in length.

Embodiment 191 the co-conjugate of any of embodiments 1 to 190, wherein the target is a polypeptide, a multiprotein complex, a nucleic acid, a carbohydrate, a glycan, a lipid molecule, a physiological metabolite, or a small molecule compound.

Embodiment 192. The co-conjugate according to any one of embodiments 1 to 190, wherein the target is an intracellular molecule, a disease marker, a neoantigen or a cell surface molecule.

Embodiment 193 the co-conjugate of embodiment 192, wherein the target molecule is a cancer antigen or a cancer marker.

Embodiment 194. The co-conjugate of any of embodiments 1-193, wherein the target is EGFR.

Embodiment 195.The co-conjugate according to any one of embodiments 1 to 194, wherein the target is expressed at a level of less than 1 x 10 ⁶ Less than 1X 10 ⁵ Less than 1X 10 ⁴ Less than 1X 10 ³ Or less than 1X 10 ² Cells.

Embodiment 196. The co-conjugate of any one of embodiments 1 to 193, wherein the target is a secreted protein.

Embodiment 197. The co-conjugate of embodiment 196, wherein the secreted protein is a growth factor, a cytokine, or a chemokine.

Embodiment 198 the co-conjugate according to embodiment 196 or 197, wherein the secreted protein is less than 1X 10 in blood ¹⁰ Less than 1X 10 ⁹ Less than 1X 10 ⁸ Less than 1X 10 ⁷ Less than 1X 10 ⁶ Less than 1X 10 ⁵ Less than 1X 10 ⁴ Less than 1X 10 ³ Or less than 1X 10 ² Each molecule/ml.

Embodiment 199. The co-conjugate of any one of embodiments 1 to 198, wherein the co-conjugate is an antagonist of the target.

Embodiment 200. The co-conjugate of any one of embodiments 1 to 198, wherein the co-conjugate is an agonist of the target.

Embodiment 201. The co-conjugate of any one of embodiments 1 to 200, wherein the co-conjugate binds to K of the target _D Less than 1X 10 ^-8 M is less than 1×10 ^-9 M is less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 M is less than 1×10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M is less than 1×10 ^-16 M is less than 1×10 ^-17 M or less than 1X 10 ^-18 M。

Embodiment 202. The co-conjugate of any one of embodiments 1 to 200, wherein the co-conjugate binds to K of the target _D Is about 1X 10 ^-8 M, about 1X 10 ^-9 M, about 1X 10 ^-10 M, about 1X 10 ^-11 M, about 1X 10 ^-12 M, about 1X 10 ^- ¹³ M, about 1X 10 ^-14 M, about 1X 10 ^-15 M, about 1X 10 ^-16 M, about 1X 10 ^-17 M or about 1X 10 ^-18 M。

Embodiment 203. The co-conjugate according to any one of embodiments 1 to 200, wherein the affinity of the co-conjugate for the target is no less than 50-fold, no less than 100-fold, no less than 150-fold, no less than 200-fold, no less than 250-fold, no less than 300-fold, no less than 350-fold, no less than 400-fold, no less than 450-fold, no less than 500-fold, no less than 600-fold, no less than 700-fold, no less than 800-fold, no less than 900-fold, no less than 1000-fold, no less than 1100-fold, no less than 1200-fold, no less than 1300-fold, no less than 1400-fold, no less than 1500-fold, no less than 1600-fold, no less than 1700-fold, no less than 1800-fold, no less than 1900-fold, no less than 2000-fold, no less than 3000-fold, no less than 4000-fold, or no less than 5000-fold, or no less than 10000-fold.

Embodiment 204. The co-conjugate of any of embodiments 1 to 6, 19 to 42, 53 to 60, 62, 65 to 94, 156 to 166, and 170 to 203, wherein said VHAb2 is a heavy chain variable region such that if conjugated to a reference Ig domain at the N-terminus of said VHAb2 via a linker comprising (GGGS) 4 (refIg-VHAb 2), the dissociation constant (KD) between said refIg-VHAb2 and said target is more than 3-fold greater than the KD between VHAb2 and said target.

Embodiment 205. The co-conjugate of any one of embodiments 7 to 18, 25 to 37, 43 to 52, 58, 60, 61, 63, 64, 95 to 149, 156 to 161, and 167 to 203 wherein the VLAb2 is a light chain variable region such that if conjugated to a reference Ig domain at the N-terminus of the VLAb2 via a linker comprising (GGGS) 4 (refIg-VLAb 2), the dissociation constant (KD) between the refIg-VLAb2 and the target is more than 3-fold greater than the KD between VLAb2 and the target.

Embodiment 206. The co-conjugate of any one of embodiments 150, 152, 154 to 166 and 170 to 203, wherein the VHAb is a heavy chain variable region such that if conjugated to a reference Ig domain at the N-terminus of the VHAb via a linker comprising (GGGS) 4 (refIg-VHAb), the dissociation constant (KD) between the refIg-VHAb and the target is more than 3-fold greater than the KD between VHAb and the target.

Embodiment 207 the co-conjugate of any one of embodiments 151, 153 to 161 and 167 to 203, wherein said VLAb is a light chain variable region such that if conjugated to a reference Ig domain via a linker comprising (GGGS) 4 (refIg-VLAb) at the N-terminus of said VLAb, the dissociation constant (KD) between said refIg-VLAb and said target is more than 3-fold greater than the KD between VLAb and said target.

Embodiment 208 a composition comprising a co-conjugate according to any one of embodiments 1 to 207.

Embodiment 209. A pharmaceutical composition comprising the co-conjugate of any one of embodiments 1 to 207 and a pharmaceutically acceptable carrier.

Embodiment 210. A detection agent comprising the co-conjugate of any one of embodiments 1 to 207.

Embodiment 211. A diagnostic agent comprising the co-conjugate of any one of embodiments 1 to 207.

Embodiment 212 a therapeutic agent comprising the co-conjugate of any one of embodiments 1-207.

Embodiment 213 a chimeric antigen receptor comprising the co-conjugate of any one of embodiments 1 to 207.

Embodiment 214. A cell expressing the co-conjugate of any one of embodiments 1 to 207.

Embodiment 215. The cell of embodiment 214, wherein the cell is an immune cell.

Embodiment 216. A complex comprising the co-conjugate of any one of embodiments 1 to 207 and a target.

Embodiment 217, a method for detecting a label in a sample comprising (i) contacting the sample with a co-conjugate of any one of embodiments 1 to 207 under conditions sufficient to form the co-conjugate and the labeled complex, and (ii) detecting the complex in the sample.

Embodiment 218. The method of embodiment 217, wherein the complex is detected by measuring a labeled reagent conjugated to the complex.

Embodiment 219 the method of embodiment 217 or 218, wherein the labeling agent is a fluorescent molecule, radioisotope, metal ion, enzyme, biotin, polymer or antibody.

Embodiment 220 the method of any one of embodiments 217-219, wherein the label is present in the sample at a concentration of no more than 1X 10 ^-10 M is not more than 1×10 ^-11 M is not more than 1×10 ^-12 M is not more than 1×10 ^-13 M is not more than 1×10 ^-14 M is not more than 1×10 ^-15 M is not more than 1×10 ^-16 M is not more than 1×10 ^-17 M is not more than 1×10 ^-18 M is not more than 1×10 ^-19 M is not more than 1×10 ^-20 M or not more than 1X 10 ^-21 M。

Embodiment 221 a method of diagnosing a disease in a subject comprising (i) contacting a sample with a co-conjugate of any one of embodiments 1 to 207 under conditions sufficient to form a complex of the co-conjugate and a disease marker, wherein the co-conjugate specifically binds the marker, and (ii) detecting the complex in the sample.

Embodiment 222. The method of embodiment 221, wherein the complex is detected by measuring a labeled reagent conjugated to the complex.

Embodiment 223 the method of embodiment 221 or 222, wherein the labeling agent is a fluorescent molecule, radioisotope, metal ion, enzyme, biotin, polymer, or antibody.

Embodiment 224 the method of any one of embodiments 221 to 223, wherein the sample is a body fluid, tissue, or cell.

Embodiment 225 the method of any one of embodiments 221 to 224, wherein the sample is blood, bone marrow, plasma, serum, urine, or cerebrospinal fluid.

Embodiment 226. Any one of embodiments 221 through 225 The method of claim, wherein the concentration of said disease marker present in said sample does not exceed 1X 10 ^-10 M is not more than 1×10 ^-11 M is not more than 1×10 ^-12 M is not more than 1×10 ^- ¹³ M is not more than 1×10 ^-14 M is not more than 1×10 ^-15 M is not more than 1×10 ^-16 M is not more than 1×10 ^-17 M is not more than 1×10 ^- ¹⁸ M is not more than 1×10 ^-19 M is not more than 1×10 ^-20 M or not more than 1X 10 ^-21 M。

Embodiment 227 a method of treating a disease in a subject comprising administering to the subject a therapeutically effective amount of the co-conjugate of any one of embodiments 1 to 207, wherein the disease is treatable by activating or inhibiting the target molecule.

Embodiment 228 a combinatorial library comprising: (i) A first variable region of a first antibody comprising an N-terminal truncation of 1 to 18 amino acids; and (ii) a plurality of polypeptide linkers; wherein the C-terminal amino acid of the linker is linked to the N-terminal amino acid of the truncated first variable region.

Embodiment 229 a combinatorial library comprising: (i) A plurality of first variable regions of a first antibody, wherein each of said first variable regions comprises an N-terminal truncation of 1 to 18 amino acids; and (ii) a plurality of polypeptide linkers; wherein the C-terminal amino acid of the linker is linked to the N-terminal amino acid of the truncated first variable region.

Embodiment 230. A combinatorial library comprising: (i) A plurality of first variable regions of a plurality of antibodies, wherein each of said first variable regions comprises an N-terminal truncation of 1 to 18 amino acids; and (ii) a plurality of polypeptide linkers; wherein the C-terminal amino acid of the linker is linked to the N-terminal amino acid of the truncated first variable region.

Embodiment 231 the combinatorial library of embodiment 228, wherein the first variable region is a heavy chain variable region.

Embodiment 232. The combinatorial library of embodiment 229 or 230, wherein the first variable region is a heavy chain variable region.

Embodiment 233. The combinatorial library of embodiment 228, wherein the first variable region is a light chain variable region.

Embodiment 234. The combinatorial library of embodiment 229 or 230, wherein the first variable region is a light chain variable region.

Embodiment 235 the combinatorial library of any one of embodiments 229-234, wherein the library comprises up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 first variable regions.

Embodiment 236 the combinatorial library of any one of embodiments 230 to 234, wherein the library comprises at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 x 10 ³ At least 1X 10 ⁴ At least 1X 10 ⁵ At least 1X 10 ⁶ At least 1X 10 ⁷ At least 1X 10 ⁸ At least 1X 10 ⁹ At least 1X 10 ¹⁰ Or at least 1X 10 ¹¹ A first variable region.

The combinatorial library of any one of embodiments 228-236, wherein the linker comprises 1 to 18 random amino acids at the C-terminus of the linker.

Embodiment 238 the combinatorial library of any one of embodiments 228-237, wherein the library comprises at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 x 10 ³ At least 1X 10 ⁴ At least 1X 10 ⁵ At least 1X 10 ⁶ At least 1X 10 ⁷ At least 1X 10 ⁸ At least 1X 10 ⁹ At least 1X 10 ¹⁰ Or at least 1X 10 ¹¹ And a plurality of connectors.

The combinatorial library of any one of embodiments 228-238, further comprising a second variable region of a second antibody, wherein the N-terminal amino acid of the linker is linked to the C-terminal amino acid of the second variable region.

Embodiment 240 the combinatorial library of any one of embodiments 228-238 further comprising a plurality of second variable regions of a second antibody, wherein the N-terminal amino acid of the linker is linked to the C-terminal amino acid of the second variable region.

Embodiment 241 the combinatorial library of any of embodiments 228-238, further comprising a plurality of second variable regions of a plurality of antibodies, wherein the N-terminal amino acid of the linker is linked to the C-terminal amino acid of the second variable region.

Embodiment 242. The combinatorial library of embodiment 239, wherein the second variable region is a heavy chain variable region.

Embodiment 243. The combinatorial library of embodiments 240 or 241, wherein said second variable region is a heavy chain variable region.

Embodiment 244 the combinatorial library of embodiment 239, wherein the second variable region is a light chain variable region

Embodiment 245. The combinatorial library of embodiments 240 or 241, wherein the second variable region is a light chain variable region.

The combinatorial library of any one of embodiments 239-245, wherein (i) the first and second variable regions bind to non-overlapping epitopes on the same target, (ii) the first and second variable regions do not bind to the same target, (iii) the first and second variable regions bind to non-overlapping epitopes on the same target, or (iv) the first and second variable regions do not bind to the same target.

Embodiment 247 a method of screening for co-binders that bind to a target comprising (i) obtaining a library according to any one of embodiments 228 to 246; and (ii) contacting the library of co-binder candidates from step (i) with the target to identify co-binders that specifically bind to the target.

Embodiment 248 a method of screening for co-binders that bind to a target comprising (i) expressing a library of expression vectors encoding the library of any one of embodiments 228 to 246; (ii) Obtaining a library according to any one of embodiments 228 to 246; and (iii) contacting the library of co-binder candidates from step (ii) with the target to identify co-binders that specifically bind to the target.

Embodiment 249. A method of screening for co-binders that bind to a target comprising (i) expressing a library of expression vectors encoding the library of any of embodiments 228-246; (ii) Obtaining a library according to any one of embodiments 228 to 246; (iii) Contacting the library of co-conjugate candidates from step (ii) with the target to form a complex between the co-conjugate that specifically binds the target and the target; (iv) Enriching complexes between co-conjugates that specifically bind to the target and the target; and (v) identifying a co-binder that specifically binds to the target and the target.

Embodiment 250 the method of any one of embodiments 247 to 249 wherein the co-conjugate binds to K of the target _D Less than 1X 10 ^-8 M is less than 1×10 ^-9 M is less than 1×10 ^-10 M is less than 1×10 ^-11 M is less than 1×10 ^-12 M is less than 1×10 ^-13 M is less than 1×10 ^-14 M is less than 1×10 ^-15 M is less than 1×10 ^-16 M is less than 1×10 ^-17 M or less than 1X 10 ^-18 M。

Embodiment 251. The method of any of embodiments 247 to 249, wherein the co-conjugate binds to K of the target _D Is about 1X 10 ^-8 M, about 1X 10 ^-9 M, about 1X 10 ^-10 M, about 1X 10 ^-11 M, about 1X 10 ^-12 M, about 1X 10 ^-13 M, about 1X 10 ^-14 M, about 1X 10 ^-15 M, about 1X 10 ^-16 M, about 1X 10 ^-17 M or about 1X 10 ^-18 M。

The method of any one of embodiments 247 to 251, wherein the co-conjugate has an affinity for the target of no less than 50-fold, no less than 100-fold, no less than 150-fold, no less than 200-fold, no less than 250-fold, no less than 300-fold, no less than 350-fold, no less than 400-fold, no less than 450-fold, no less than 500-fold, no less than 600-fold, no less than 700-fold, no less than 800-fold, no less than 900-fold, no less than 1000-fold, no less than 1100-fold, no less than 1200-fold, no less than 1300-fold, no less than 1400-fold, no less than 1500-fold, no less than 1600-fold, no less than 1700-fold, no less than 1800-fold, no less than 1900-fold, no less than 2000-fold, no less than 3000-fold, no less than 4000-fold, or no less than 5000-fold, or no less than 10000-fold.

Certain watches

This section provides some of the lengthy tables described herein.

Table 3: sequences of framework 1 region (FR 1, framework region 1) of isotype Ig heavy chain variable region. The description in the left column contains a database identifier based on which immunoglobulins can be obtained

The full length sequence of the sequence record, which is incorporated by reference herein in its entirety.

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Table 4: sequences of framework 1 region (FR 1, framework region 1) of isotype Ig kappa (kappa) light chain variable region. The description in the left column contains a database identifier based on which the full-length sequence of the immunoglobulin sequence record can be obtained, which is incorporated herein by reference in its entirety.

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Table 5: sequence of framework 1 region (FR 1, framework region 1) of isotype Ig lambda (λ) light chain variable region. The description in the left column contains a database identifier based on which the full-length sequence of the immunoglobulin sequence record can be obtained, which is incorporated herein by reference in its entirety.

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Examples

The examples in this section are provided as illustrations of the applications taught herein and are not intended to limit the disclosure of the application. Many embodiments have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, the following examples are intended to illustrate and not limit the scope of what is described in the claims.

Example 1: affinity and specificity increase in co-conjugates

To determine the affinity and specificity increase in the co-binders, the affinity of the co-binders is expressed as K _D ＝K _D1 ×K _D2 X K', where K _D Is the affinity of the co-conjugate, K _D1 、K _D2 Is the corresponding binding affinity (i.e., dissociation constant) of the two separate binding moieties, and K' is the factor that will be caused by the loss of binding energy from the two binders linking together. Thus, the increase in affinity of the co-binders relative to the individual single binders depends on K'. Such affinity terms may be converted into energy terms according to the laws of thermodynamics. The binding free energy of the co-conjugate can be expressed as: ΔG _CB ＝ΔG _B1 +ΔG _B2 +ΔG', where ΔG _CB Is the binding free energy of the co-conjugate, ΔG _B1 And ΔG _B2 Is the corresponding binding free energy of the two linked individual binding moieties, and Δg' is the loss of binding energy caused by linking the two binders together. Affinity of the co-binders to the co-binders during ligation (K _D ) The objective of maximization is to minimize the binding energy loss Δg ', or to minimize the linker-related energy loss, i.e., to minimize the K' factor.

The specificity of the conjugate is expressed as: sp=k _D，NT /K _D，T Wherein K is _D，NT Is affinity for non-target molecules, and K _D，T Is the affinity for the target molecule. Specificity is described as the ratio of non-target binding to target binding, which is a number greater than 1. For co-conjugates, the binding affinity for non-target and target can be further expressed as: k (K) _D，CB，NT ＝K _D，B1，NT ·K _D，B2，NT ·K′ _NT The method comprises the steps of carrying out a first treatment on the surface of the And K _D，CB，T ＝K _D，B1，T ·K _D，B2，T ·K′ _T Wherein K is _D，CB Is the affinity of the co-conjugate, K _D，B1 And K _D，B2 Is the corresponding binding affinity (i.e., dissociation constant) of the two linked individual binding moieties, K' is the affinity-losing factor that links the two binders together, T represents the target, and NT represents the non-target. Co-bondingThe specificity of the substance can then be expressed as: SP (service provider) _CB ＝SP _B1 ·SP _B2 ·(K′ _NT /K′ _T ). For target and homologous non-target, affinity loses factor K due to structural similarity _D，NT And K _D，T Should be similar. Thus, SP _CB ≈SP _B1 ·SP _B2 It is the product of the specificity of the individual binding moieties. Based on this equation, linking two single conjugates to form a co-conjugate results in an increase in specificity.

Figures 3A-3D show the main cases where energy loss may occur. In fig. 3A, even without a linker, two single bonds may interfere with each other's binding. This may be due to the size of the binding moiety, the relative position and/or orientation of the two epitopes, etc. In fig. 3B, the bond of one or more portions may be broken by a joint having an improper length and/or rigidity. In fig. 3C, too long a joint length may increase the entropy of the system. In fig. 3D, the linker may invade and/or interfere with the binding of one or both binding moieties. The present disclosure provides a solution for these and other situations.

To demonstrate the improvement in binding affinity and specificity as taught herein, two anti-EGFR VHHs were selected as exemplary demonstration of single binders. 7D12 and 9G8 were previously discovered from llama immunity and showed inhibitory activity against tumor growth (Roovers, laeremans et al Cancer Immunol Immunother (3): 303-317; roovers, vosjan et al Int J cancer.2011, 10 months 15; 129 (8): 2013-24). From their co-crystal Structure with EGFR, the binding epitopes of these two VHHs do not overlap each other (PDB codes 4KRM and 4 KRP), so the study can focus on the effect of the ligation on increased binding affinity and specificity (FIG. 3E) (Schmitz, bagchi et al Structure 21 (7): 1214-1224).

By inserting a flexibility (GGGS) between the C-terminus of 7D12 and the N-terminus of 9G8 ₄ The linker (designated 7D12-GS-9G8 or 7D12- (GGGS) 4-9G 8) to produce the co-conjugate 7D12-GS-9G8. Information about 7D12 and 9G8 may be in respectively<https://www.ebi.ac.uk/pdbe/entry/pdb/4krm/protein/2>And<https://www.ebi.ac.uk/pdbe/entry/pdb/4krp/protein/2>where found. The co-binders were cloned, expressed and purified and their binding affinities to human and mouse EGFR were determined by Biacore, as shown in table 12 below. Affinity of Co-binders for human EGFR (K _D ) Is 0.4pM, improved by more than 1000-fold over the single conjugate 7D12 (KD, 0.5 nM) or 9G8 (KD, 1.1 nM). The K' factor of human EGFR is 727,000. In contrast, the same co-conjugate has an affinity for mouse EGFR of (K _D ) 120nM, which is weaker than the stronger 9G8 of the two single binders. The K' factor of mouse EGFR is 570,000. Thus, the results indicate that if the linker-related energy loss is less than the binding energy obtained with the additional conjugate, the co-conjugate can indeed achieve synergistic co-binding, thereby improving affinity, as in the case of human EGFR. However, if the energy loss is greater than the binding energy obtained with the additional binder, there may be a net loss of binding affinity, as in the case of mouse EGFR. The specificity of the co-conjugate was approximately the same as the multiplication of the specificity of 7D12 and 9G8. When compared using the lower specific conjugate 9G8, the specificity improved by more than a factor of 10,000. If human EGFR is considered as the target and mouse EGFR is considered as the cognate non-target, KD, NT and KD, T are similar as shown in the previous paragraph, which results in a co-conjugate specificity of 7D12-GS-9G8 that is close to the product of the specificities of 7D12 and 9G8. Thus, the results indicate that in addition to the increased affinity, the co-conjugates can indeed also have improved specificity.

Biacore measurements were also performed on wild-type human EGFR and mutant human EGFR containing double substitutions of L325A and S340A at 37 ℃ (table 13). The higher temperature results in faster kinetics, but weaker affinity for the same co-and single binders. For wild-type and mutant EGFR, the co-binders showed stronger affinity than single binders. The wild-type K' factor was measured as 130,000 and the mutant was measured as 185,000. Here, the wild-type and mutant K' factors are also similar to each other, since there are only 2 mutations between the two. The co-conjugate was 288-fold more specific for wild type than mutant EGFR, which is close to the product of the specificities of 7D12 and 9G8. Again, the results demonstrate the increased affinity and specificity obtained by forming the co-conjugate.

TABLE 12 free 7D12, 9G8 and Co-conjugate 7D12- (GGGS) measured by SPR at 25 ℃ ₄ Kinetic parameters, K of 9G8 _D K' and specificity for human and mouse EGFR-Fc.

TABLE 13 7D12, 9G8 and Co-conjugates measured by Biacore at 37 ℃

Kinetic parameters, K, of 7D12-GS-9G8 _D K' and its use against human and mutant EGFR

(L325V, S340A).

Example 2: factor affecting linker-related energy loss in Co-conjugates (K')

Interestingly, it was observed that although 7D12-GS-9G8 bound more strongly to mouse EGFR than 7D12, it was 4-fold weaker than 9G8 (Table 12). In this case, linking the two binders together resulted in non-optimal binding of 9G8, which significantly counteracts the increase in binding affinity by forming a co-binder.

The crystal structures of 7D12 and 9G8 were first studied in detail, and their N-termini were found to be in close contact with the EGFR protein surface. The shortest distance of the C.alpha.atom of the first residue in 9G8 to EGFR isWhereas 7D12 has a shortest distance of +.>In both cases, the first amino acid is in direct contact with the antigen EGFR. Thus, the addition of linkers at their N-terminal ends may impair binding. To investigate whether the source of loss of affinity is the linker, we generated 7D12 and 9G8 variants with pass (GGGS) ₄ An anti-human lysozyme VHH HuL6 linker was attached to its N-terminus. Information about HuL6Can be in<https://www.ebi.ac.uk/pdbe/entry/pdb/1op9/protein/1>Where found. Although the linker is considered flexible, both HuL6-GS-7D12 and HuL6-GS-9G8 show a significant loss of binding affinity. N-terminal linker attachment resulted in 26-fold and 15-fold reduction in KD for human EGFR, respectively, compared to free VHH (Table 14). Construct 7D12-GS-HuL6, with opposite orientation, showed similar binding affinity to free 7D12, indicating that the loss of affinity was not due to any interference of HuL6, but rather was due to the linker at its N-terminus.

Table 14 binding affinities of free 7D12, 9G8 (as individual single conjugates) and their HuL6 linked variants as measured by Biacore at 25 ℃.

In the case of co-conjugate 7D12-GS-9G8, the linker-related loss of affinity became more discernable as mouse EGFR bound much weaker than human EGFR (tables 12 and 15). Similar to the HuL6 fusion construct, 9G8 in this co-binder construct also lost affinity due to linker attachment, which resulted in weaker affinity than 9G8 alone. In addition, the reverse co-conjugate 9G8-GS-7D12 was also constructed and its affinity for mouse EGFR was measured (Table 15). Binding affinity was 2-fold weaker than 9G8 alone. In this case 7D12 was provided as the second conjugate, its contribution to the binding was negligible, possibly also due to the impaired binding of the linker, which is consistent with the affinity loss seen in HuL6-GS-7D12 measurements.

TABLE 15 measurement of binding of the single binders 7D12, 9G8 and co-binders to human and mouse EGFR by Biacore at 25 ℃

Constructs	KD (muEGFR (mouse EGFR), M)
		7D12	7.2x10 ^-6
9G8	2.9x10 ^-8
		7D12-GS-9G8	1.6x10 ^-7
9G8-GS-7D12	5.8x10 ^-8

Example 3: strategies for limiting energy (and therefore affinity) loss (K') in co-binders

Various linker designs were explored that can minimize linker-related energy loss (even if the K' factor was minimized) so that lower affinity binders could be used for co-binder formation and still obtain maximum affinity gain.

To address the problem of linker proximity interference, three strategies are devised herein to address the linker-related energy loss, the core idea of which is to physically move the N-terminal linker attachment point away from the antigen binding interface. To achieve this, the amino acids can be trimmed from the N-terminus of the conjugate linked by the N-terminus (fig. 4A); alternatively, an amino acid motif may be inserted that will create a structural element, such as a hairpin, that will move the N-terminus away from the antigen binding interface; alternatively, two of the foregoing strategies may be combined, a portion of the original binding amino acid sequence trimmed and the amino acid motif inserted.

A series of HuL6-7D12 and HuL6-9G8 constructs were created, with 1 to 15 residues truncated at the N-terminus of the second conjugate. The proteins were expressed from yeast secretory strains, purified, and then analyzed by SDS-PAGE (FIGS. 4B and 4C). All co-conjugates containing truncations were similar in mobility to the non-truncated co-conjugate, indicating that the protein produced was intact.

It is important to emphasize that in the above strategy the exact number of amino acids trimmed from the N-terminus of the conjugate or the exact amino acid sequence of the insertion motif need not be known in advance. Instead, one can create a combinatorial library of solutions using the described strategy for engineering the linker attachment points, where the most successful binders can be selected using library screening methods such as phage display, yeast display, ribosome display, or equivalent methods. This does not exclude the possibility of creating universal, engineered linker attachment points within the same class of antibody scaffold or within many or all classes of antibody scaffolds.

To further investigate the linker-related affinity loss, the crystal structures of 7D12 and 9G8 were examined in detail. Both the N-termini of 7D12 and 9G8 were found to be very close to the EGFR protein surface (fig. 3E). The C.alpha.atom of the first residue in 9G8 is at a distance from EGFR ofAnd 7D12 is +.>Similar to the length of one amino acid, there is no room left for free attachment of the linker peptide. Thus, the addition of linkers at their N-terminus is expected to result in binding interference and loss of affinity.

Where the source of affinity loss is understood to be the proximity of the linker to the antigen, truncations from the N-terminus of the second conjugate are designed to move the linker anchor point away from the antigen surface.

Above the truncation of the N-terminus of the second conjugate, an amino acid motif may be inserted prior to the truncation of the second conjugate, which will introduce structural flexibility, such as glycine residues, or create structural elements, such as hairpins, that move the N-terminus away from the antigen surface.

To demonstrate that these strategies can be used to minimize linker-related affinity loss, identical N-terminally modified co-conjugates of HuL 6-fusion and second conjugate were constructed and compared for their affinity to unmodified constructs. For each single binding compound modified at the second position, the first residue is cleaved and ligated to the Phe/Thr/Gly motif prior to ligation to the C-terminus of the (GGGS) 4 linker. For 7D12, the modified HuL6 fusion HuL6-GS-FTG- [ -1AA ]7D12 had a 7-fold increase in binding affinity compared to the unmodified HuL6-GS-7D12 (Table 16). For 9G8, the modified HuL6 fusion HuL6-GS-FTG- [ -1AA ]9G8 had a 31-fold improvement over HuL6-GS-9G8 (Table 16). In the case of the co-conjugate, the N-terminally modified construct 7D12-GS-FTG- [ -1AA ]9G8 showed a 133-fold improvement in affinity for mouse EGFR compared to the unmodified reference 7D12-GS-9G8 (Table 16).

TABLE 16 measurement of binding of human or mouse EGFR by Biacore N-terminally modified and unmodified HuL6 fusions and co-conjugates at 25 ℃

Example 4: joint contributing to a reduction of energy loss (K')

To demonstrate that the energy loss and thus K' caused by adaptor proximity interference can be minimized, four unique libraries of co-binders were created, including a first VHH binder, in particular HuL6 (Dumoulin et al, protein Sci.2002, month 3; 11 (3): 500-15), followed by four GGGS repeated adaptors, followed by three fully randomized amino acids, and ending with a second VHH binder, 9G8 or 7D12, where the first amino acid of the second binder was unchanged or removed (FIG. 5).

The first and second single conjugates have intentionally different specificities. HuL6 is specific for human lysozyme and does not bind detectably to EGFR. In this example, huL6 was used as a nonfunctional conjugate to highlight the affinity improvement of EGFR binding for 7D12 and 9G 8. Four resulting libraries, each with 8000 diversity, were subjected to conventional yeast display screens (Chao et al, nat Protoc 1 (2): 755-768 (2006)). The engineered adaptor DNA sequence was read using next generation sequencing and translated into amino acid sequences and unique sequences were counted. The sequence enrichment factor is then calculated by dividing the count of each unique sequence in the last round of selection by the count of the previous round. Table 17 lists the top 20 sequences most enriched in each library and library combination.

Table 17: the first 20 triamino motifs from the N terminus of anti-EGFR VHH (9G 8 or 7D 12), with or without the first amino acid of VHH, pass (GGGS) ₄ The linker was attached to an anti-lysozyme VHH (HuL 6) and enriched in the screen to improve binding to EGFR.

Due to the proximity of the engineered linker sequence to the antigen, it is possible to inadvertently improve binding, not by reducing the energy loss associated with the linker, but by increasing the binding energy of the individual binders, i.e. affinity maturation of the binders is performed. To limit this possibility, sequences common to different binders were searched and identified, as the same sequences are unlikely to transmit specificity to different epitopes (Table 17, columns 9-14). In the case of comparing two or more libraries, the enrichment value of the lowest enrichment member is used. Motif analysis of the first 20 binders per library found that the frequency of repetition of certain amino acids was higher than expected at random. In addition, the same is true when comparing two or more libraries, such as where libraries 1 and 2 9g8 and 7D12 contain conjugates that retain the first amino acid (fig. 6A), libraries 4 and 5 g8 and 7D12 contain conjugates that remove the first amino acid (fig. 6A), and where all libraries are combined (fig. 6A). These findings indicate that the selection strategy has determined a limited number of solutions for the linker ligation site.

Example 5: properties of Co-conjugates engineered according to strategies for reducing energy loss (K')

To further verify that the identified solution translates into improved affinityDissociation constants of selected clones were measured using yeast display and SPR (fig. 6B). These measurements clearly show that linking the functional conjugates (9G 8 and 7D 12) to the non-functional conjugates (HuL 6) results in a loss of binding energy (compare the red and green bars in fig. 6B). Furthermore, if the conjugate is linked by its C-terminus remote from its antigen binding region, K is not caused _D Attenuation (compare green bar and gray bar in fig. 6B). Finally, by choosing the majority of solutions found resulted in improved binding, the best solution completely eliminated the observed energy loss due to interference in proximity of the linker (compare Huang Setiao with red and green bars in fig. 6B).

Example 6: directed evolution of linker libraries of co-binders

To further demonstrate that engineering of the linker linking sequence can be used to mitigate linker-induced energy loss, two anti-EGFR VHH binders (7D 12 and 9G 8) were linked to create a co-binder. To mitigate the negative splice effect, the same strategy described in fig. 4 was applied. Briefly, at the N-terminus of the second conjugate, the first amino acid is removed and a XXG randomization sequence is inserted, wherein X represents any amino acid and G represents glycine. The choice of glycine in this position is determined by the preference of this amino acid at this position which is not found in fig. 6A. In addition, the effect of linker length and rigidity on co-binder affinity was studied. For this purpose, four different linkers were selected: two alpha-helix forming motifs EAAAK and E ₄ K ₄ Repeat sequence, AP repeat sequence and G _3-4 S repeat sequence. As indicated in fig. 3B, rigid joints (i.e., EAAAK, E ₄ K ₄ And AP repeats) need to be of the correct length to not interfere with binding, for which the crossover is designed to spanIs provided for the different joint lengths of the connector. The C-terminus of 7D12 VHH is approximately +.about.>The pitch of the alpha helix is about +.>And AP repeat linker per amino acid +.>The length of the flexible linker does not appear to affect the radius of gyration of the co-conjugate (Klein et al Protein Eng Des sel.2014, 10 months; 27 (10): 325-30), therefore G _3-4 S repeat flexible linkers are designed to be long enough, but have limited length diversity (i.e., 24-25 amino acids). Furthermore, three additional fully randomized amino acids were inserted at the C-terminus of the first VHH to investigate the effect of C-terminal linker ligation on co-binder affinity (fig. 7A). A total of 4 libraries were created, 6X 10 each ⁶ -1×10 ⁷ The members: library 7-7D12-XXX- (EAAAK) _5-8 -XXG[-AA]9G8, library 8-7D12-XXX- (E) ₄ K ₄ ) _5-6 -XXG[-AA]9G8, library 9-7D12-XXX- (AP) _11-13- XXG[-AA]9G8 and library 11-7D12-XXX- (G) _3-4 S) _6-5 -XXG[-AA]9G8。

The resulting library was subjected to conventional yeast display selection (Chao et al, nat Protoc 1 (2): 755-768 (2006)). The selection of the strongest binders was performed by 3-4 rounds of consecutive selection, reducing the EGFR antigen concentration in each subsequent round. The DNA sequence of the engineered linker was read using next generation sequencing. The DNA sequence was trimmed, translated into amino acid sequences, and the unique sequences were counted. The sequence enrichment factor is calculated by dividing the count of the single unique sequence of the previous round by the count of the previous round.

Table 18: the first 20 triamino motifs from the N-and C-terminal of the anti-EGFR VHH 7D12-9G8 co-conjugate, through EAAAK, E ₄ K ₄ AP and G _3-4 S repeat ligation, enriched in selection to improve binding to EGFR.

In Table 18, the top 20 sequences most enriched in each library are listed. For all libraries, a number of different solutions were found from the point of view of the N-terminal and C-terminal ligation sites (FIG. 7B) (regarding the N-terminal and C-terminal ends of the linker), the linker length (FIG. 7C) and the linker composition (FIG. 8). No significant enrichment of any particular sequence was observed at the C-terminal ligation site.

Example 7: improvement of affinity and specificity by engineering of the N-terminus of the linker and the second binding moiety

To verify that the observed enrichment translates into improved binding properties, the 7D12-9G8 co-binders were selected for expression and their kinetic properties were measured using SPR. Because the affinity of these co-conjugates for human EGFR has exceeded the limit of detection of SPR, EGFR mouse homologs with significantly reduced binding strength were used instead for affinity measurements (compare fig. 8 green bars with fig. 6B green bars). To establish a baseline, the dissociation constants of several 7D12-9G8 co-conjugates with different linkers but without N-terminal or C-terminal linker ligation engineering were measured (fig. 8, red bars). Co-binders lacking either the N-terminal or C-terminal linker attachment sites did not improve the affinity of the antibodies, whereas binding was attenuated 2-21 fold relative to the stronger binder of the two single binders (9G 8). This loss is likely to be caused by the order of the two single bonds. In the 7D12-9G8 pair, the stronger conjugate 9G8 is linked by its N-terminus, which increases the likelihood of the linker interfering with binding. To determine if this problem can be solved by linker ligation site engineering, several solutions were expressed randomly chosen from all four libraries (fig. 8, orange bars). Most engineering solutions lead to binding improvement, of which the best two 7D12-FAS- (AP) ₁₁ -VGG-[-AA]9G8 and 7D12-ITA- (E4K 4) ₄ -IGG-[-AA]The 9G8 achieved a 300-900 fold improvement over the appropriate co-conjugate and a 40-50 fold improvement over the stronger conjugate (9G 8) of the two single conjugates. To further test the effect of N-terminal and C-terminal engineered ligation sites, a single N-terminal sequence-VSG [ -AA ] that was repeated in all 4 libraries was selected and tested](Table 18), and also tested for FTG [ -AA ] identified in FIG. 6A]Sequence (fig. 8, yellow bars). These N-terminally engineered 7D12-9G8 co-conjugates (FIG. 8, yellow bars) perform and perform bestRandomly selected N-terminal and C-terminal engineered co-conjugates (FIG. 8, orange bars) were comparable. In addition, VSG [ -AA]The increase in binding energy between co-conjugates is similar and independent of the linker used. Different types and lengths of linkers were observed from the co-conjugates, which showed improved binding affinity (table 19). To further verify the results, mutant human EGFR with reduced binding affinity was created by engineering, which can be measured more accurately by Biacore. Likewise, the same linker design achieves similar binding affinity enhancement as murine EGFR. As another indicator of improved joint design, the K' factors can be measured and they are significantly reduced in the case of engineered joints. These findings highlight the importance of engineering the linker ligation site (especially for sites near the antigen binding pocket), as linker interference may be the only greatest source of energy loss of the ligated co-conjugate. The results indicate that engineering of the linker sequence provided herein for the co-conjugate and the N-terminal sequence of the second binding moiety can result in excellent improvements in binding energy, greatly reduced K' factor, and thus excellent improvements in binding affinity and specificity.

Table 19 shows the linkers used for the co-conjugates with improved binding affinity.

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Example 8: screening method

Workflow

The general workflow is depicted in fig. 9. Single binder library SB0 is an unselected library comprising a single binding moiety. The sequence diversity in SB0 is high, at least 10 ⁵ But it is expected that most members will not bind to the target of interest. Thus, SB0 will first undergo selection for target binding, which results in a single binder library SB1 enriched in binders. A library of co-binders (CB 0) was then constructed by inserting a library of linkers (L) between randomly paired single binders, followed by SB1. CB0 was then further screened to generate the high affinity co-binder pool CB1. CB1 can then be paired by NGSThe co-binders in the library are sequenced and top enriched co-binders can be selected for protein production and affinity determination by SPR.

Single binders and sorted libraries

The initial single conjugate library SB0 may be a VHH or any protein conjugate used with display technology, including but not limited to well known antibodies and antibody fragments such as Fab and Fd, single chain variable fragments (scFv), single domain antibodies (sdAb)/heavy chain antibodies (HCAb)/V _H H. Affibody, affilin, affimer, affitin, alpha body, anticalin, affibody, DARPin, fork related domain, fynomer, kunitz domain peptide, monomer (Adnectin), minibody, nanoCLAMP, any peptide, peptidomimetic, antibody mimetic, or other binding scaffold. The display technology may also vary between ribosome display, bacterial display, yeast surface display, insect cell surface display, mammalian cell surface display, and the like. General Methods for constructing various SB0 libraries can be found in publications (Akamatsu et al J Immunol Methods 327:40-52 (2007); chao et al Nat Protoc 1 (2): 755-768 (2006); daugherty Cur Opin Struc Biol 17:474-480 (2007); ernst et al Nucleic Acids Res 26:1718-1723 (1998); ho et al Proc National Acad Sci 103:9637-9642 (2006); zahnd et al Nat Methods 4:269:279 (2007)). By way of example, RNA was isolated and VHH genes were extracted from Peripheral Blood Mononuclear Cells (PBMC) from either raw llamas or llamas immunized with EGFR according to the protocol described (Pardon et al, nat Protoc 9:674-693 (2014)). The SB0 library was constructed by cloning the VHH gene into a yeast surface display vector (Wang et al Protein Eng Des Sel (7): 337-343 (2005)). The induced SB0 yeast display library is stained with antigen and single binding compound, e.g., sorted by Fluorescence Activated Cell Sorting (FACS), as shown in fig. 10A.

In addition to FACS-based sorting methods, other methods can be used to separate binders from non-binders. For example, the target protein may be immobilized to a solid surface, such as a plastic surface or a magnetic bead surface in an ELISA plate, by direct conjugation, hydrophobic adsorption, or by capture through modification on a protein (such as biotin, protein or peptide tag, etc.). Library members that recognize the target protein will remain bound to the solid surface until the non-binding members are washed away. The binding member may then be eluted from the solid surface. Magnetic Activated Cell Sorting (MACS) can also be used for isolation. Using these methods, conjugates that recognize the target protein can be separated from other non-conjugates. The isolated population is enriched in conjugates and has only a small portion of SB0 diversity. To further enrich the binders and reduce diversity, it is often necessary to proliferate the isolated population and make multiple rounds of selection. Since it is expected that high affinity co-binders could be found by using the single binders selected, 2-3 rounds of selection may be preferred to generate a reasonably diverse SB1 library. Table 20 shows the first 32 enriched human EGFR binders from the immunized VHH library.

Table 20: the SB1 VHH yeast surface displayed the frequency of the most abundant CDR3 sequences in the library.

Co-conjugate library

Co-conjugate library CB0 can be constructed from single-conjugate library SB1 and linker library L. DNA sequences encoding the binding members of SB1 are extracted and amplified into a first single conjugate (B1) and a second single conjugate (B2), respectively. B1 is linked to the N-terminus of the linker and B2 is linked to the C-terminus. B1 and B2 may share the same pool of binder sequences, but differ in the region overlapping the yeast surface display vector, which is used for insertion of the co-binder gene.

The library of L sequences can be rationally designed to reduce linker-related energy loss and minimize K' factors. In the first example of linker library construction, a portion of the design rules known from the model system studies described above are applied by removing multiple N-terminal residues of the second conjugate (B2). In addition, rigid and flexible linkers with different lengths were used in library L. For flexible linkers, the number of amino acids ranged from 6 to 34, with combinations of GGS, GGGS, and GGGGS followed by 2 randomized positions (table 21). For rigid joints, the length varies between 12 and 32 (table 21). Different types of proline-rich sequences were used as building blocks, including XP and XPP motifs. Interspersed with the building blocks are two random residues that connect conjugate B1 and conjugate B2. Randomization was limited to a defined subset of amino acids to limit the sequence diversity of library CB0.

Table 21: linker sequences for constructing library L.

Watch 21 (Xue)

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* Amino acid code for randomized positions

The method for CB0 library enrichment is similar to that used for SB0 library selection, but the stringency of the selection needs to be controlled so that high affinity co-binders can be distinguished from weaker co-binders. The selection stringency can be controlled in several ways. First, as an example, the concentration of the target protein may be changed as shown in fig. 10B. The lower the concentration, the higher the stringency. High affinity binders can maintain good target binding at high stringency, but weaker binders cannot. Second, the washing time may vary. After incubating the library members with the target protein, unbound library members or proteins need to be washed away. The longer the washing time, the higher the stringency. In general, weaker binders can dissociate from the target protein during the washing step, but high affinity binders do not. Third, the time of incubation of the target protein may also vary. Strong binders tend to bind targets faster than weak binders. By limiting the incubation time, high affinity binders are better enriched than low affinity binders. After 3-4 rounds of more stringent selection, the resulting library CB1 will be enriched in high affinity co-binders.

Two methods can be used to determine which co-binders in CB1 will be selected for further analysis. First, clones in the CB1 library may be randomly picked and their sequences determined (e.g., table 22 below). The affinity of the selected co-binders can be analyzed by FACS. In addition, co-conjugate proteins may be produced and their binding affinities may be analyzed by SPR (e.g., table 23 below). Alternatively, NGS analysis may be performed for the last two rounds of CB1 selection, and the sequence enrichment factor may be calculated. The most enriched sequences will be selected for co-binder protein production and SPR analysis to determine their affinities.

Properties of the Co-conjugates screened

Co-conjugate clones selected from the CB1 library were sequenced by sanger sequencing. Each co-binder sequence contains a single binding domain at the N-terminus and a different binding domain at the C-terminus, which are linked by a linker sequence designed from the linker library shown in table 21. It was found that the co-conjugate was not formed by two single binding domains comprising the same CDR region, indicating that high affinity co-conjugates are most likely formed by single conjugates targeting different epitopes. Table 22: selected sequences of single binder B1 CDR sequences, linker sequences and single binder B2 CDR sequences of the unique co-binder clones enriched in library CB 1.

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The co-binder gene and its constituent single binder genes are subcloned into a yeast expression vector. The co-binder protein and the component single binder protein are expressed and purified. The binding affinity of each co-binder and component single binders was measured using a surface plasmon resonance method using purified binder proteins (table 23). The affinity range of the selected clones was 0.09 to 12nM, while the affinity range of the component single binders was weaker and more broadly 1.2 to 990nM, indicating that the co-binder screening methods provided herein were effective in producing co-binders with synergistic binding. For example, co-conjugate 3B7 with KD of 1.2nM consisted of two single conjugates with KD in the 100nM range, indicating a 100-fold improvement in affinity. Several co-binders with KD in the 100pM range include single binders with KD in the 10nM range. Thus, the screening methods provided herein do allow for the use of low affinity single binders to generate co-binders having affinities and specificities suitable for therapeutic and diagnostic applications. It is also interesting to know that those component single conjugates do not exist in a single conjugate library at very high frequencies. Thus, the single binding compound with the highest affinity is not necessarily the most suitable binding compound for producing a co-binding compound. This is consistent with previous observations that linking two high affinity single binders does not necessarily result in a co-binder with greatly improved affinity.

Table 23: affinity characterization of selected co-binders enriched from library CB1 and constituent single binders thereof.

In summary, provided and validated herein are screening methods that are capable of robustly, scalable, and predictably producing co-conjugates with synergistic co-binding.

Since the screening method incorporates a linker design that minimizes linker-related energy loss, it can utilize low affinity (e.g., 100 nM) single-binding compounds that target an epitope expansion pool in the antigen. Thus, the method has an unexpected and higher success rate in finding synergistic co-binders than the traditional method of first searching for high affinity single binders and then ligating them together with GS linkers.

Example 9: co-conjugate application

A co-binder with synergistic co-binding has a higher binding affinity and specificity than its constituent individual binders (e.g., antibody variable domains). Thus, the co-conjugate may be used in any application where a single conjugate may be used, including therapeutic, diagnostic, and research applications known to those skilled in the art.

Conjugated proteins

Co-conjugates can be modified by ligating protein domains, peptide tags and additional cysteine residues, or by substituting amino acids with those that carry specific chemical groups by artificial synthesis. These modified co-conjugates are conjugated to chemical molecules such as dyes, enzymes, cytotoxic agents, toxins, radioisotopes, nucleic acids, and the like.

Activity determination

The physical or chemical properties and biological functions of the co-conjugates can be characterized by various assays known in the art.

Diagnosis and imaging

The co-conjugate may be used to determine protein levels in a biological sample using classical immunohistological methods known to those skilled in the art. Overexpression of one or more antigens of interest can also be studied by measuring one or more antigens in biological fluids such as serum, cerebrospinal fluid, urine, saliva, or other body fluids. In particular, the synergistic co-conjugates can be used in pairs in sandwich immunoassays for detection of disease markers in biological fluids. Synergistic co-conjugates of the markers are also useful in medical imaging, such as Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI).

Therapeutic use

Co-conjugates with synergistic co-binding may be useful in therapeutic applications. For example, they are useful in the treatment of cancer, allergic or inflammatory diseases, ocular diseases, neurodegenerative diseases, and the like. Co-conjugates can be used to enhance specificity of therapeutic targeting to reduce off-target binding. This applies broadly to any treatment and is particularly useful when effective therapeutic agents such as chimeric antigen receptor T cells or NK cells and antibody drug conjugates are used. The co-conjugate may further be combined with additional functional conjugates to enhance delivery of therapeutic activity into the brain by crossing the blood brain barrier.

In an exemplary study, two anti-EGFR co-conjugates showed inhibition of EGF-induced signaling in tumor cells. A431 cells were cultured in serum-free medium for 24 hours and then treated with the drug for 1 hour. Cells were then stimulated with EGF for 15 min. The stimulated cells were then washed and lysed with cold PBS, followed by western blot experiments to detect the total phosphorylated EGFR molecules. An exemplary result is shown in fig. 11. The results demonstrate that the anti-EGFR co-conjugates identified herein can inhibit EGFR signaling.

Enhancement of therapeutic efficacy

Co-conjugates can be used to enhance therapeutic efficacy. For example, a single binding compound may perform a weak but active biological function, but not exceed an effective or specific threshold. Combining them with other inactive single binders to form co-binders can enhance binding affinity and specificity as well as biological function, crossing the threshold of effectiveness and specificity.

Elimination of synergistic effects of Co-conjugates

The synergistic effect of the co-conjugates is produced by linking two single conjugates. This enhanced binding synergy can be eliminated by specifically degrading the linker portion of the co-conjugate. For example, the linker may contain a sequence motif that is recognized by endopeptidases. The co-conjugate is treated with endopeptidase to cleave the linker peptide and convert the strong co-conjugate into two weaker single conjugates. Such treatment may be useful where modulation of co-conjugate activity is desired, e.g., chimeric antigen receptor therapy, imaging, etc.

Example 10: searching for non-overlapping epitope pairs

Any conventional method can be used to screen for non-interfering pairs of protein conjugates, including single binding moieties and co-conjugates. FIGS. 12A-12C show examples of screening of non-interfering pairs of protein conjugates. Briefly, three EGFR conjugates were screened by surface plasmon resonance with their cognate antigen EGFR as ligand. EGFR conjugates flowed one after the other and the signal changes of the sensorgram were observed. Figures 12A-12B show two examples of non-interfering EGFR conjugates. Namely, 1E10 and 15E2 form a pair (FIG. 12A), and 7D12-9G8 and 15E2 form a pair (FIG. 12B). However, the 7D12-9G8 and 1E10 protein conjugates did not form a pair as shown in fig. 12C, because no additional binding (no additional response signal) was detected when the second EGFR conjugate was injected.

Example 11: n-terminal proximity is widely observed in antibodies

Typically, the paratope of an antigen binding fragment, including the VHH and VH or VL of a Fab, is near the N-terminus of such an antigen binding fragment. Ligating a linker peptide to the N-terminus of such an antigen binding fragment can result in energy loss due to the linker interfering with the binding site of the antibody. To determine the likelihood of the linker interfering with epitope-complementary phase interactions, the distance of the N-terminus of the antibody to the antigen surface was measured. 133 unique structures of the VHH antigen complex and 60 structures of Fab and its cognate antigen were selected from the protein database. The distance from the first C alpha atom (N-terminal) of VHH or Fab (including VH and VL) to the nearest non-hydrogen atom on the antigen surface was measured and plotted (fig. 13). Notably, about 25% of the VHH has an N-terminal distance antigen less than This is considered to be the contact made. Assuming that the linker can make a tight beta or gamma turn when attached to the N-terminus of the 2 nd single conjugate, a minimum radius of +.>Therefore, there is a need for +.>The minimum gap is used to attach the 2 nd single bond without interference from the linker. However, 42% of the VHHs (56 out of 133) are less than +.>With a risk of collision. According to the same threshold, 9% of the heavy chains and 15% of the light chains in Fab are so close to their antigens. Due to structural conservation, this problem can be generalized to all immunoglobulin (Ig) folds. Ig folding is a common and very conserved scaffold for antigen receptors, including standard antibodies from vertebrates (e.g. IgA, igD, igE, igG, igM, igY, igW, etc.), VHH domains (HCAbs) of camelid heavy chain antibodies, V-NAR domains (IgNAR) of cartilage fish immunoglobulin neoantigen receptors, and T Cell Receptors (TCRs).

Example 12: generation of anti-HIV p24 co-conjugates

By using the screening method in example 8, high affinity anti-HIV p24 co-binders were found. Briefly, an initial and immunized single-binding yeast surface display library SB0 was constructed and used to select p 24-binding clones. Based on the single binder library SB1 selected, a co-binder library CB0 was constructed by inserting the same linker library L between B1 and N-terminally truncated B2 (Table 21). To avoid binding interference and thus enhance affinity improvement, zero to three N-terminal residues of B2 are deleted prior to ligation to the linker sequence. The pool CB0 was subjected to multiple rounds of sorting to enrich for high affinity binders. Individual yeast clones expressing the anti-p 24 co-binders were selected and characterized (table 24).

Table 24: anti-HIV p24 co-binder sequences as measured by Biacore and binding kinetics and affinity thereof.

Example 13: affinity improvement of co-binders containing N-terminal truncations

The anti-HIV p24 co-binder gene and its component single binders B1 and B2 were subcloned into yeast expression vectors. The co-binder protein and the component single binder protein are expressed and purified. The binding affinity of each co-binder and component single binders was measured using a surface plasmon resonance method using purified binder proteins (table 25). In summary, the co-conjugates generated by the N-terminal truncation method showed greatly improved affinity over single conjugates (FIG. 14). The median of the combined anti-EGFR and anti-HIV p24 co-conjugate was 215pM, with 200-fold stronger affinity than the median of the combined single conjugate (48 nM).

Table 25: affinity measurements were performed on selected anti-HIV p24 co-binders and their constituent single binders by Biacore.

Example 14: other examples of high affinity co-binders discovered via screening methods

Libraries and screening methods based on N-terminally truncated co-binders are generally applicable. By applying the method, high affinity co-conjugates were generated for 14 different targets including EGFR and HIV p24 targets, cancer biomarkers, neurological disease biomarkers, cytokines, and a pair of therapeutic targets for treatment of neurodegenerative diseases (fig. 15). The median affinity of the strongest co-binder for each target, measured by Biacore, was 4pM. It should be noted that due to instrument limitations, these measurements are limited to 10 ^-6 s ^-1 Is not limited by the dissociation rate of the polymer. Thus, the actual affinity may be higher than the measured affinity. When compared with the median antibody affinity from the structural antibody database (doi: 10.1093/nar/gkt 1043), the co-binders were 1,350-fold stronger than conventional non-engineered antibodies from the database, demonstrating the effectiveness of generating high affinity co-binders by the N-terminal truncation mechanism and related screening methods.

Claims

1. A co-conjugate comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site,

wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation ("N-terminal truncation").

Wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the N-terminally truncated antibody variable region.

2. The co-conjugate of claim 1, wherein the affinity of the co-conjugate for binding to the second target site is at least about 3-fold greater than the affinity of a control co-conjugate comprising an antibody variable domain without the N-terminal truncation.

3. The co-conjugate of claim 1 or claim 2, wherein the first target site and the second target site are non-overlapping binding sites on a target molecule.

4. A co-conjugate according to claim 3, wherein the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold greater than the affinity of a control co-conjugate comprising an antibody variable domain without the N-terminal truncation.

5. The co-conjugate of any one of claims 1-4, wherein the first binding moiety is a first antibody moiety.

6. The co-conjugate of claim 5, wherein the first antibody moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragments.

7. The co-conjugate of claim 5, wherein the first antibody moiety is a single domain antibody.

8. The co-conjugate of any one of claims 1-7, wherein the second antibody moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragments.

9. The co-conjugate of claim 8, wherein the N-terminal truncated antibody variable domain is a truncated VH or truncated VL domain.

10. The co-conjugate of any one of claims 1-7, wherein the second antibody moiety is a single domain antibody.

11. The co-conjugate of claim 10, wherein the N-terminally truncated antibody variable domain is a truncated V _H H domain.

12. The co-conjugate of any one of claims 5-11, wherein the first binding moiety comprises a first V _H An H domain; wherein the second binding moiety comprises a second V having an N-terminal truncation _H H domain (truncated V) _H H domain "),

wherein the first V _H The C-terminal end of the H domain is connected to the second V via a linker _H N-terminal ligation of H domains.

13. The co-conjugate of any one of claims 1-12, wherein the N-terminal truncation of the N-terminally truncated antibody variable domain is from about 1 to about 25 amino acids.

14. The co-conjugate of claim 13, wherein the N-terminal truncation of the N-terminal truncated antibody variable domain is 1 amino acid.

15. The co-conjugate of any one of claims 1-14, wherein the linker is a peptide linker.

16. The co-conjugate of claim 15, wherein the C-terminal amino acid of the peptide linker directly linked to the N-terminal truncated antibody variable domain is G.

17. The co-conjugate of claim 15 or 16, wherein the three C-terminal amino acids of the peptide linker directly linked to the N-terminal truncated antibody variable domain are X ₁ -X ₂ -X ₃ ，

Wherein X is ₁ V, L, W, P, S, G, K, D, F, M, T, N or R;

X ₂ v, A, L, S, G, R, K, M, C, F, T, P or E; and is also provided with

X ₃ Is G.

18. The co-conjugate of any one of claims 15-17, wherein the linker comprises (G _x S _y ) _n Wherein x is 1 to 5, y is 0 to 5, and n is 1 or greater.

19. The co-conjugate of any one of claims 15-17, wherein the linker comprises [ EAAAK] _n Or [ EEEEKKKK] _n Wherein n is 1 or greater.

20. The co-conjugate of any one of claims 15-17, wherein the linker comprises [ AP] _n Wherein n is 1 or greater.

21. The co-conjugate of any one of claims 15-20, wherein the linker is no more than about 40 amino acids in length.

22. The co-conjugate of any one of claims 1-21, wherein the truncated variable domain is from an antibody variable domain of any one of IgG, igA, igE, igM or IgD types.

23. The co-conjugate of any one of claims 1-22, wherein the co-conjugate further comprises a third binding moiety that specifically recognizes a third target site.

24. The co-conjugate of claim 23, wherein the third binding moiety is a third antibody moiety.

25. The co-conjugate of claim 24, wherein the third antibody portion comprises an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain").

26. The co-conjugate of claim 25, wherein the third antibody moiety is linked to the second antibody moiety via a linker through the N-terminus of the N-terminal truncated antibody variable domain of the third antibody moiety.

27. The co-conjugate of claim 25, wherein the third antibody moiety is linked to a fourth binding moiety via a linker through the N-terminus of the N-terminal truncated antibody variable domain of the third antibody moiety.

28. The co-conjugate of any one of claims 1-27, wherein the co-conjugate is an antibody comprising an Fc region.

29. The co-conjugate of any one of claims 1-27, wherein the co-conjugate is a chimeric antigen receptor ("CAR").

30. A co-conjugate comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site,

wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain;

Wherein the first binding moiety is linked to the second binding moiety via a peptide linker through the N-terminus of the antibody variable domain;

wherein the three amino acids at the C-terminal end of the peptide linker directly linked to the antibody variable domain of the second binding moiety is X ₁ -X ₂ -X ₃ ，

Wherein X is ₁ Is any amino acid;

X ₂ k, R, Y, M, G or N; and is also provided with

X ₃ R, G, Y or P.

31. The co-conjugate of claim 30, wherein the affinity of the co-conjugate to bind to the second target site is at least about 3-fold greater than the affinity of a linker control co-conjugate.

32. The co-conjugate of claim 30 or claim 31, wherein the first target site and the second target site are non-overlapping binding sites on a target molecule.

33. The co-conjugate of claim 32, wherein the affinity of the co-conjugate for binding to the target molecule is at least about 3-fold greater than the affinity of a linker control co-conjugate.

34. The co-conjugate of any one of claims 30-33, wherein the first binding moiety is a first antibody moiety.

35. The co-conjugate of claim 34, wherein the first antibody moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragments.

36. The co-conjugate of claim 34, wherein the first antibody moiety is a single domain antibody.

37. The co-conjugate of any one of claims 30-36, wherein the second antibody moiety is selected from the group consisting of: fab, fv, scFv, dsFv, fab 'or (Fab') 2 fragments.

38. The co-conjugate of claim 37, wherein the antibody variable domain is a VH or VL domain.

39. The co-conjugate of any one of claims 30-36, wherein the second antibody moiety is a single domain antibody.

40. The co-conjugate of claim 39, wherein the antibody variable domain is V _H H domain.

41. The co-conjugate of any one of claims 30-40, wherein the first binding moiety comprises a first V _H An H domain; wherein the second binding moiety comprises a second V _H The H domain is a sequence of amino acids,

wherein the first V _H The C-terminal end of the H domain is connected to the second V via the peptide linker _H N-terminal ligation of H domains.

42. The co-conjugate of any one of claims 30-41, wherein the linker comprises (G _x S _y ) _n Wherein x is 1 to 5, y is 0 to 5, and n is 1 or greater.

43. The co-conjugate of any one of claims 30-41, wherein the linker comprises [ EAAAK ] _n Or [ EEEEKKKK] _n Wherein n is 1 or greater.

44. The co-conjugate of any one of claims 30-41, wherein the linker comprises [ AP ]] _n Wherein n is 1 or greater.

45. The co-conjugate of any one of claims 30-44, wherein the linker is no more than about 40 amino acids in length.

46. The co-conjugate of any one of claims 30-45, wherein the co-conjugate further comprises a third binding moiety that specifically recognizes a third target site.

47. The co-conjugate of claim 46, wherein the third binding moiety is a third antibody moiety.

48. The co-conjugate according to claim 47, wherein the third antibody moiety comprises an antibody variable domain having an N-terminal truncation ("N-terminal truncated antibody variable domain").

49. The co-conjugate of claim 48, wherein the third antibody moiety is linked to the second antibody moiety via a linker through the N-terminus of the N-terminal truncated antibody variable domain of the third antibody moiety.

50. The co-conjugate of claim 48, wherein the third antibody moiety is linked to the fourth binding moiety via a linker through the N-terminus of the N-terminal truncated antibody variable domain of the third antibody moiety.

51. The co-conjugate of any one of claims 1-50, wherein the co-conjugate is an antibody comprising an Fc region.

52. The co-conjugate of any one of claims 1-50, wherein the co-conjugate is a chimeric antigen receptor ("CAR").

53. A library comprising a plurality of co-binders or a plurality of polynucleotides encoding a plurality of co-binders, each co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, wherein the first binding moiety is linked to the second binding moiety via a peptide linker through the N-terminus of the antibody variable domain, wherein at least two co-binders in the library differ from each other in peptide linker sequence.

54. The library of claim 53, wherein said first binding moiety is a first antibody moiety comprising an antibody variable domain.

55. The library of claim 53 or claim 54, wherein the diversity of the second binding moiety is at least about 20.

56. The library of claim 55, wherein the diversity of the first binding moiety is at least about 20.

57. The library of claim 56, wherein said library has a diversity of at least about 4000.

58. The library of any one of claims 53-57, wherein the antibody variable domain has an N-terminal truncation ("N-terminal truncated antibody variable domain").

59. A method of screening for co-binders that specifically bind to a second target site with a desired affinity, the method comprising:

(1) Contacting the library of any one of claims 53-58 with a target molecule comprising the second target site to form a complex between a co-conjugate that specifically binds to the target molecule and the target molecule, and

(2) Identifying a co-binder that binds to the second target site with a desired affinity.

60. A method of screening for co-conjugates that specifically bind to a target molecule with a desired affinity, the method comprising:

(1) Contacting the library of any one of claims 53-58 with the target molecule to form a complex between the co-conjugate that specifically binds to the target molecule and the target molecule, and

(2) Identifying a co-binder that binds to the target molecule with a desired affinity.

61. A method of increasing the binding affinity of a control co-binder that specifically binds to a target molecule, wherein the control co-binder comprises a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, wherein the first binding moiety is linked to the second binding moiety via a linker through the N-terminus of the antibody variable domain, wherein the control co-binder comprises a full-length antibody variable domain, wherein the binding affinity of the control co-binder to the second target site is lower than the affinity of the second antibody moiety in the free state, the method comprising obtaining a co-binder having an N-terminal truncation at the antibody variable region of the second antibody moiety compared to the control co-binder.

62. The method of claim 61, wherein the first target site and the second target site are non-overlapping binding sites on a target molecule.