WO2019183406A1

WO2019183406A1 - Multispecific antibody purification with ch1 resin

Info

Publication number: WO2019183406A1
Application number: PCT/US2019/023447
Authority: WO
Inventors: Qufei LI; Lucas Bailey; Bryan Glaser; Roland Green
Original assignee: Invenra Inc.
Priority date: 2018-03-21
Filing date: 2019-03-21
Publication date: 2019-09-26

Abstract

Methods for purifying multivalent antibody constructs, antibody constructs purified using the methods, and pharmaceutical compositions comprising the purified constructs, and methods of treatment of the purified constructs are presented.

Description

Multispecific Antibody Purification with CHI Resin

1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Not Applicable

2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated herein by reference in its entirety. Said ASCII copy, created on Month XX, 20XX, is named XXXXXWO_sequencelisting.txt, and is

X, XXX, XXX bytes in size.

3. BACKGROUND

[0003] Antibodies are an invaluable tool in the medical field. In particular, the importance of monoclonal antibodies, including their roles in scientific research and medical diagnostics, have been widely recognized for several decades. However, the full potential of antibodies, especially their successful use as therapeutic agents, has only more recently been demonstrated, as demonstrated by the successful therapies adalimumab (Humira), rituximab (Rituxan), infliximab (Remicade), bevacizumab (Avastin), trastuzumab

(Herceptin), pembrolizumab (Keytruda), and ipilimumab (Yervoy). Following these clinical successes, interest in antibody therapies will likely only continue to increase. Therefore, a need for efficient generation and manufacturing of antibodies exists in the field, both in the research drug development and downstream clinical settings.

[0004] An area of active research in the antibody therapeutic field is the design and use of multispecific antibodies, i.e. a single antibody engineered to recognize multiple targets. These antibodies offer the promise of greater therapeutic control. For example, a need exists to improve target specificity in order to reduce the off-target effects associated with many antibody therapies, particularly in the case of antibody-based immunotherapies. In addition, multispecific antibodies offer new therapeutic strategies, such as synergistic targeting of multiple cell receptors, especially in an immunotherapy context.

[0005] Despite the promise of multispecific antibodies, their production and use has been plagued by numerous constraints that have limited their practical implementation. In general, all multispecific antibody platforms must solve the problem of ensuring high fidelity pairing between cognate heavy and light chain pairs. However, a multitude of issues exist across the various platforms. For example, antibody chain engineering can result in poor stability of assembled antibodies, poor expression and folding of the antibody chains, and/or generation of immunogenic peptides. Other approaches suffer from impractical manufacturing processes, such as complicated in vitro assembly reactions or purification methods. In addition, several platforms suffer from the inability to easily and efficiently plug in different antibody binding domains. These various problems associated with multispecific antibody manufacturing limit the applicability of many platforms, especially their use in high-throughput screens necessary for many therapeutic drug pipelines.

[0006] There is, therefore, a need for an antibody platform capable of high-level expression and efficient purification. In particular, there is a need for a multispecific antibody platform that improves the manufacturing capabilities of multispecific antibodies with direct applicability in both research and therapeutic settings.

4. SUMMARY

[0007] We have developed a novel method of purifying multivalent antibody constructs that have specific antibody architectures. The architecture of these antigen-binding CH1- substituted proteins drives high fidelity pairing of the cognate polypeptide chains that together form the antigen binding sites of monospecific, bispecific, trispecific, and tetraspecific constructs. We have found that these constructs can be purified in a single- step with CH1 affinity resins. The binding molecules are readily expressed using conventional antibody expression systems, including in vitro cell-free translation systems and mammalian transient transfection systems. High fidelity assembly, high level in vitro and mammalian expression, and the ability to purify expression products in a single step make these constructs well-suited to high throughput screening of variable region libraries

[0008] According, in a first aspect, disclosed herein is a method of purifying an antigen binding CH1 -substituted protein, the method comprising the steps of: i) contacting a sample comprising the antigen-binding CH1 -substituted protein with a CH1 binding reagent, wherein the antigen-binding CH1- substituted protein comprises at least a first, a second, a third, and a fourth polypeptide chain associated in a complex, wherein the complex comprises at least one CH1 domain, or portion thereof, and wherein the number of CH1 domains in the complex is at least one fewer than the valency of the complex, and wherein the contacting is performed under conditions sufficient for the CH1 binding reagent to bind the CH1 domain, or portion thereof; and ii) purifying the complex away from one or more incomplete complexes, wherein the incomplete complexes do not comprise the first, the second, the third, and the fourth polypeptide chain. [0009] In certain aspects, the antigen-binding CH1- substituted protein has a single CH1 domain, or portion thereof.

[0010] In certain aspects, (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C- terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and wherein domain B, domain D, and domain E have a constant region domain amino acid sequence; (b) the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence; (c) the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C-terminus, in a H-I-J-K orientation, and wherein domain H has a variable region domain amino acid sequence, domain I has a CL amino acid sequence, and domains J and K have a constant region domain amino acid sequence; (d) the fourth polypeptide chain comprises a domain L and a domain M, wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and wherein domain L has a variable region domain amino acid sequence, and wherein domain M is the single CH1 domain, or portion thereof; (e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains; (f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains; (g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen-binding CH1 -substituted protein.

[0011] In certain aspects, (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C- terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and wherein domain B, domain D, and domain E have a constant region domain amino acid sequence; (b) the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence; (c) the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C-terminus, in a H-I-J-K orientation, and wherein domain I is the single CH1 domain, or portion thereof, domain H has a variable region domain amino acid sequence, and domains J and K have a constant region domain amino acid sequence; (d) the fourth polypeptide chain comprises a domain L and a domain M, wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and wherein domain L has a variable region domain amino acid sequence, and wherein domain M has a CL amino acid sequence; (e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains; (f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains; (g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen-binding CH1 -substituted protein.

[0012] In certain aspects, domain B and domain G have a CH3 amino acid sequence. In certain aspects, the amino acid sequences of the B and the G domains are identical, wherein the sequence is an endogenous CH3 sequence

[0013] In certain aspects, the amino acid sequences of the B and the G domains are different and separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, wherein the B domain interacts with the G domain, and wherein neither the B domain nor the G domain significantly interacts with a CH3 domain lacking the orthogonal modification In certain aspects, the orthogonal modifications of the B and the G domains comprise mutations that generate engineered disulfide bridges between domain B and G. In certain aspects, the mutations of the B and the G domains that generate engineered disulfide bridges are a S354C mutation in one of the B domain and G domains, and a 349C in the other domain. In certain aspects, the orthogonal modifications of the B and the G domains comprise knob-in-hole mutations. In certain aspects, the knob-in hole mutations of the B and the G domains are a T366W mutation in one of the B domain and G domain, and a T366S, L368A, and aY407V mutation in the other domain. In certain aspects, wherein the orthogonal modifications of the B and the G domains comprise charge- pair mutations. In certain aspects, the charge-pair mutations of the B and the G domains are a T366K mutation in one of the B domain and G domain, and a L351D mutation in the other domain. [0014] In certain aspects, domain B and domain G have an IgM CH2 amino acid sequence or an IgE CH2 amino acid sequence. In certain aspects, the IgM CH2 amino acid sequence or the IgE CH2 amino acid sequence comprise orthogonal modifications. In certain aspects, domain E and domain K have a CH3 amino acid sequence. In certain aspects, the amino acid sequences of the E and K domains are identical, wherein the sequence is an

endogenous CH3 sequence. In certain aspects, the amino acid sequences of the E and the K domains are different. In certain aspects, the different sequences of the E and the K domains separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, wherein the E domain interacts with the K domain, and wherein neither the E domain nor the K domain significantly interacts with a CH3 domain lacking the orthogonal modification. In certain aspects, the orthogonal modifications of the E and the K domains comprise mutations that generate engineered disulfide bridges between domain E and K. In certain aspects, wherein the mutations of the E and the K domains that generate engineered disulfide bridges are a S354C mutation in one of the E domain and K domain, and a 349C in the other domain.

[0015] In certain aspects, the orthogonal modifications in the E and K domains comprise knob-in-hole mutations. In certain aspects, the knob-in hole mutations of the E and the K domains are a T366W mutation in one of the E domain or K domain and a T366S, L368A, and aY407V mutation in the other domain.

[0016] In certain aspects, the orthogonal modifications of the E and the K domains comprise charge-pair mutations. In certain aspects, the charge-pair mutations of the E and the K domains are a T366K mutation in one of the E domain or K domain and a

corresponding L351D mutation in the other domain.

[0017] In certain aspects, (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C- terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, wherein domain B and domain D have a constant region domain amino acid sequence, and wherein domain E is the single CH1 domain, or portion thereof; (b) the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence; (c) the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C- tenninus, in a H-I-J-K orientation, and wherein domain H has a variable region domain amino acid sequence, and domains I and J have a constant region domain amino acid sequence, and wherein domain K has a CL amino acid sequence; (d) the fourth polypeptide chain comprises a domain L and a domain M, wherein the domains are arranged, from N- terminus to C-terminus, in a L-M orientation, and wherein domain L has a variable region domain amino acid sequence, and wherein domain M has a constant region domain amino acid sequence; (e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains; (f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains; (g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen binding CH1- substituted protein.

[0018] In certain aspects, (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C- terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and wherein domain B and domain D have a constant region domain amino acid sequence, and wherein domain E has a CL amino acid sequence, (b) the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence; (c) the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C- terminus, in a H-I-J-K orientation, and wherein domain H has a variable region domain amino acid sequence, and domains I and J have a constant region domain amino acid sequence, and wherein domain K is the single CH1 domain, or portion thereof; (d) the fourth polypeptide chain comprises a domain L and a domain M, wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and wherein domain L has a variable region domain amino acid sequence, and wherein domain M has a constant region domain amino acid sequence; (e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains; (f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains; (g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen-binding CH1 -substituted protein.

[0019] In certain aspects, domain A has a VL amino acid sequence and domain F has a VH amino acid sequence. In certain aspects, domain A has a VH amino acid sequence and domain F has a VL amino acid sequence. In certain aspects, domain H has a VL amino acid sequence and domain L has a VH amino acid sequence. In certain aspects, domain H has a VH amino acid sequence and domain L has a VL amino acid sequence.

[0020] In certain aspects, domain D and domain J have a CH2 amino acid sequence.

[0021] In certain aspects, the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, and the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen.

[0022] In certain aspects, the first polypeptide chain or the third polypeptide chain further comprises a domain N and a domain O, wherein domain N has a variable region domain amino acid sequence, wherein domain O has a constant region amino acid sequence, wherein domains N and O are arranged, from N-terminus to C-terminus, in a N-0 orientation, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain A of the first polypeptide chain or to the N-terminus of domain H of the third polypeptide chain; the binding molecule further comprises a fifth polypeptide chain, comprising: a domain P and a domain Q, wherein the domains are arranged, from N- terminus to C-terminus, in a P-Q orientation, and domain P has a variable region domain amino acid sequence and domain Q has a constant region amino acid sequence; and either the first or third polypeptide chain is associated with the fifth polypeptide chain through an interaction between the N and the P domains and an interaction between the O and the Q domains to form the binding molecule. In some embodiments, the first polypeptide chain further comprises domain N and domain O, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain A of the first polypeptide chain. In some embodiments, the third polypeptide chain further comprises domain N and domain O, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain H of the third polypeptide chain.

[0023] In some embodiments, (a) the amino acid sequences of domain N and domain A are identical, the amino acid sequences of domain H is different from domains N and A, the amino acid sequences of domain O and domain B are identical, the amino acid sequences of domain I is different from domains O and B, the amino acid sequences of domain P and domain F are identical, the amino acid sequences of domain L is different from domains P and F, the amino acid sequences of domain Q and domain G are identical, the amino acid sequences of domain M is different from domains Q and G; and (b) wherein the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for the first antigen.

[0024] In some embodiments, (a) the amino acid sequences of domain N, domain A, and domain H are different, the amino acid sequences of domain O, domain B, and domain I are different, the amino acid sequences of domain P, domain F, and domain L are different, and the amino acid sequences of domain Q, domain G, and domain M are different; and (b) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for a third antigen.

[0025] In some embodiments, domain N, domain A, and domain H each comprise a VL amino acid sequence, domain P, domain F, and domain L each comprise a VH amino acid sequence, domain O and domain Q each comprise a CH3 amino acid sequence, domain B and domain I each comprise a CL amino acid sequence, and domain G and domain M each comprise a CH1 amino acid sequence.

[0026] In some embodiments, domain N, domain A, and domain H are VL domains, domain P, domain F, and domain L are VH domains, domain O and domain Q are CH3 domains, domain B and domain I are CL domains, and domain G and domain M are CH1 domains.

[0027] In some embodiments, domain N, domain A, and domain H each comprise a VL amino acid sequence; domain P, domain F, and domain L each comprise a VH amino acid sequence; domain O and domain Q each comprise a CH3 amino acid sequence; domain B and domain G each comprise a CH3 amino acid sequence; domain I comprises a CL amino acid sequence; and domain M comprises a CH1 amino acid sequence.

[0028] In some embodiments, domain N, domain A, and domain H are VL domains;

domain P, domain F, and domain L are VH domains; domain O and domain Q are CH3 domains; domain B and domain G are CH3 domains; domain l is a CL domain; and domain M is a CH1 domain.

[0029] In some embodiments, the amino acid sequences of the O and the Q domains are identical, and the sequences of the O and the Q domains are endogenous CH3 sequences.

[0030] In some embodiments, the amino acid sequences of the O and the Q domains are different and separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, the O domain interacts with the Q domain, and neither the O domain nor the Q domain significantly interacts with a CH3 domain lacking the orthogonal modification.

[0031] In some embodiments, the orthogonal modifications of the O and the Q domains comprise mutations that generate engineered disulfide bridges between domain O and G.

In some embodiments, the mutations of the O and the Q domains that generate engineered disulfide bridges are a S354C mutation in one of the O domain and Q domains, and a 349C in the other domain.

[0032] In some embodiments, the orthogonal modifications of the O and the Q domains comprise knob-in-hole mutations. In some embodiments, the knob-in hole mutations of the O and the Q domains are a T366W mutation in one of the O domain and Q domain, and a T366S, L368A, and aY407V mutation in the other domain.

[0033] In some embodiments, the orthogonal modifications of the O and the Q domains comprise charge-pair mutations. In some embodiments, the charge-pair mutations of the O and the Q domains are a T366K mutation in one of the O domain and Q domain, and a L351D mutation in the other domain.

[0034] In certain aspects, the antigen-binding CH1- substituted protein further comprises: a sixth polypeptide chain, wherein: (a) the third polypeptide chain further comprises a domain R and a domain S, wherein the domains are arranged, from N-terminus to C- terminus, in a R-S-H-I-J-K orientation, and wherein domain R has a variable region domain amino acid sequence and domain S has a constant domain amino acid sequence; (b) the binding molecule further comprises a sixth polypeptide chain, comprising: a domain T and a domain U, wherein the domains are arranged, from N-terminus to C-terminus, in a T- U orientation, and wherein domain T has a variable region domain amino acid sequence and domain U has a constant domain amino acid sequence; and (c) the third and the sixth polypeptides are associated through an interaction between the R and the T domains and an interaction between the S and the U domains to form the binding molecule. [0035] In certain aspects, (a) the amino acid sequences of domain R and domain A are identical, the amino acid sequences of domain H is different from domain R and A, the amino acid sequences of domain S and domain B are identical, the amino acid sequences of domain I is different from domain S and B, the amino acid sequences of domain T and domain F are identical, the amino acid sequences of domain L is different from domain T and F, the amino acid sequences of domain U and domain G are identical, the amino acid sequences of domain M is different from domain U and G and (b) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for the first antigen.

[0036] In certain aspects, the antigen-binding CH1- substituted protein further comprises a second CH1 domain, or portion thereof.

[0037] In certain aspects, (a) the amino acid sequences of domain R and domain H are identical, the amino acid sequences of domain A is different from domain R and H, the amino acid sequences of domain S and domain I are identical, the amino acid sequences of domain B is different from domain S and I, the amino acid sequences of domain T and domain L are identical, the amino acid sequences of domain F is different from domain T and L, the amino acid sequences of domain U and domain M are identical, the amino acid sequences of domain G is different from domain U and M and (b) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for the second antigen. In certain aspects, the amino acid sequences of domain S and domain I are CH1 sequences. In certain aspects, the amino acid sequences of domain U and domain M are CH1 sequences.

[0038] In certain aspects, (a) the amino acid sequences of domain R, domain A, and domain H are different, the amino acid sequences of domain S, domain B, and domain I are different, the amino acid sequences of domain T, domain F, and domain L are different, and the amino acid sequences of domain U, domain G, and domain M are different; and (b) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for a third antigen. In certain aspects, the antigen binding CH1 -substituted protein further comprises a second CH1 domain, or portion thereof. In certain aspects, the amino acid sequences of domain S and domain I are CH1 sequences. In certain aspects, the amino acid sequences of domain U and domain M are CH1 sequences.

[0039] In certain aspects, the sequence that forms the junction between the A domain and the B domain is IKRTPREP or IKRTVREP. In certain aspects, the sequence that forms the junction between the F domain and the G domain is SSASPREP.

[0040] In certain aspects, at least one CH3 amino acid sequence has a C-terminal tripeptide insertion connecting the CH3 amino acid sequence to a hinge amino acid sequence, wherein the tripeptide insertion is selected from the group consisting of PGK, KSC, and GEC.

[0041] In certain aspects, the sequences are human sequences.

[0042] In certain aspects, at least one CH3 amino acid sequence is an IgG sequence. In certain aspects, the IgG sequences are IgGl sequences.

[0043] In certain aspects, at least one CH3 amino acid sequence has one or more isoallotype mutations. In certain aspects, the isoallotype mutations are D356E and L358M.

[0044] In certain aspects, at least one of the at least one CH1 domain comprises a human CH1 amino acid sequence, and wherein the CH1 binding reagent binds to a human CH1 epitope. In certain aspects, at least one of the at least one CH1 domain comprises an CH1 amino acid sequence selected from the group consisting of: an IgG CH1, an IgA CH1, an IgE CH1, an IgM CH1, and an IgD CH1. In certain aspects, at least one of the at least one CH1 domain comprises an IgG CH1 amino acid sequence. In certain aspects, the IgG CH1 amino acid sequence comprises an IgGl CH1 amino acid sequence.

[0045] In certain aspects, at least one of the at least one CH1 domain comprises an IgA CH1 amino acid sequence.

[0046] In certain aspects, at least one of the at least one CH1 domain comprises SEQ ID NO:23.

[0047] In certain aspects, at least one of the at least one CH1 domain comprises one or more orthogonal modifications.

[0048] In certain aspects, the orthogonal modifications comprise mutations that generate engineered disulfide bridges between the at least one CH1 domain and a CL domain, the mutations selected from the group consisting of: an engineered cysteine at position 138 of the CH1 sequence and position 116 of the CL sequence; an engineered cysteine at position 128 of the CH1 sequence and position 119 of the CL sequence, and an engineered cysteine at position 129 of the CH1 sequence and position 210 of the CL sequence. In certain aspects, the orthogonal modifications comprise mutations that generate engineered disulfide bridges between the at least one CH1 domain and a CL domain, wherein the mutations comprise and engineered cysteines at position 128 of the CH1 sequence and position 118 of a CL Kappa sequence. In certain aspects, the orthogonal modifications comprise mutations that generate engineered disulfide bridges between the at least one CH1 domain and a CL domain, the mutations selected from the group consisting of: a Fl 18C mutation in the CL sequence with a corresponding A141C in the CH1 sequence; a Fl 18C mutation in the CL sequence with a corresponding L128C in the CH1 sequence; and a S162C mutations in the CL sequence with a corresponding P171C mutation in the CH1 sequence.

[0049] In certain aspects, the orthogonal modifications comprise charge-pair mutations between the at least one CH1 domain and a CL domain, the charge-pair mutations selected from the group consisting of: a Fl 18S mutation in the CL sequence with a corresponding A141L in the CH1 sequence; a Fl 18A mutation in the CL sequence with a corresponding A141L in the CH1 sequence; a Fl 18V mutation in the CL sequence with a corresponding A141L in the CH1 sequence; and a T129R mutation in the CL sequence with a

corresponding K147D in the CH1 sequence. In certain aspects, the orthogonal

modifications comprise charge-pair mutations between the at least one CH1 domain and a CL domain, the charge-pair mutations selected from the group consisting of: a N138K mutation in the CL sequence with a corresponding G166D in the CH1 sequence,; and a N138D mutation in the CL sequence with a corresponding G166K in the CH1 sequence.

[0050] In certain aspects, the CH1 binding reagent comprises an anti-CHl antigen binding site. In certain aspects, the CH1 binding reagent comprises an anti-CHl antibody. In certain aspects, the anti-CHl antibody comprises a single-domain antibody. In certain aspects, the single-domain antibody comprises a Camelid-derived antibody.

[0051] In certain aspects, the CH1 binding reagent is attached to a surface of a solid support. In certain aspects, the solid support is selected from the group consisting of: an agarose bead, a magnetic bead, and a resin. In certain aspects, the CH1 binding reagent is attached to the surface prior to step (ii). In certain aspects, the CH1 binding reagent is attached to the surface subsequent to step (ii). [0052] In certain aspects, the purifying step is selected from the group consisting of:

magnetic isolation, column purification, bead centrifugation, resin centrifugation, flow cytometry, and combinations thereof.

[0053] In certain aspects, the method further comprises an elution step following step (ii) generating an eluate comprising antigen-binding CH1 -substituted protein. In certain aspects, the elution step comprises contacting the antigen-binding CH1- substituted protein bound to the CH1 binding reagent with a low-pH solution. In certain aspects, the low-pH solution comprises 0.1 M acetic acid pH 4.0.

[0054] In certain aspects, the method further comprises an additional purification step following the elution step. In certain aspects, the additional purification step comprises an ion exchange chromatography purification. In certain aspects, the ion exchange

chromatography purification comprises cation exchange chromatography.

[0055] In certain aspects, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% (w/w) of the total protein in the eluate is the antigen-binding CH1- substituted protein. In certain aspects, the greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% (w/w) of the total protein in the eluate is obtained following a single iteration of steps (i)-(iii In certain aspects, the greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% (w/w) of the total protein in the eluate is obtained using any of the above methods wherein the purifying step does not comprise use of a binding reagent other than the CH1 binding reagent of the methods disclosed or described herein.

[0056] In certain aspects, less than 5% of the first and the third polypeptides are

unassociated in the eluate. In certain aspects, less than 5% of the first and the second polypeptides are unassociated in the eluate. In certain aspects, less than 5% of the third and the fourth polypeptides are unassociated in the eluate. In certain aspects, the less than less than 5% of the first and the third polypeptides, the less than 5% of the first and the second polypeptides, or the less than 5% of the third and the fourth polypeptides unassociated in the eluate is obtained following a single iteration of steps (i)-(iii). In certain aspects, the less than less than 5% of the first and the third polypeptides, the less than 5% of the first and the second polypeptides, or the less than 5% of the third and the fourth polypeptides unassociated in the eluate is obtained using any of the above methods wherein the purifying step does not comprise use of a binding reagent other than the CH1 binding reagent of the methods disclosed or described herein.

[0057] In certain aspects, the sample is a supernatant or a lysate of an expression system. In certain aspects, the expression system is selected from the group consisting of: a cell free expression system, a mammalian cell culture, a bacterial cell culture, a yeast cell culture. In certain aspects, the mammalian cell culture comprises an immortalized cell line. In certain aspects, the immortalized cell line is a chine hamster ovary (CHO) or human 293 derived cell line. In certain aspects, the expression system stably expresses the polypeptide chains of the antigen-binding CH1 -substituted protein. In certain aspects, the expression system is a serum-free expression system.

[0058] Also disclosed herein is an antigen-binding CH1 -substituted protein purified by any of the methods disclosed or described herein.

[0059] Also disclosed herein is a pharmaceutical composition comprising the antigen binding CH1 -substituted protein purified by any of the methods disclosed or described herein.

[0060] Also disclosed herein is a method of treatment, comprising administering to a subject in need of treatment the pharmaceutical composition comprising any of the antigen binding CH1 -substituted protein purified by any of the methods disclosed or described herein and any of the pharmaceutically acceptable carriers disclosed or described herein.

5. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Fig. 1 shows SDS-PAGE analysis of bispecific antibodies comprising standard knob-hole orthogonal mutations introduced into CH3 domains found in their native positions within the Fc portion of the bispecific antibody that have been purified using a single-step CH1 affinity purification step (Capture Select™ CH1 affinity resin).

[0062] FIG. 2 presents schematic architectures, with respective naming conventions, for various antigen-binding CH1 -substituted proteins (also called antibody constructs) described herein.

[0063] FIG. 3 presents a higher resolution schematic of polypeptide chains and their domains, with respective naming conventions, for the bivalent lxl antibody constructs described herein.

[0064] FIG. 4 illustrates features of an exemplary bivalent lxl bispecific antigen-binding CH1- substituted protein,“BC1”. [0065] FIG. 5A shows size exclusion chromatography (SEC) analysis of“BC1”, demonstrating that a single-step CH1 affinity purification step (CaptureSelect™ CH1 affinity resin) yields a single, monodisperse peak via gel filtration in which >98% is unaggregated bivalent protein. FIG. 5B shows comparative literature data of SEC analysis of a CrossMab bivalent antibody construct [data from Schaefer el al. ( Proc Natl Acad Sci USA. 2011 Jul 5; 108(27): 11187-92)].

[0066] FIG. 6A is a cation exchange chromatography elution profile of“BC1” following one-step purification using the CaptureSelect™ CH1 affinity resin, showing a single tight peak. FIG. 6B is a cation exchange chromatography elution profile of“BC1” following purification using standard Protein A purification.

[0067] FIG. 7 shows non-reducing SDS-PAGE gels of“BC1” at various stages of purification.

[0068] FIGS. 8A and 8B compare SDS-PAGE gels of“BC1” after single-step CH1- affmity purification under both non-reducing and reducing conditions (FIG. 8A) with SDS- PAGE gels of a CrossMab bispecific antibody under non-reducing and reducing conditions as published in the referenced literature (FIG. 8B).

[0069] FIGS. 9A and 9B show mass spec analysis of“BC1”, demonstrating two distinct heavy chains (FIG. 9A) and two distinct light chains (FIG. 9B) under reducing conditions.

[0070] FIG. 10 presents a mass spectrometry analysis of purified“BC1” under non reducing conditions, confirming the absence of incomplete pairing after purification.

[0071] FIG. 11 illustrates features of an exemplary bivalent lxl bispecific antigen-binding CH1- substituted protein,“BC6”, further described in Example 3.

[0072] FIG. 12A presents size exclusion chromatography (SEC) analysis of“BC6” following one-step purification using the CaptureSelect™ CH1 affinity resin,

demonstrating that the single step CH1 affinity purification yields a single monodisperse peak and the absence of non-covalent aggregates. FIG. 12B shows a SDS-PAGE gel of “BC6” under non-reducing conditions.

[0073] FIG. 13 illustrates features of an exemplary bivalent bispecific antigen-binding CH1- substituted protein,“BC28”, further described in Example 4.

[0074] FIG. 14 shows SEC analysis of“BC28” and“BC30”, each following one-step purification using the CaptureSelect™ CH1 affinity resin.

[0075] FIG. 15 presents a schematic of polypeptide chains and their domains, with respective naming conventions, for the trivalent 2x1 antibody constructs described herein. [0076] FIG. 16 illustrates features of an exemplary trivalent 2x1 bispecific antigen-binding CH1- substituted protein,“BCl-2xl”, further described in Example 7.

[0077] FIG. 17 shows non-reducing SDS-PAGE of“BC1” and“BCl-2xl” protein expressed using the ThermoFisher Expi293 transient transfection system, at various stages of purification.

[0078] FIG. 18 presents a schematic of polypeptide chains and their domains, with respective naming conventions, for the trivalent 1x2 antibody constructs described herein.

[0079] FIG. 19 illustrates features of an exemplary trivalent 1x2 trispecific construct, “BC28-lxlxla”, further described in Example 11.

[0080] FIG. 20 shows size exclusion chromatography of“BC28-lxlxla” following transient expression and single step CH1 affinity resin purification, demonstrating a single well-defined peak.

[0081] FIG. 21 presents a schematic of polypeptide chains and their domains, with respective naming conventions, for certain tetravalent 2x2 constructs described herein.

[0082] FIG. 22 illustrates certain salient features of the exemplary tetravalent 2x2 construct,“BC22-2x2” further described in Example 14.

[0083] FIG. 23 is a non-reducing SDS-PAGE gel comparing the 2x2 tetravalent“BC22- 2x2” construct to a 1x2 trivalent construct“BC 12-1x2” and a 2x1 trivalent construct “BC21-2x1” at different stages of purification.

[0084] FIG. 24 shows SDS-PAGE results with bivalent and trivalent constructs, each after transient expression and one-step purification using the CaptureSelect™ CH1 affinity resin, under non-reducing and reducing conditions, as further described in Example 17.

[0085] FIG. 25 shows supernatant of the Expi293 Expression system transiently transfected with different ratios of vectors encoding the four polypeptide chains of a BC28 antibody and run directly on an native SDS-PAGE gel.

[0086] FIG. 26A shows the architecture of trivalent, bispecific constructs that benefit from CH1 purification. FIG. 26B shows SDS-PAGE gel of the purification products of the constructs depicted in FIG. 26A, following expression using the Expi293 system and one- step purification using the CaptureSelect™ CH1 affinity resin.

[0087] FIG. 27A the architecture of trivalent bispecific constructs that were expressed using the ExpiCHO system. FIG. 27B shows an SDS-PAGE gel of the resulting purification products of the constructs depicted in FIG. 27A, following expression using the ExpiCHO system and one-step purification using the CaptureSelect™ CH1 affinity resin. FIG. 27C shows size exclusion chromatography results following ExpiCHO expression and one-step purification of the constructs depicted in FIG. 27A.

[0088] The figures depict various embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.

6. DETAILED DESCRIPTION

6.1. Definitions

[0089] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

[0090] By“antigen binding site” is meant a region of an antigen-binding CH1 -substituted protein that specifically recognizes or binds to a given antigen or epitope.

[0091]“B-Body” means any of the antigen-binding CH1- substituted protein constructs described herein.

[0092] As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of multiple sclerosis, arthritis, or cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0093] By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

[0094] The term“sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell. [0095] The term“therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a

“prophylactically effective amount” as prophylaxis can be considered therapy.

6.2. Other interpretational conventions

[0096] Unless otherwise specified, all references to sequences herein are to amino acid sequences.

[0097] Unless otherwise specified, antibody constant region residue numbering is according to the Eu index as described at

www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html #refs (accessed Aug. 22, 2017) and in Edelman et al. , Proc. Natl. Acad. USA , 63 :78-85 (1969), which are hereby incorporated by reference in their entireties, and identifies the residue according to its location in an endogenous constant region sequence regardless of the residue’s physical location within a chain of the antigen-binding CH1 -substituted proteins described herein. By“endogenous sequence” or“native sequence” is meant any sequence, including both nucleic acid and amino acid sequences, which originates from an organism, tissue, or cell and has not been artificially modified or mutated.

[0098] In this disclosure, "comprises," "comprising," "containing," "having,"“includes,” “including,” and linguistic variants thereof have the meaning ascribed to them in U.S. Patent law, permitting the presence of additional components beyond those explicitly recited.

[0099] Ranges provided herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[00100] Unless specifically stated or apparent from context, as used herein the term "or" is understood to be inclusive. Unless specifically stated or apparent from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[00101] Unless specifically stated or otherwise apparent from context, as used herein the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

6.3. Methods of purification

[00102] In a first aspect, a method of purifying an antigen-binding CH1- substituted protein is provided.

[00103] In a first series of embodiments, the method comprises the steps of: i) contacting a sample comprising the antigen-binding CH1- substituted protein with a CH1 binding reagent, wherein the antigen-binding CH1- substituted protein comprises at least a first, a second, a third, and a fourth polypeptide chain associated in a complex, wherein the complex comprises at least one CH1 domain, or portion thereof, and wherein the number of CH1 domains in the complex is at least one fewer than the valency of the complex, and wherein the contacting is performed under conditions sufficient for the CH1 binding reagent to bind the CH1 domain, or portion thereof; and ii) purifying the complex from one or more incomplete complexes, wherein the incomplete complexes do not comprise the first, the second, the third, and the fourth polypeptide chain.

[00104] In a typical, naturally occurring, antibody, two heavy chains are associated, each of which has a CH1 domain as the second domain, numbering from N-terminus to C- terminus. Thus, a typical antibody has two CH1 domains. CH1 domains are described in more detail in Section 6.4.1. An“antigen-binding CHI-substituted protein” as described herein, refers to an antigen-binding protein wherein a CH1 domain typically found in the protein has been substituted with another domain, such that the number of CH1 domains in the protein is effectively reduced. In a non-limiting illustrative example, the CH1 domain of a typical antibody can be substituted with a CH3 domain, generating an antigen-binding protein having only a single CH1 domain.

[00105] Antigen-binding proteins can also refer to molecules based on antibody architectures that have been engineered such that they no longer possess a typical antibody architecture. For example, an antibody can be extended at its N or C terminus to increase the valency (described in more detail in Section 6.4.14.1) of the antigen-binding protein, and in certain instances the number of CH1 domains is also increased beyond the typical two CH1 domains. Such molecules can also have one or more of their CH1 domains substituted, such that the number of CH1 domains in the protein is at least one fewer than the valency of the antigen-binding protein. In some embodiments, the number of CH1 domains that are substituted by other domains generates an antigen-binding CH1- substituted protein having only a single CH1 domain. In other embodiments, the number of CH1 domains substituted by another domain generates an antigen-binding CH1 -substituted protein having two or more CH1 domains, but at least one fewer than the valency of the antigen-binding protein. In particular embodiments, where an antigen-binding CH1- substituted protein has two or more CH1 domains, the multiple CH1 domains can all be in the same polypeptide chain. In other particular embodiments, where an antigen-binding CH1- substituted protein has two or more CH1 domains, the multiple CH1 domains can be a single CH1 domain in multiple copies of the same polypeptide chain present in the complete complex.

6.3.1. CHI Binding Reagents

[00106] In the method of purifying antigen-binding CH1 -substituted proteins, a sample comprising the antigen-binding CH1- substituted proteins is contacted with CH1 binding reagents . CH1 binding reagents, as described herein, can be any molecule that specifically binds a CH1 epitope. The various CH1 sequences that provide the CH1 epitope are described in more detail in Section 6.4.1, and specific binding is described in more detail in Section 6.4.14.1.

[00107] In some embodiments, CH1 binding reagents are derived from

immunoglobulin proteins and have an antigen binding site (ABS) that specifically binds the CH1 epitope. In particular embodiments, the CH1 binding reagent is an antibody, also referred to as an“anti-CHl antibody.” The anti-CHl antibody can be derived from a variety of species. In particular embodiments, the anti-CHl antibody is a mammalian antibody, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human antibodies. In specific embodiments, the anti-CHl antibody is a single-domain antibody. Single-domain antibodies, as described herein, have a single variable domain that forms the ABS and specifically binds the CH1 epitope. Exemplary single-domain antibodies include, but are not limited to, heavy chain antibodies derived from camels and sharks, as described in more detail in international application WO 2009/011572, herein incorporated by reference for all it teaches. In a preferred embodiment, the anti-CHl antibody is a camel derived antibody (also referred to as a“camelid antibody”). Exemplary camelid antibodies include, but are not limited to, human IgG-CHl CaptureSelect™ (Therm oFisher,

#194320010) and human IgA-CHl (Therm oFisher, #194311010). In some embodiments, the anti-CHl antibody is a monoclonal antibody. Monoclonal antibodies are typically produced from cultured antibody-producing cell lines. In other embodiments, the anti-CHl antibody is a polyclonal antibody, i.e., a collection of different anti-CHl antibodies that each recognize the CH1 epitope. Polyclonal antibodies are typically produced by collecting the antibody containing serum of an animal immunized with the antigen of interest, or fragment thereof, here CH1.

[00108] In some embodiments, CH1 binding reagents are molecules not derived from immunoglobulin proteins. Examples of such molecules include, but are not limited to, aptamers, peptoids, and affibodies, as described in more detail in Perret and Boschetti ( Biochimie , Feb. 2018, Vol 145:98-112).

6.3.2. Solid Supports

[00109] In the method of purifying antigen-binding CH1 -substituted proteins, the CH1 binding reagent can be attached to a solid support in various embodiments of the invention. Solid supports, as described herein, refers to a material to which other entities can be attached or immobilized, e.g ., the CH1 binding reagent. Solid supports, also referred to as “carriers,” are described in more detail in international application WO 2009/011572.

[00110] In specific embodiments, the solid support comprises a bead or nanoparticle. Examples of beads and nanoparticles include, but are not limited to, agarose beads, polystyrene beads, magnetic nanoparticles (e.g., Dynabeads™, ThermoFisher), polymers (e.g, dextran), synthetic polymers (e.g, Sepharose™), or any other material suitable for attaching the CH1 binding reagent. In particular embodiments, the solid support is modified to enable attachment of the CH1 binding reagent. Example of solid support modifications include, but are not limited to, chemical modifications that form covalent bonds with proteins (e.g, activated aldehyde groups) and modifications that specifically pair with a cognate modification of a CH1 binding reagent (e.g, biotin-streptavidin pairs, disulfide linkages, polyhistidine-nickel, or“click-chemistry” modifications such as azido-alkynyl pairs).

[00111] In certain embodiments, the CH1 binding reagent is attached to the solid support prior to the CH1 binding reagent contacting the antigen-binding CH1- substituted proteins, herein also referred to as an“anti-CHl resin.” In some embodiments, anti-CHl resins are dispersed in a solution. In other embodiments, anti-CHl resins are“packed” into a column. The anti-CHl resin is then contacted with the antigen-binding CH1 -substituted proteins and the CH1 binding reagents specifically bind the antigen-binding CH1- substituted proteins. [00112] In other embodiments, the CH1 binding reagent is attached to the solid support after the CH1 binding reagent contacts the antigen-binding CH1 -substituted proteins. As a non-limiting illustration, a CH1 binding reagent with a biotin modification can be contacted with the antigen-binding CH1- substituted proteins, and subsequently the CH1 binding reagent/antigen-binding CH1- substituted protein mixture can be contacted with streptavidin modified solid support to attach the CH1 binding reagent to the solid support, including CH1 binding reagents specifically bound to the antigen-binding CH1 -substituted proteins.

[00113] In methods wherein the CH1 binding reagents are attached to solid supports, in a variety of embodiments, the bound antigen-binding CH1- substituted proteins are released, or“eluted,” from the solid support forming an eluate having the antigen-binding CH1- substituted proteins. In some embodiments, the bound antigen-binding CH1- substituted proteins are released through reversing the paired modifications ( e.g ., reduction of the disulfide linkage), adding a reagent to compete off the antigen-binding CH1- substituted proteins (e.g., adding imidazole that competes with a polyhistidine for binding to nickel), cleaving off the antigen-binding CH1 -substituted proteins (e.g, a cleavable moiety can be included in the modification), or otherwise interfering with the specific binding of the CH1 binding reagent for the antigen-binding CH1- substituted protein.

Methods that interfere with specific binding include, but are not limited to, contacting antigen-binding CH1- substituted proteins bound to CH1 binding reagents with a low-pH solution. In preferred embodiment, the low-pH solution comprises 0.1 M acetic acid pH 4.0. In other embodiments, the bound antigen-binding CH1 -substituted proteins can be contacted with a range of low-pH solutions, i.e., a“gradient.”

6.3.3. Further Purification

[00114] In some embodiments of the method, a single iteration of the method using the steps of contacting the antigen-binding CH1 -substituted proteins with the CH1 binding reagents, followed by eluting the antigen-binding CH1- substituted proteins, is used to purify the antigen-binding CH1- substituted proteins from the one or more incomplete complexes. In particular embodiments, no other purifying step is performed. In other embodiments, one or more additional purification steps are performed to further purify the antigen-binding CH1 -substituted proteins from the one or more incomplete complexes. The one or more additional purification steps include, but are not limited to, purifying the antigen-binding CH1- substituted proteins based on other protein characteristics, such as size (e.g, size exclusion chromatography), charge (e.g, ion exchange chromatography), or hydrophobicity ( e.g ., hydrophobicity interaction chromatography). In a preferred embodiment, an additional cation exchange chromatograph is performed. Additionally, the antigen-binding CH1- substituted proteins can be further purified repeating contacting the antigen-binding CH1- substituted proteins with the CH1 binding reagents as described above, as well as modifying the CH1 purification method between iterations, e.g., using a step elution for the first iteration and a gradient elution for a subsequent elution.

6.3.4. Assembly and Purity of Complexes

[00115] In the embodiments of the present invention, at least four distinct polypeptide chains associate together to form a complete complex, i.e., the antigen-binding CH1- substituted protein. However, incomplete complexes can also form that do not contain the at least four distinct polypeptide chains. For example, incomplete complexes may form that only have one, two, or three of the polypeptide chains. In other examples, an incomplete complex may contain more than three polypeptide chains, but does not contain the at least four distinct polypeptide chains, e.g, the incomplete complex inappropriately associates with more than one copy of a distinct polypeptide chain. The method of the invention purifies the complex, i.e., the completely assembled antigen-binding CH1- substituted protein, from incomplete complexes.

[00116] Methods to assess the efficacy and efficiency of the purification steps are well known to those skilled in the art and include, but are not limited to, SDS-PAGE analysis, ion exchange chromatography, size exclusion chromatography, and mass spectrometry. Purity can also be assessed according to a variety of criteria. Examples of criterion include, but are not limited to: 1) assessing the percentage of the total protein in an eluate that is provided by the completely assembled antigen-binding CH1 -substituted protein, 2) assessing the fold enrichment or percent increase of the method for purifying the desired products, e.g., comparing the total protein provided by the completely assembled antigen binding CH1 -substituted protein in the eluate to that in a starting sample, 3) assessing the percentage of the total protein or the percent decrease of undesired products, e.g, the incomplete complexes described above, including determining the percent or the percent decrease of specific undesired products (e.g, unassociated single polypeptide chains, dimers of any combination of the polypeptide chains, or trimers of any combination of the polypeptide chains). Purity can be assessed after any combination of methods described herein. For example, purity can be assessed after a single iteration of using the anti-CHl binding reagent, as described herein, or after additional purification steps, as described in more detail in Section 6.3.3. The efficacy and efficiency of the purification steps may also be used to compare the methods described using the anti-CHl binding reagent to other purification methods known to those skilled in the art, such as Protein A purification.

6.4. Antigen-binding CHI-substituted Proteins

[00117] Further aspects of the antigen-binding CH1 -substituted proteins useful for the invention are provided.

[00118] With reference to FIG. 3, in a first series of embodiments, the antigen-binding CH1- substituted proteins comprise a first and a second polypeptide chain, wherein: (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and wherein domain B, domain D, and domain E have a constant region domain amino acid sequence; (b) the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence; (c) the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C-terminus, in a H-I-J-K orientation, and wherein domain H has a variable region domain amino acid sequence, domain I has a CL amino acid sequence, and domains J and K have a constant region domain amino acid sequence; (d) the fourth polypeptide chain comprises a domain L and a domain M, wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and wherein domain L has a variable region domain amino acid sequence, and wherein the fourth polypeptide chain comprises the CH1 domains and domain M is the CH1 domain, or portion thereof; (e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains; (f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains; (g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen-binding CH1 -substituted protein.

[00119] In a second series of embodiments, (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and wherein domain B, domain D, and domain E have a constant region domain amino acid sequence; (b) the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence; (c) the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C-terminus, in a H-I-J-K orientation, and wherein the third polypeptide chain comprises the CH1 domain and domain I is the CH1 domain, or portion thereof, domain H has a variable region domain amino acid sequence, and domains J and K have a constant region domain amino acid sequence; (d) the fourth polypeptide chain comprises a domain L and a domain M, wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and wherein domain L has a variable region domain amino acid sequence, and wherein domain M has a CL amino acid sequence; (e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains; (f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains; (g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen binding CH1- substituted protein.

6.4.1. CHI and CL Regions

[00120] CH1 amino acid sequences, as described herein, are antibody heavy chain constant domain sequences. In some embodiments, CH1 sequences are sequences of the second domain of a native IgG antibody heavy chain, with reference from the N-terminus to C-terminus. In certain embodiments, the CH1 sequences are endogenous sequences. In a variety of embodiments, the CH1 sequences are mammalian sequences, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH1 sequences are human sequences. In certain embodiments, the CH1 sequences are from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM isotype. In a preferred embodiment, the CH1 sequences are from an IgGl isotype. In preferred embodiments, the CH1 sequence is ETniProt accession number P01857 amino acids 1-98. [00121] The CL amino acid sequences useful in the antigen-binding CH1- substituted proteins described herein are light chain constant domain sequences. In some

embodiments, Cl sequences are sequences of the second domain of a native IgG antibody light chain. In certain embodiments, the CL sequences are endogenous sequences. In a variety of embodiments, the CL sequences are mammalian sequences, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, CL sequences are human sequences.

[00122] In certain embodiments, the CL amino acid sequences are lambda (l) light chain constant domain sequences. In particular embodiments, the CL amino acid sequences are human lambda light chain constant domain sequences. In preferred embodiments, the lambda (l) light chain sequence is UniProt accession number P0CG04.

[00123] In certain embodiments, the CL amino acid sequences are kappa (K) light chain constant domain sequences. In a preferred embodiment, the CL amino acid sequences are human kappa (K) light chain constant domain sequences. In a preferred embodiment, the kappa light chain sequence is UniProt accession number P01834.

[00124] In certain embodiments, the CH1 sequence and the CL sequence are both endogenous sequences. In some embodiments, the CH1 sequence, the CL sequence, or both the CH1 and Cl sequence are modified sequences. For example, the CH1 sequence and the CL sequences may separately comprise respectively orthogonal modifications in endogenous CH1 and CL sequences, as discussed below in greater detail in Section 6.4.1.1. It is to be understood that orthogonal mutations in the CH1 sequence do not eliminate the specific binding interaction between the CH1 binding reagent and the CH1 domain.

However, in some embodiments, the orthogonal mutations may reduce, though not eliminate, the specific binding interaction. CH1 and CL sequences can also be portions thereof, either of an endogenous or modified sequence, such that a domain having the CH1 sequence, or portion thereof, can associate with a domain having the CH1 sequence, or portion thereof. Furthermore, the antigen-binding CH1- substituted protein having a portion of the CH1 sequences described above can be bound by the CH1 binding reagent.

[00125] Without wishing to be bound by theory, the CH1 domain is also unique in that it’s folding is typically the rate limiting step in the secretion of IgG (Feige et al. Mol Cell. 2009 Jun l2;34(5):569-79; herein incorporated by reference in its entirety). Thus, purifying the antigen-binding CH1- substituted proteins based on the rate limiting component of CH1 comprising polypeptide chains can provide a means to purify complete complexes from incomplete chains, i.e., purifying complexes have the limiting CH1 domain from complexes only having the one or more non-CHl comprising chains.

[00126] While the CH1 limiting expression may be a benefit in some aspects, as discussed, there is the potential for CH1 to limit overall expression of the complete antigen binding CH1 -substituted proteins. Thus, in certain embodiments, the expression of the polypeptide chain comprising the CH1 sequence(s) is adjusted to improve the efficiency of the antigen-binding CH1- substituted proteins forming complete complexes. In an illustrative example, the ratio of a plasmid vector constructed to express the polypeptide chain comprising the CH1 sequence(s) can be increased relative to the plasmid vectors constructed to express the other polypeptide chains. In another illustrative example, the polypeptide chain comprising the CH1 sequence(s) when compared to the polypeptide chain comprising the CL sequence(s) can be the smaller of the two polypeptide chains. In another specific embodiment, the expression of the polypeptide chain comprising the CH1 sequence(s) can be adjusted by controlling which polypeptide chain has the CH1 sequence(s). For example, engineering the antigen-binding CH1 -substituted protein such that the CH1 domain is present in a two-domain polypeptide chain ( e.g ., the 4^th polypeptide chain described herein), instead of the CH1 sequence’s native position in a four-domain polypeptide chain (e.g., the 3^rd polypeptide chain described herein), can be used to control the expression of the polypeptide chain comprising the CH1 sequence(s). However, in other aspects, a relative expression level of CH1 containing chains that is too high compared to the other chains can result in incomplete complexes the have the CH1 chain, but not each of the other chains. Thus, in certain embodiments, the expression of the polypeptide chain comprising the CH1 sequence(s) is adjusted to both reduce the formation incomplete complexes without the CH1 containing chain, and to reduce the formation incomplete complexes with the CH1 containing chain but without the other chains present in a complete complex.

6.4.1.1.CH1 and CL Orthogonal Modifications

[00127] In certain embodiments, the CH1 sequence and the CL sequences separately comprise respectively orthogonal modifications in endogenous CH1 and CL sequences. Orthogonal mutations, in general, are described in more detail below in Sections 6.4.15.1- 6.4.15.3. [00128] In particular embodiments, the orthogonal modifications in endogenous CH1 and CL sequences are an engineered disulfide bridge selected from engineered cysteines at position 138 of the CH1 sequence and position 116 of the CL sequence, at position 128 of the CH1 sequence and position 119 of the CL sequence, or at position 129 of the CH1 sequence and position 210 of the CL sequence, as numbered and discussed in more detail in U.S. Pat. No. 8,053,562 and U.S. Pat. No. 9,527,927, each incorporated herein by reference in its entirety. In a preferred embodiment, the engineered cysteines are at position 128 of the CH1 sequence and position 118 of the CL Kappa sequence, as numbered by the Eu index.

[00129] In a series of preferred embodiments, the mutations that provide non- endogenous cysteine amino acids are a Fl 18C mutation in the CL sequence with a corresponding A141C in the CH1 sequence, or a Fl 18C mutation in the CL sequence with a corresponding L128C in the CH1 sequence, or a S162C mutations in the CL sequence with a corresponding P171C mutation in the CH1 sequence, as numbered by the Eu index.

[00130] In a variety of embodiments, the orthogonal mutations in the CL sequence and the CH1 sequence are charge-pair mutations. In specific embodiments the charge-pair mutations are a Fl 18S, Fl 18A or Fl 18V mutation in the CL sequence with a corresponding A141L in the CH1 sequence, or a T129R mutation in the CL sequence with a corresponding K147D in the CH1 sequence, as numbered by the Eu index and described in greater detail in Bonisch et al. (Protein Engineering, Design & Selection , 2017, pp. 1-12), herein incorporated by reference for all that it teaches. In a series of preferred embodiments the charge-pair mutations are a N138K mutation in the CL sequence with a corresponding G166D in the CH1 sequence, or a N138D mutation in the CL sequence with a

corresponding G166K in the CH1 sequence, as numbered by the Eu index.

6.4.2. Domain A (Variable Region)

[00131] In some embodiments of the antigen-binding CH1- substituted proteins, domain A has a variable region domain amino acid sequence. Variable region domain amino acid sequences, as described herein, are variable region domain amino acid sequences of an antibody including VL and VH antibody domain sequences. VL and VH sequences are described in greater detail below in Sections 6.4.2.1 and 6.4.2.4, respectively. In a preferred embodiment, domain A has a VL antibody domain sequence and domain F has a VH antibody domain sequence. 6.4.2.1.VL Regions

[00132] The VL amino acid sequences useful in the antigen-binding CH1- substituted proteins described herein are antibody light chain variable domain sequences. In a typical arrangement in both natural antibodies and the antibody constructs described herein, a specific VL amino acid sequence associates with a specific VH amino acid sequence to form an antigen-binding site. In various embodiments, the VL amino acid sequences are mammalian sequences, including human sequences, synthesized sequences, or

combinations of human, non-human mammalian, mammalian, and/or synthesized sequences, as described in further detail below in Sections 6.4.2.2 and 6.4.2.3.

[00133] In various embodiments, VL amino acid sequences are mutated sequences of naturally occurring sequences. In certain embodiments, the VL amino acid sequences are lambda (l) light chain variable domain sequences. In certain embodiments, the VL amino acid sequences are kappa (K) light chain variable domain sequences. In a preferred embodiment, the VL amino acid sequences are kappa (K) light chain variable domain sequences.

[00134] In the antigen-binding CH1- substituted proteins described herein, the C- terminus of domain A is connected to the N-terminus of domain B. In certain

embodiments, domain A has a VL amino acid sequence that is mutated at its C-terminus at the junction between domain A and domain B, as described in greater detail below in Section 6.4.20.1 and in Example 6.

6.4.2.2. Complementarity Determining Regions

[00135] The VL amino acid sequences comprise highly variable sequences termed “complementarity determining regions” (CDRs), typically three CDRs (CDR1, CD2, and CDR3). In a variety of embodiments, the CDRs are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CDRs are human sequences. In various embodiments, the CDRs are naturally occurring sequences. In various embodiments, the CDRs are naturally occurring sequences that have been mutated to alter the binding affinity of the antigen binding site for a particular antigen or epitope. In certain embodiments, the naturally occurring CDRs have been mutated in an in vivo host through affinity maturation and somatic hypermutation. In certain embodiments, the CDRs have been mutated in vitro through methods including, but not limited to, PCR-mutagenesis and chemical mutagenesis. In various embodiments, the CDRs are synthesized sequences including, but not limited to, CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries.

6.4.2.3. Framework Regions and CDR Grafting

[00136] The VL amino acid sequences comprise“framework region” (FR) sequences. FRs are generally conserved sequence regions that act as a scaffold for interspersed CDRs (see Section 6.4.2.2.), typically in a FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 arrangement (from N-terminus to C-terminus). In a variety of embodiments, the FRs are mammalian sequences, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the FRs are human sequences. In various embodiments, the FRs are naturally occurring sequences. In various embodiments, the FRs are synthesized sequences including, but not limited, rationally designed sequences.

[00137] In a variety of embodiments, the FRs and the CDRs are both from the same naturally occurring variable domain sequence. In a variety of embodiments, the FRs and the CDRs are from different variable domain sequences, wherein the CDRs are grafted onto the FR scaffold with the CDRs providing specificity for a particular antigen. In certain embodiments, the grafted CDRs are all derived from the same naturally occurring variable domain sequence. In certain embodiments, the grafted CDRs are derived from different variable domain sequences. In certain embodiments, the grafted CDRs are synthesized sequences including, but not limited to, CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries. In certain embodiments, the grafted CDRs and the FRs are from the same species. In certain embodiments, the grafted CDRs and the FRs are from different species. In a preferred grafted CDR embodiment, an antibody is “humanized”, wherein the grafted CDRs are non-human mammalian sequences including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, and goat sequences, and the FRs are human sequences. Humanized antibodies are discussed in more detail in U.S. Pat. No. 6,407,213, the entirety of which is hereby incorporated by reference for all it teaches.

In various embodiments, portions or specific sequences of FRs from one species are used to replace portions or specific sequences of another species’ FRs.

6.4.2.4.VH Regions

[00138] The VH amino acid sequences in the antigen-binding CH1 -substituted proteins described herein are antibody heavy chain variable domain sequences. In a typical antibody arrangement in both nature and in the antigen-binding CH1 -substituted proteins described herein, a specific VH amino acid sequence associates with a specific VL amino acid sequence to form an antigen-binding site. In various embodiments, VH amino acid sequences are mammalian sequences, including human sequences, synthesized sequences, or combinations of non-human mammalian, mammalian, and/or synthesized sequences, as described in further detail above in Sections 6.4.2.2 and 6.4.2.3. In various embodiments, VH amino acid sequences are mutated sequences of naturally occurring sequences.

6.4.3. Domain B (Constant Region)

[00139] In some embodiments, domain B has a constant region domain sequence. In some embodiments, domain B has a constant region domain sequence that is not a CH1 sequence. Constant region domain amino acid sequences, as described herein, are sequences of a constant region domain of an antibody.

[00140] In a variety of embodiments, the constant region sequences are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the constant region sequences are human sequences. In certain embodiments, the constant region sequences are from an antibody light chain. In particular embodiments, the constant region sequences are from a lambda or kappa light chain. In certain embodiments, the constant region sequences are from an antibody heavy chain, except for the CH1 region of a heavy chain. In particular embodiments, the constant region sequences are an antibody heavy chain sequence that is an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM isotype. In a specific

embodiment, the constant region sequences are from an IgG isotype. In a preferred embodiment, the constant region sequences are from an IgGl isotype. In preferred specific embodiments, the constant region sequence is a CH3 sequence. CH3 sequences are described in greater detail below in Section 6.4.3.1. In other preferred embodiments, the constant region sequence is an orthologous CH2 sequence. Orthologous CH2 sequences are described in greater detail below in Section 6.4.3.2.

[00141] In particular embodiments, for example wherein the valency of the binding molecule is three or greater than three, the constant region sequence is a CH1 or Cl sequence. In some embodiments, the constant region sequence is a Cl sequence. CH1 and Cl sequences are described herein. In some embodiments, the CH1 or Cl sequence comprises one or more CH1 or Cl orthogonal modifications described herein.

[00142] In particular embodiments, the constant region sequence has been mutated to include one or more orthogonal mutations. In a preferred embodiment, domain B has a constant region sequence that is a CH3 sequence comprising knob-hole (synonymously, “knob-in-hole,”“KIH”) orthogonal mutations, as described in greater detail below in Section 6.4.15.2, and either a S354C or a Y349C mutation that forms an engineered disulfide bridge with a CH3 domain containing an orthogonal mutation, as described in in greater detail below in Section 6.4.15.1. In some preferred embodiments, the knob-hole orthogonal mutation is a T366W mutation.

6.4.3.1.CH3 Regions

[00143] CH3 amino acid sequences, as described herein, are sequences of the C- terminal domain of an antibody heavy chain.

[00144] In a variety of embodiments, the CH3 sequences are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH3 sequences are human sequences. In certain embodiments, the CH3 sequences are from an IgAl, IgA2, IgD, IgE, IgM, IgGl, IgG2, IgG3, IgG4 isotype or CH4 sequences from an IgE or IgM isotype. In a specific

embodiment, the CH3 sequences are from an IgG isotype. In a preferred embodiment, the CH3 sequences are from an IgGl isotype.

[00145] In certain embodiments, the CH3 sequences are endogenous sequences. In particular embodiments, the CH3 sequence is ETniProt accession number P01857 amino acids 224-330. In various embodiments, a CH3 sequence is a segment of an endogenous CH3 sequence. In particular embodiments, a CH3 sequence has an endogenous CH3 sequence that lacks the N-terminal amino acids G224 and Q225. In particular

embodiments, a CH3 sequence has an endogenous CH3 sequence that lacks the C-terminal amino acids P328, G329, and K330. In particular embodiments, a CH3 sequence has an endogenous CH3 sequence that lacks both the N-terminal amino acids G224 and Q225 and the C-terminal amino acids P328, G329, and K330. In preferred embodiments, an antigen binding CH1 -substituted protein has multiple domains that have CH3 sequences, wherein a CH3 sequence can refer to both a full endogenous CH3 sequence as well as a CH3 sequence that lacks N-terminal amino acids, C-terminal amino acids, or both.

[00146] In certain embodiments, the CH3 sequences are endogenous sequences that have one or more mutations. In particular embodiments, the mutations are one or more orthogonal mutations that are introduced into an endogenous CH3 sequence to guide specific pairing of specific CH3 sequences, as described in more detail below in Sections 6.4.15.1-6.4.15.3. [00147] In certain embodiments, the CH3 sequences are engineered to reduce immunogenicity of the antibody by replacing specific amino acids of one allotype with those of another allotype and referred to herein as isoallotype mutations, as described in more detail in Stickler et al. {Genes Immun. 2011 Apr; 12(3): 213-221), which is herein incorporated by reference for all that it teaches. In particular embodiments, specific amino acids of the Glml allotype are replaced. In a preferred embodiment, isoallotype mutations D356E and L358M are made in the CH3 sequence.

[00148] In a preferred embodiment, domain B has a human IgGl CH3 amino acid sequence with the following mutational changes: P343V; Y349C; and a tripeptide insertion, 445P, 446G, 447K. In other preferred embodiments, domain B has a human IgGl CH3 sequence with the following mutational changes: T366K; and a tripeptide insertion, 445K, 446S, 447C. In still other preferred embodiments, domain B has a human IgGl CH3 sequence with the following mutational changes: Y349C and a tripeptide insertion, 445P, 446G, 447K.

[00149] In certain embodiments, domain B has a human IgGl CH3 sequence with a 447C mutation incorporated into an otherwise endogenous CH3 sequence.

[00150] In the antigen-binding CH1- substituted proteins described herein, the N- terminus of domain B is connected to the C-terminus of domain A. In certain embodiments, domain B has a CH3 amino acid sequence that is mutated at its N-terminus at the junction between domain A and domain B, as described in greater detail below in Section 6.4.20.1 and Example 6.

[00151] In some embodiments of the antigen-binding CH1- substituted proteins, the C- terminus of domain B is connected to the N-terminus of domain D. In certain embodiments, domain B has a CH3 amino acid sequence that is extended at the C-terminus at the junction between domain B and domain D, as described in greater detail below in Section 6.4.20.3.

6.4.3.2. Orthologous CH2 Regions

[00152] CH2 amino acid sequences, as described herein, are sequences of the third domain of an antibody heavy chain, with reference from the N-terminus to C-terminus.

CH2 amino acid sequences, in general, are discussed in more detail below in section 6.4.4. In a series of embodiments, an antigen-binding CH1 -substituted protein has more than one paired set of CH2 domains that have CH2 sequences, wherein a first set has CH2 amino acid sequences from a first isotype and one or more orthologous sets of CH2 amino acid sequences from another isotype. The orthologous CH2 amino acid sequences, as described herein, are able to interact with CH2 amino acid sequences from a shared isotype, but not significantly interact with the CH2 amino acid sequences from another isotype present in the antigen-binding CH1- substituted protein. In particular embodiments, all sets of CH2 amino acid sequences are from the same species. In preferred embodiments, all sets of CH2 amino acid sequences are human CH2 amino acid sequences. In other embodiments, the sets of CH2 amino acid sequences are from different species. In particular embodiments, the first set of CH2 amino acid sequences is from the same isotype as the other non-CH2 domains in the antigen-binding CH1 -substituted protein. In a specific embodiment, the first set has CH2 amino acid sequences from an IgG isotype and the one or more orthologous sets have CH2 amino acid sequences from an IgM or IgE isotype. In certain embodiments, one or more of the sets of CH2 amino acid sequences are endogenous CH2 sequences. In other embodiments, one or more of the sets of CH2 amino acid sequences are endogenous CH2 sequences that have one or more mutations. In particular embodiments, the one or more mutations are orthogonal knob-hole mutations, orthogonal charge-pair mutations, or orthogonal hydrophobic mutations. Orthologous CH2 amino acid sequences useful for the antigen-binding CH1- substituted proteins are described in more detail in international PCT applications W02017/011342 and WO2017/106462, herein incorporated by reference in their entirety

6.4.4. Domain D (Constant Region)

[00153] In some embodiments of the antigen-binding CH1- substituted proteins, domain D has a constant region amino acid sequence. Constant region amino acid sequences are described in more detail, e.g., in Section 6.4.3.

[00154] In a preferred series of embodiments, domain D has a CH2 amino acid sequence. CH2 amino acid sequences, as described herein, are CH2 amino acid sequences of the third domain of a native antibody heavy chain, with reference from the N-terminus to C-terminus. In a variety of embodiments, the CH2 sequences are mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH2 sequences are human sequences. In certain embodiments, the CH2 sequences are from a IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM isotype. In a preferred embodiment, the CH2 sequences are from an IgGl isotype.

[00155] In certain embodiments, the CH2 sequences are endogenous sequences. In particular embodiments, the sequence is UniProt accession number P01857 amino acids 111-223. In a preferred embodiment, the CH2 sequences have a N-terminal hinge region peptide that connects the N-terminal variable domain-constant domain segment to the CH2 domain, as discussed in more detail below in Section 6.4.20.3.

[00156] In some embodiments of the antigen-binding CH1- substituted proteins, the N- terminus of domain D is connected to the C-terminus of domain B. In certain

embodiments, domain B has a CH3 amino acid sequence that is extended at the C-terminus at the junction between domain D and domain B, as described in greater detail below in Section 6.4.20.3.

6.4.5. Domain E (Constant Region)

[00157] In some embodiments of the antigen-binding CH1- substituted proteins, domain E has a constant region domain amino acid sequence. Constant region amino acid sequences are described in more detail, e.g., in Section 6.4.3.

[00158] In certain embodiments, the constant region sequence is a CH3 sequence.

CH3 sequences are described in greater detail above in Section 6.4.3.1. In particular embodiments, the constant region sequence has been mutated to include one or more orthogonal mutations. In a preferred embodiment, domain E has a constant region sequence that is a CH3 sequence comprising knob-hole (synonymously,“knob-in-hole,”“KIH”) orthogonal mutations, as described in greater detail below in Section 6.4.15.2, and either a S354C or a Y349C mutation that forms an engineered disulfide bridge with a CH3 domain containing an orthogonal mutation, as described in in greater detail below in Section 6.4.15.1. In some preferred embodiments, the knob-hole orthogonal mutation is a T366W mutation.

[00159] In certain embodiments, the constant region domain sequence is a CH1 sequence. In particular embodiments, the CH1 amino acid sequence of domain E is the only CH1 amino acid sequence in the antigen-binding CH1 -substituted protein. In certain embodiments, the N-terminus of the CH1 domain is connected to the C-terminus of a CH2 domain, as described in greater detail below in 6.4.20.5. In certain embodiments, the constant region sequence is a CL sequence. In certain embodiments, the N-terminus of the CL domain is connected to the C-terminus of a CH2 domain, as described in greater detail below in 6.4.20.5. CH1 and CL sequences are described in further detail in Section 6.4.1.

6.4.6. Domain F (Variable Region)

[00160] In some embodiments of the antigen-binding CH1- substituted proteins, domain F has a variable region domain amino acid sequence. Variable region domain amino acid sequences, as discussed in greater detail in Section 6.4.2, are variable region domain amino acid sequences of an antibody including VL and VH antibody domain sequences. VL and VH sequences are described in greater detail above in Sections 6.4.2.1 and 6.4.2.4, respectively. In a preferred embodiment, domain F has a VH antibody domain sequence.

6.4.7. Domain G (Constant Region)

[00161] In some embodiments, domain G has a constant region domain sequence. In some embodiments, domain G has a constant region domain sequence that is not a CH1 sequence.

[00162] In some embodiments of the antigen-binding CH1- substituted proteins, domain G has a CH3 amino acid sequence. CH3 sequences are described in greater detail above in Section 6.4.3.1.

[00163] In certain preferred embodiments, domain G has a human IgGl CH3 sequence with the following mutational changes: S354C; and a tripeptide insertion, 445P, 446G, 447K. In some preferred embodiments, domain G has a human IgGl CH3 sequence with the following mutational changes: S354C; and 445P, 446G, 447K tripeptide insertion. In some preferred embodiments, domain G has a human IgGl CH3 sequence with the following changes: L351D, and a tripeptide insertion of 445G, 446E, 447C.

[00164] In some embodiments, domain G comprises an orthologous CH2 amino acid sequence described herein.

[00165] In particular embodiments, for example wherein the valency of the binding molecule is three or greater than three, the constant region sequence is a CH1 or Cl sequence. In some embodiments wherein domain B is a Cl sequence, domain G is a CH1 sequence. CH1 and Cl sequences are described herein. In some embodiments, the CH1 or Cl sequence comprises one or more CH1 or Cl orthogonal modifications described herein.

6.4.8. Domain H (Variable Region)

[00166] In some embodiments of the antigen-binding CH1- substituted proteins, domain H has a variable region domain amino acid sequence. Variable region domain amino acid sequences, as discussed in greater detail in Section 6.4.2, are variable region domain amino acid sequences of an antibody including VL and VH antibody domain sequences. VL and VH sequences are described in greater detail above in Sections 6.4.2.1. and 6.4.2.4, respectively. In a preferred embodiment, domain H has a VL antibody domain sequence. 6.4.9. Domain I (Constant Region)

[00167] In some embodiments of the antigen-binding CH1- substituted proteins, domain I has a constant region domain amino acid sequence. Constant region domain amino acid sequences are described in greater detail above, e.g., in Section 6.4.3. In a series of preferred embodiments of the antigen-binding CH1 -substituted proteins, domain I has a CL amino acid sequence. In another series of embodiments, domain I has a CH1 amino acid sequence. CH1 and CL amino acid sequences are described in further detail in Section 6.4.1.

6.4.10. Domain J (CH2)

[00168] In some embodiments of the antigen-binding CH1- substituted proteins, domain J has a CH2 amino acid sequence. CH2 amino acid sequences are described in greater detail above in Section 6.4.4. In a preferred embodiment, the CH2 amino acid sequence has a N-terminal hinge region that connects domain J to domain I, as described in more detail below in Section 6.4.20.4.

[00169] In some embodiments of the antigen-binding CH1- substituted proteins, the C- terminus of domain J is connected to the N-terminus of domain K. In particular

embodiments, domain J is connected to the N-terminus of domain K that has a CH1 amino acid sequence or CL amino acid sequence, as described in further detail below in Section 6.4.20.5.

6.4.11. Domain K (Constant Region)

[00170] In some embodiments of the antigen-binding CH1- substituted proteins, domain K has a constant region domain amino acid sequence. Constant region domain amino acid sequences are described in greater detail above in Section 6.4.3. In a preferred embodiment, domain K has a constant region sequence that is a CH3 sequence comprising knob-hole orthogonal mutations, as described in greater detail below in Section 6.4.15.2; isoallotype mutations, as described in more detail above in 6.4.3.1.; and either a S354C or a Y349C mutation that forms an engineered disulfide bridge with a CH3 domain containing an orthogonal mutation, as described in in greater detail below in Section 6.4.15.1. In some preferred embodiments, the knob-hole orthogonal mutations combined with isoallotype mutations are the following mutational changes: D356E, L358M, T366S, L368A, and Y407V.

[00171] In certain embodiments, the constant region domain sequence is a CH1 sequence. In particular embodiments, the CH1 amino acid sequence of domain K is the only CH1 amino acid sequence in the antigen-binding CH1- substituted protein. In certain embodiments, the N-terminus of the CH1 domain is connected to the C-terminus of a CH2 domain, as described in greater detail below in 6.4.20.5. In certain embodiments, the constant region sequence is a CL sequence. In certain embodiments, the N-terminus of the CL domain is connected to the C-terminus of a CH2 domain, as described in greater detail below in 6.4.20.5. CH1 and CL sequences are described in further detail in Section 6.4.1.

6.4.12. Domain L (Variable Region)

[00172] In some embodiments of the antigen-binding CH1- substituted proteins, domain L has a variable region domain amino acid sequence. Variable region domain amino acid sequences, as discussed in greater detail in Section 6.4.2, are variable region domain amino acid sequences of an antibody including VL and VH antibody domain sequences. VL and VH sequences are described in greater detail above in Sections

6.4.2. Land 6.4.2.4, respectively. In a preferred embodiment, domain L has a VH antibody domain sequence.

6.4.13. Domain M (Constant Region)

[00173] In some embodiments of the antigen-binding CH1- substituted proteins, domain M has a constant region domain amino acid sequence. Constant region domain amino acid sequences are described in greater detail above, e.g., in Section 6.4.3. In a series of preferred embodiments wherein domain I has a CL amino acid sequence, domain M has a CH1 amino acid sequence. In another series of preferred embodiments of the antigen binding CH1- substituted proteins wherein domain I has a CH1 amino acid sequence, domain M has a Cl domain sequence. CH1 and CL amino acid sequences are described in further detail in Section 6.4.1.

6.4.14. Pairing of Domains A & F

[00174] In some embodiments of the antigen-binding CH1- substituted proteins, a domain A VL or VH amino acid sequence and a cognate domain F VL or VH amino acid sequence are associated and form an antigen binding site (ABS). The A:F antigen binding site (ABS) is capable of specifically binding an epitope of an antigen. Antigen binding by an ABS is described in greater detail below in Section 6.4.14.1.

[00175] In a variety of multivalent embodiments, the ABS formed by domains A and F (A:F) is identical in sequence to one or more other ABSs within the antigen-binding CH1- substituted protein and therefore has the same recognition specificity as the one or more other sequence-identical ABSs within the antigen-binding CH1 -substituted protein.

[00176] In a variety of multivalent embodiments, the A:F ABS is non-identical in sequence to one or more other ABSs within the antigen-binding CH1- substituted protein.

In certain embodiments, the A:F ABS has a recognition specificity different from that of one or more other sequence-non-identical ABSs in the antigen-binding CH1 -substituted protein. In particular embodiments, the A:F ABS recognizes a different antigen from that recognized by at least one other sequence-non-identical ABS in the antigen-binding CH1- substituted protein. In particular embodiments, the A:F ABS recognizes a different epitope of an antigen that is also recognized by at least one other sequence-non-identical ABS in the antigen-binding CH1- substituted protein. In these embodiments, the ABS formed by domains A and F recognizes an epitope of antigen, wherein one or more other ABSs within the antigen-binding CH1- substituted protein recognizes the same antigen but not the same epitope.

6.4.14.1. Binding of Antigen by ABS

[00177] An ABS, and the antigen-binding CH1 -substituted protein comprising such ABS, is said to“recognize” the epitope (or more generally, the antigen) to which the ABS specifically binds, and the epitope (or more generally, the antigen) is said to be the

“recognition specificity” or“binding specificity” of the ABS.

[00178] The ABS is said to bind to its specific antigen or epitope with a particular affinity. As described herein,“affinity” refers to the strength of interaction of non-covalent interm olecular forces between one molecule and another. The affinity, i.e. the strength of the interaction, can be expressed as a dissociation equilibrium constant (KD), wherein a lower KD value refers to a stronger interaction between molecules. KD values of antibody constructs are measured by methods well known in the art including, but not limited to, bio layer interferometry (e.g. Octet/FORTEBIO^®), surface plasmon resonance (SPR) technology (e.g. Biacore^®), and cell binding assays. For purposes herein, affinities are dissociation equilibrium constants measured by bio-layer interferometry using

Octet/FORTEBIO^®.

[00179] “Specific binding,” as used herein, refers to an affinity between an ABS and its cognate antigen or epitope in which the KD value is below 10 ⁶M, 10 ⁷M, 10 ⁸M, 10 ⁹M, or 10 ¹⁰M. [00180] The number of ABSs in an antigen-binding CH1 -substituted protein as described herein defines the“valency” of the antigen-binding CH1- substituted protein. As schematized in FIG. 2, an antigen-binding CH1- substituted protein having a single ABS is “monovalent”. An antigen-binding CH1 -substituted protein having a plurality of ABSs is said to be“multivalent”. A multivalent antigen-binding CH1 -substituted protein having two ABSs is“bivalent.” A multivalent antigen-binding CH1 -substituted protein having three ABSs is“trivalent” A multivalent antigen-binding CH1 -substituted protein having four ABSs is“tetravalent.”

[00181] In various multivalent embodiments, all of the plurality of ABSs have the same recognition specificity. As schematized in FIG. 2, such an antigen-binding CH1- substituted protein is a“monospecific”“multivalent” binding construct. In other multivalent embodiments, at least two of the plurality of ABSs have different recognition specificities. Such antigen-binding CH1- substituted proteins are multivalent and

“multispecific”. In multivalent embodiments in which the ABSs collectively have two recognition specificities, the antigen-binding CH1 -substituted protein is“bispecific.” In multivalent embodiments in which the ABSs collectively have three recognition

specificities, the antigen-binding CH1 -substituted protein is“trispecific.”

[00182] In multivalent embodiments in which the ABSs collectively have a plurality of recognition specificities for different epitopes present on the same antigen, the antigen binding CH1 -substituted protein is“multiparatopic” Multivalent embodiments in which the ABSs collectively recognize two epitopes on the same antigen are“biparatopic”

[00183] In various multivalent embodiments, multivalency of the antigen-binding CH1- substituted protein improves the avidity of the antigen-binding CH1- substituted protein for a specific target. As described herein,“avidity” refers to the overall strength of interaction between two or more molecules, e.g. a multivalent antigen-binding CH1- substituted protein for a specific target, wherein the avidity is the cumulative strength of interaction provided by the affinities of multiple ABSs. Avidity can be measured by the same methods as those used to determine affinity, as described above. In certain embodiments, the avidity of an antigen-binding CH1- substituted protein for a specific target is such that the interaction is a specific binding interaction, wherein the avidity between two molecules has a KD value below 10 ⁶M, 10 ⁷M, 10 ⁸M, 10 ⁹M, or 10 ¹⁰M. In certain embodiments, the avidity of an antigen-binding CH1- substituted protein for a specific target has a KD value such that the interaction is a specific binding interaction, wherein the one or more affinities of individual ABSs do not have has a KD value that qualifies as specifically binding their respective antigens or epitopes on their own. In certain embodiments, the avidity is the cumulative strength of interaction provided by the affinities of multiple ABSs for separate antigens on a shared specific target or complex, such as separate antigens found on an individual cell. In certain embodiments, the avidity is the cumulative strength of interaction provided by the affinities of multiple ABSs for separate epitopes on a shared individual antigen.

6.4.15. Pairing of Domains B & G

[00184] In the antigen-binding CH1- substituted proteins described herein, a domain B constant region amino acid sequence and a domain G constant region amino acid sequence are associated. Constant region domain amino acid sequences are described in greater detail above in Section 6.4.3. Other constant region domain amino acid sequences, including CH1 and Cl amino acid sequences, are described herein in Section 6.4.1.

[00185] In a series of preferred embodiments, domain B and domain G have CH3 amino acid sequences. CH3 sequences are described in greater detail above in Section 6.4.3.1. In various embodiments, the amino acid sequences of the B and the G domains are identical. In certain of these embodiments, the sequence is an endogenous CH3 sequence.

[00186] In a variety of embodiments, the amino acid sequences of the B and the G domains are different, and separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, wherein the B domain interacts with the G domain, and wherein neither the B domain nor the G domain significantly interacts with a CH3 domain lacking the orthogonal modification.

[00187] “Orthogonal modifications” or synonymously“orthogonal mutations” as described herein are one or more engineered mutations in an amino acid sequence of an antibody domain that increase the affinity of binding of a first domain having orthogonal modification for a second domain having a complementary orthogonal modification. In certain embodiments, the orthogonal modifications decrease the affinity of a domain having the orthogonal modifications for a domain lacking the complementary orthogonal modifications. In certain embodiments, orthogonal modifications are mutations in an endogenous antibody domain sequence. In a variety of embodiments, orthogonal modifications are modifications of the N-terminus or C-terminus of an endogenous antibody domain sequence including, but not limited to, amino acid additions or deletions. In particular embodiments, orthogonal modifications include, but are not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge-pair mutations, as described in greater detail below in Sections 6.4.15.1-6.4.15.3. In particular embodiments, orthogonal modifications include a combination of orthogonal modifications selected from, but not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge-pair mutations. In particular embodiments, the orthogonal modifications can be combined with amino acid substitutions that reduce immunogenicity, such as isoallotype mutations, as described in greater detail above in Section 6.4.3.1.

6.4.15.1. Orthogonal Engineered Disulfide Bridges

[00188] In a variety of embodiments, the orthogonal modifications comprise mutations that generate engineered disulfide bridges between a first and a second domain. As described herein,“engineered disulfide bridges” are mutations that provide non- endogenous cysteine amino acids in two or more domains such that a non-native disulfide bond forms when the two or more domains associate. Engineered disulfide bridges are described in greater detail in Merchant et al. (. Nature Biotech (1998) 16:677-681), the entirety of which is hereby incorporated by reference for all it teaches. In certain embodiments, engineered disulfide bridges improve orthogonal association between specific domains. In a particular embodiment, the mutations that generate engineered disulfide bridges are a K392C mutation in one of a first or second CH3 domains, and a D399C in the other CH3 domain. In a preferred embodiment, the mutations that generate engineered disulfide bridges are a S354C mutation in one of a first or second CH3 domains, and a Y349C in the other CH3 domain. In another preferred embodiment, the mutations that generate engineered disulfide bridges are a 447C mutation in both the first and second CH3 domains that are provided by extension of the C-terminus of a CH3 domain incorporating a KSC tripeptide sequence.

6.4.15.2. Orthogonal Knob-Hole Mutations

[00189] In a variety of embodiments, orthogonal modifications comprise knob-hole (synonymously, knob-in-hole) mutations. As described herein, knob-hole mutations are mutations that change the steric features of a first domain’s surface such that the first domain will preferentially associate with a second domain having complementary steric mutations relative to association with domains without the complementary steric mutations. Knob-hole mutations are described in greater detail in U.S. Pat. No. 5,821,333 and U.S. Pat. No. 8,216,805, each of which is incorporated herein in its entirety. In various

embodiments, knob-hole mutations are combined with engineered disulfide bridges, as described in greater detail in Merchant et al. (. Nature Biotech (1998) 16:677-681)), incorporated herein by reference in its entirety. In various embodiments, knob-hole mutations, isoallotype mutations, and engineered disulfide mutations are combined.

[00190] In certain embodiments, the knob-in-hole mutations are a T366Y mutation in a first domain, and a Y407T mutation in a second domain. In certain embodiments, the knob- in-hole mutations are a F405A in a first domain, and a T394W in a second domain. In certain embodiments, the knob-in-hole mutations are a T366Y mutation and a F405A in a first domain, and a T394W and a Y407T in a second domain. In certain embodiments, the knob-in-hole mutations are a T366W mutation in a first domain, and a Y407A in a second domain. In certain embodiments, the combined knob-in-hole mutations and engineered disulfide mutations are a S354C and T366W mutations in a first domain, and a Y349C, T366S, L368A, and aY407V mutation in a second domain. In a preferred embodiment, the combined knob-in-hole mutations, isoallotype mutations, and engineered disulfide mutations are a S354C and T366W mutations in a first domain, and a Y349C, D356E, L358M, T366S, L368A, and aY407V mutation in a second domain.

6.4.15.3. Orthogonal Charge-pair Mutations

[00191] In a variety of embodiments, orthogonal modifications are charge-pair mutations. As used herein, charge-pair mutations are mutations that affect the charge of an amino acid in a domain’s surface such that the domain will preferentially associate with a second domain having complementary charge-pair mutations relative to association with domains without the complementary charge-pair mutations. In certain embodiments, charge-pair mutations improve orthogonal association between specific domains. Charge- pair mutations are described in greater detail in U.S. Pat. No. 8,592,562, U.S. Pat. No. 9,248,182, and U.S. Pat. No. 9,358,286, each of which is incorporated by reference herein for all they teach. In certain embodiments, charge-pair mutations improve stability between specific domains. In a preferred embodiment, the charge-pair mutations are a T366K mutation in a first domain, and a L351D mutation in the other domain.

6.4.16. Pairing of Domains E & K

[00192] In various embodiments, the E domain has a CH3 amino acid sequence.

[00193] In various embodiments, the K domain has a CH3 amino acid sequence.

[00194] In a variety of embodiments, the amino acid sequences of the E and K domains are identical, wherein the sequence is an endogenous CH3 sequence.

[00195] In a variety of embodiments, the sequences of the E and K domains are different. In a variety of embodiments, the different sequences separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, wherein the E domain interacts with the K domain, and wherein neither the E domain nor the K domain significantly interacts with a CH3 domain lacking the orthogonal modification. In certain embodiments, the orthogonal modifications include, but are not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge-pair mutations, as described in greater detail above in sections 6.4.15.1-6.4.15.3. In particular embodiments, orthogonal modifications include a combination of orthogonal modifications selected from, but not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge-pair mutations. In particular embodiments, the orthogonal modifications can be combined with amino acid substitutions that reduce immunogenicity, such as isoallotype mutations.

6.4.17. Pairing of Domains I & M and Domains H & L

[00196] In a variety of embodiments, domain I has a CL sequence and domain M has a CH1 sequence. In a variety of embodiments, domain H has a VL sequence and domain L has a VH sequence. In a preferred embodiment, domain H has a VL amino acid sequence, domain I has a CL amino acid sequence, domain L has a VH amino acid sequence, and domain M has a CH1 amino acid sequence. In another preferred embodiment, domain H has a VL amino acid sequence, domain I has a CL amino acid sequence, domain L has a VH amino acid sequence, domain M has a CH1 amino acid sequence, and domain K has a CH3 amino acid sequence.

[00197] In a variety of embodiments, the amino acid sequences of the I domain and the M domain separately comprise respectively orthogonal modifications in an endogenous sequence, wherein the I domain interacts with the M domain, and wherein neither the I domain nor the M domain significantly interacts with a domain lacking the orthogonal modification. In a series of embodiments, the orthogonal mutations in the I domain are in a CL sequence and the orthogonal mutations in the M domain are in CH1 sequence.

Orthogonal mutations are in CH1 and CL sequences are described in more detail above in Section 6.4.1.1.

[00198] In a variety of embodiments, the amino acid sequences of the H domain and the L domain separately comprise respectively orthogonal modifications in an endogenous sequence, wherein the H domain interacts with the L domain, and wherein neither the H domain nor the L domain significantly interacts with a domain lacking the orthogonal modification. In a series of embodiments, the orthogonal mutations in the H domain are in a VL sequence and the orthogonal mutations in the L domain are in VH sequence. In specific embodiments, the orthogonal mutations are charge-pair mutations at the VH/VL interface. In preferred embodiments, the charge-pair mutations at the VH/VL interface are a Q39E in VH with a corresponding Q38K in VL, or a Q39K in VH with a corresponding Q38E in VL, as described in greater detail in Igawa et al. {Protein Eng. Des. Sel ., 2010, vol. 23, 667-677), herein incorporated by reference for all it teaches.

[00199] In certain embodiments, the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, and the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen. In certain embodiments, the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, and the interaction between the H domain and the L domain form a second antigen binding site specific for the first antigen.

6.4.18. Trivalent antigen-binding CHI-substituted proteins

[00200] In another series of embodiments, the antigen-binding CH1 -substituted proteins have three antigen binding sites and are therefore termed“trivalent.”

[00201] In some embodiments of the trivalent antigen-binding CH1 -substituted protein, the first polypeptide chain or the third polypeptide chain further comprises a domain N and a domain O, wherein domain N has a variable region domain amino acid sequence, wherein domain O has a constant region amino acid sequence, wherein domains N and O are arranged, from N-terminus to C-terminus, in a N-0 orientation, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain A of the first polypeptide chain or to the N-terminus of domain H of the third polypeptide chain; the binding molecule further comprises a fifth polypeptide chain, comprising: a domain P and a domain Q, wherein the domains are arranged, from N-terminus to C- terminus, in a P-Q orientation, and domain P has a variable region domain amino acid sequence and domain Q has a constant region amino acid sequence; and either the first or third polypeptide chain is associated with the fifth polypeptide chain through an interaction between the N and the P domains and an interaction between the O and the Q domains to form the binding molecule. In some embodiments, the first polypeptide chain further comprises domain N and domain O, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain A of the first polypeptide chain. In some embodiments, the third polypeptide chain further comprises domain N and domain O, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain H of the first polypeptide chain.

[00202] In some embodiments, (a) the amino acid sequences of domain N and domain A are identical, the amino acid sequences of domain H is different from domains N and A, the amino acid sequences of domain O and domain B are identical, the amino acid sequences of domain I is different from domains O and B, the amino acid sequences of domain P and domain F are identical, the amino acid sequences of domain L is different from domains P and F, the amino acid sequences of domain Q and domain G are identical, the amino acid sequences of domain M is different from domains Q and G; and (b) wherein the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for the first antigen.

[00203] In some embodiments, (a) the amino acid sequences of domain N, domain A, and domain H are different, the amino acid sequences of domain O, domain B, and domain I are different, the amino acid sequences of domain P, domain F, and domain L are different, and the amino acid sequences of domain Q, domain G, and domain M are different; and (b) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for a third antigen.

[00204] In some embodiments, domain N , domain A, and domain H each comprise a VL amino acid sequence, domain P, domain F, and domain L each comprise a VH amino acid sequence, domain O and domain Q each comprise a CH3 amino acid sequence, domain B and domain I each comprise a CL amino acid sequence, and domain G and domain M each comprise a CH1 amino acid sequence.

[00205] In some embodiments, domain N, domain A, and domain H are VL domains, domain P, domain F, and domain L are VH domains, domain O and domain Q are CH3 domains, domain B and domain I are CL domains, and domain G and domain M are CH1 domains.

[00206] In some embodiments, the amino acid sequences of the O and the Q domains are identical, and the sequences of the O and the Q domains are endogenous CH3 sequences. [00207] In some embodiments, the amino acid sequences of the O and the Q domains are different and separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, the O domain interacts with the Q domain, and neither the O domain nor the Q domain significantly interacts with a CH3 domain lacking the orthogonal modification.

[00208] In some embodiments, the orthogonal modifications of the O and the Q domains comprise mutations that generate engineered disulfide bridges between domain O and G. In some embodiments, the mutations of the O and the Q domains that generate engineered disulfide bridges are a S354C mutation in one of the O domain and Q domains, and a 349C in the other domain.

[00209] In some embodiments, the orthogonal modifications of the O and the Q domains comprise knob-in-hole mutations. In some embodiments, the knob-in hole mutations of the O and the Q domains are a T366W mutation in one of the O domain and Q domain, and a T366S, L368A, and aY407V mutation in the other domain.

[00210] In some embodiments, the orthogonal modifications of the O and the Q domains comprise charge-pair mutations. In some embodiments, the charge-pair mutations of the O and the Q domains are a T366K mutation in one of the O domain and Q domain, and a L351D mutation in the other domain.

[00211] With reference to FIG. 15, in various trivalent embodiments, (a) the first polypeptide chain further comprises domain N and domain O, wherein the domains are arranged, from N-terminus to C-terminus, in a N-O-A-B-D-E orientation, and wherein domain N has a VL amino acid sequence, domain O has a CH3 amino acid sequence; (b) the antigen-binding CH1- substituted protein further comprises a fifth polypeptide chain, comprising: a domain P and a domain Q, wherein the domains are arranged, from N- terminus to C-terminus, in a P-Q orientation, and wherein domain P has a VH amino acid sequence and domain Q has a CH3 amino acid sequence; and (c) the first and the fifth polypeptides are associated through an interaction between the N and the P domains and an interaction between the O and the Q domains to form the antigen-binding CH1 -substituted protein. As schematized in FIG. 2, these trivalent embodiments are termed“2x1” trivalent constructs.

[00212] With reference to FIG. 18, in a further series of trivalent embodiments, the antigen-binding CH1- substituted proteins further comprise a sixth polypeptide chain, wherein (a) the third polypeptide chain further comprises a domain R and a domain S, wherein the domains are arranged, from N-terminus to C-terminus, in a R-S-H-I-J-K orientation, and wherein domain R has a VL amino acid sequence and domain S has a constant domain amino acid sequence; (b) the antigen-binding CH1- substituted protein further comprises a sixth polypeptide chain, comprising: a domain T and a domain U, wherein the domains are arranged, from N-terminus to C-terminus, in a T-U orientation, and wherein domain T has a VH amino acid sequence and domain U has a constant domain amino acid sequence; and (c) the third and the sixth polypeptides are associated through an interaction between the R and the T domains and an interaction between the S and the U domains to form the antigen-binding CH1 -substituted protein. As schematized in FIG. 2, these trivalent embodiments are termed“1x2” trivalent constructs.

[00213] In a variety of embodiments, the domain O is connected to domain A through a peptide linker. In a variety of embodiments, the domain S is connected to domain H through a peptide linker. In a preferred embodiment, the peptide linker connecting either domain O to domain A or connecting domain S to domain H is a 6 amino acid GSGSGS peptide sequence, as described in more detail in Section 6 4 20 6

6.4.18.1. Trivalent 2x1 Bispecific Constructs [2(A-A)xl(B)]

[00214] With reference to FIG. 15, in a variety of embodiments the amino acid sequences of domain N and domain A are identical, the amino acid sequences of domain H is different from domains N and A, the amino acid sequences of domain O and domain B are identical, the amino acid sequences of domain I is different from domains O and B, the amino acid sequences of domain P and domain F are identical, the amino acid sequences of domain L is different from domains P and F, the amino acid sequences of domain Q and domain G are identical, the amino acid sequences of domain M is different from domains Q and G; and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for the first antigen.

6.4.18.2. Trivalent 2x1 Bispecific Constructs [2(A-B)xl(A)]

[00215] With reference to FIG. 15, in a variety of embodiments the amino acid sequences of domain N and domain H are identical, the amino acid sequences of domain A is different from domains N and H, the amino acid sequences of domain O and domain I are identical, the amino acid sequences of domain B is different from domains O and I, the amino acid sequences of domain P and domain L are identical, the amino acid sequences of domain F is different from domains P and L, the amino acid sequences of domain Q and domain M are identical, the amino acid sequences of domain G is different from domains Q and M; and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for the second antigen.

6.4.18.3. Trivalent 2x1 Trispecific Constructs [2(A-B)xl(C)]

[00216] With reference to FIG. 15, in a variety of embodiments, the amino acid sequences of domain N, domain A, and domain H are different, the amino acid sequences of domain O, domain B, and domain I are different, the amino acid sequences of domain P, domain F, and domain L are different, and the amino acid sequences of domain Q, domain G, and domain M are different; and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for a third antigen.

[00217] In certain embodiments, domain O has a constant region sequence that is a CL from a kappa light chain and domain Q has a constant region sequence that is a CH1 from an IgGl isotype, as discussed in more detail in Section 6.4.1. In a preferred embodiment, domain O and domain Q have CH3 sequences such that they specifically associate with each other, as discussed in more detail above in Section 6.4.15.

6.4.18.4. Trivalent 1x2 Bispecific Constructs [l(A)x2(B-A)]

[00218] With reference to FIG. 18, in a variety of embodiments, the amino acid sequences of domain R and domain A are identical, the amino acid sequences of domain H is different from domain R and A, the amino acid sequences of domain S and domain B are identical, the amino acid sequences of domain I is different from domain S and B, the amino acid sequences of domain T and domain F are identical, the amino acid sequences of domain L is different from domain T and F, the amino acid sequences of domain U and domain G are identical, the amino acid sequences of domain M is different from domain U and G and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for the first antigen. 6.4.18.5. Trivalent 1x2 Bispecific Constructs [l(A)x2(B-B)]

[00219] In a variety of embodiments, the antigen-binding CH1- substituted protein further comprises a second CH1 domain, or portion thereof. With reference to FIG. 18, in specific embodiments, the amino acid sequences of domain R and domain H are identical, the amino acid sequences of domain A is different from domain R and H, the amino acid sequences of domain S and domain I are identical, the amino acid sequences of domain B is different from domain S and I, the amino acid sequences of domain T and domain L are identical, the amino acid sequences of domain F is different from domain T and L, the amino acid sequences of domain U and domain M are identical, the amino acid sequences of domain G is different from domain U and M and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for the second antigen.

[0100] In particular embodiments, the amino acid sequences of domain S and domain I are

CH1 sequences. In particular embodiments, the amino acid sequences of domain U and domain M are CH1 sequences.

6.4.18.6. Trivalent 1x2 Trispecific Constructs [l(A)x2(B-C)]

[00220] With reference to FIG. 18, in a variety of embodiments, the amino acid sequences of domain R, domain A, and domain H are different, the amino acid sequences of domain S, domain B, and domain I are different, the amino acid sequences of domain T, domain F, and domain L are different, and the amino acid sequences of domain U, domain G, and domain M are different; and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for a third antigen.

[00221] In particular embodiments, domain S has a constant region sequence that is a CL from a kappa light chain and domain U has a constant region sequence that is a CH1 from an IgGl isotype, as discussed in more detail in Section 6.4.1. In a preferred embodiment, domain S and domain U have CH3 sequences such that they specifically associate with each other, as discussed in more detail above in Section 6.4.15. [00222] In certain embodiments, the antigen-binding CH1 -substituted protein further comprises a second CH1 domain, or portion thereof. In particular embodiments, the amino acid sequences of domain S and domain I are CH1 sequences. In particular embodiments, the amino acid sequences of domain U and domain M are CH1 sequences.

6.4.19. Tetravalent 2x2 antigen-binding CHI-substituted proteins

[00223] In a variety of embodiments, the antigen-binding CH1- substituted proteins have 4 antigen binding sites and are therefore termed“tetravalent.”

[00224] With reference to FIG. 21, in a further series of embodiments, the antigen binding CH1 -substituted proteins further comprise a fifth and a sixth polypeptide chain, wherein (a) the first polypeptide chain further comprises a domain N and a domain O, wherein the domains are arranged, from N-terminus to C-terminus, in a N-O-A-B-D-E orientation; (b) the third polypeptide chain further comprises a domain R and a domain S, wherein the domains are arranged, from N-terminus to C-terminus, in a R-S-H-I-J-K orientation; (c) the antigen-binding CH1 -substituted protein further comprises a fifth and a sixth polypeptide chain, wherein the fifth polypeptide chain comprises a domain P and a domain Q, wherein the domains are arranged, from N-terminus to C-terminus, in a P-Q orientation, and the sixth polypeptide chain comprises a domain T and a domain U, wherein the domains are arranged, from N-terminus to C-terminus, in a T-U orientation; and (d) the first and the fifth polypeptides are associated through an interaction between the N and the P domains and an interaction between the O and the Q domains, and the third and the sixth polypeptides are associated through an interaction between the Rand the T domains and an interaction between the S and the U domains to form the antigen-binding CH1 -substituted protein.

[00225] In a variety of embodiments, the domain O is connected to domain A through a peptide linker and the domain S is connected to domain H through a peptide linker. In a preferred embodiment, the peptide linker connecting domain O to domain A and connecting domain S to domain H is a 6 amino acid GSGSGS peptide sequence, as described in more detail in Section 6.4.20.6.

6.4.19.1. Tetravalent 2x2 Bispecific Constructs

[00226] With reference to FIG. 21, in a series of tetravalent 2x2 bispecific antigen binding CH1 -substituted proteins, the amino acid sequences of domain N and domain A are identical, the amino acid sequences of domain H and domain R are identical, the amino acid sequences of domain O and domain B are identical, the amino acid sequences of domain I and domain S are identical, the amino acid sequences of domain P and domain F are identical, the amino acid sequences of domain L and domain T are identical, the amino acid sequences of domain Q and domain G are identical, the amino acid sequences of domain M and domain U are identical; and wherein the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the domain N and domain P form a second antigen binding site specific for the first antigen, the interaction between the H domain and the L domain form a third antigen binding site specific for a second antigen, and the interaction between the R domain and the T domain form a fourth antigen binding site specific for the second antigen.

[00227] With reference to FIG. 21, in another series of tetravalent 2x2 bispecific antigen-binding CH1- substituted proteins, the amino acid sequences of domain H and domain A are identical, the amino acid sequences of domain N and domain R are identical, the amino acid sequences of domain I and domain B are identical, the amino acid sequences of domain O and domain S are identical, the amino acid sequences of domain L and domain F are identical, the amino acid sequences of domain P and domain T are identical, the amino acid sequences of domain M and domain G are identical, the amino acid sequences of domain Q and domain U are identical; and wherein the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the domain N and domain P form a second antigen binding site specific for a second antigen, the interaction between the H domain and the L domain form a third antigen binding site specific for the first antigen, and the interaction between the R domain and the T domain form a fourth antigen binding site specific for the second antigen.

6.4.20. Domain Junctions

6.4.20.1. Junctions Connecting VL and CH3 Domains

[00228] In a variety of embodiments, the amino acid sequence that forms a junction between the C-terminus of a VL domain and the N-terminus of a CH3 domain is an engineered sequence. In certain embodiments, one or more amino acids are deleted or added in the C-terminus of the VL domain. In certain embodiments, the junction connecting the C-terminus of a VL domain and the N-terminus of a CH3 domain is one of the sequences described in Table 3 below in Section 6.13.6. In particular embodiments, Al 11 is deleted in the C-terminus of the VL domain. In certain embodiments, one or more amino acids are deleted or added in the N-terminus of the CH3 domain. In particular

embodiments, P343 is deleted in the N-terminus of the CH3 domain. In particular embodiments, P343 and R344 are deleted in the N-terminus of the CH3 domain. In certain embodiments, one or more amino acids are deleted or added to both the C-terminus of the VL domain and the N-terminus of the CH3 domain. In particular embodiments, Al 11 is deleted in the C-terminus of the VL domain and P343 is deleted in the N-terminus of the CH3 domain. In a preferred embodiment, Al 11 and VI 10 are deleted in the C-terminus of the VL domain. In another preferred embodiment, Al 11 and VI 10 are deleted in the C- terminus of the VL domain and the N-terminus of the CH3 domain has a P343 V mutation.

6.4.20.2. Junctions Connecting VH and CH3 Domains

[00229] In a variety of embodiments, the amino acid sequence that forms a junction between the C-terminus of a VH domain and the N-terminus of a CH3 domain is an engineered sequence. In certain embodiments, one or more amino acids are deleted or added in the C-terminus of the VH domain. In certain embodiments, the junction connecting the C-terminus of a VH domain and the N-terminus of the CH3 domain is one of the sequences described in Table 4 below in Section 6.13.6. In particular embodiments, K177 and Gl 18 are deleted in the C-terminus of the VH domain. In certain embodiments, one or more amino acids are deleted or added in the N-terminus of the CH3 domain. In particular embodiments, P343 is deleted in the N-terminus of the CH3 domain. In particular embodiments, P343 and R344 are deleted in the N-terminus of the CH3 domain. In particular embodiments, P343, R344, and E345 are deleted in the N-terminus of the CH3 domain. In certain embodiments, one or more amino acids are deleted or added to both the C-terminus of the VH domain and the N-terminus of the CH3 domain. In a preferred embodiment, T166, K177, and Gl 18 are deleted in the C-terminus of the VH domain.

6.4.20.3. Junctions Connecting CH3 C-terminus to CH2 N- terminus (Hinge)

[00230] In the antigen-binding CH1- substituted proteins described herein, the N- terminus of the CH2 domain has a“hinge” region amino acid sequence. As used herein, hinge regions are sequences of an antibody heavy chain that link the N-terminal variable domain-constant domain segment of an antibody and a CH2 domain of an antibody. In addition, the hinge region typically provides both flexibility between the N-terminal variable domain-constant domain segment and CH2 domain, as well as amino acid sequence motifs that form disulfide bridges between heavy chains (e.g. the first and the third polypeptide chains). As used herein, the hinge region amino acid sequence is SEQ ID NO: 56. [00231] In a variety of embodiments, a CH3 amino acid sequence is extended at the C- terminus at the junction between the C-terminus of the CH3 domain and the N-terminus of a CH2 domain. In certain embodiments, a CH3 amino acid sequence is extended at the C- terminus at the junction between the C-terminus of the CH3 domain and a hinge region, which in turn is connected to the N-terminus of a CH2 domain. In a preferred embodiment, the CH3 amino acid sequence is extended by inserting a PGK tripeptide sequence followed by the DKTHT motif of an IgGl hinge region.

[00232] In a particular embodiment, the extension at the C-terminus of the CH3 domain incorporates amino acid sequences that can form a disulfide bond with orthogonal C-terminal extension of another CH3 domain. In a preferred embodiment, the extension at the C-terminus of the CH3 domain incorporates a KSC tripeptide sequence that is followed by the DKTHT motif of an IgGl hinge region that forms a disulfide bond with orthogonal C-terminal extension of another CH3 domain that incorporates a GEC motif of a kappa light chain.

6.4.20.4. Junctions Connecting CL C-Terminus and CH2 N- Terminus (Hinge)

[00233] In a variety of embodiments, a CL amino acid sequence is connected through its C-terminus to a hinge region, which in turn is connected to the N-terminus of a CH2 domain. Hinge region sequences are described in more detail above in Section 6.4.20.3. In a preferred embodiment, the hinge region amino acid sequence is SEQ ID NO:56.

6.4.20.5. Junctions Connecting CH2 C-terminus to Constant

Region Domain

[00234] In a variety of embodiments, a CH2 amino acid sequence is connected through its C-terminus to the N-terminus of a constant region domain. Constant regions are described in more detail above in Section 6.4.5. In a preferred embodiment, the CH2 sequence is connected to a CH3 sequence via its endogenous sequence. In other

embodiments, the CH2 sequence is connected to a CH1 or CL sequence. Examples discussing connecting a CH2 sequence to a CH1 or CL sequence are described in more detail in LT.S. Pat. No. 8,242,247, which is hereby incorporated in its entirety. 6.4.20.6. Junctions Connecting Domain O to Domain A or

Domain S to Domain H on Trivalent and Tetravalent Molecules

[00235] In a variety of embodiments, heavy chains of antibodies (e.g. the first and third polypeptide chains) are extended at their N-terminus to include additional domains that provide additional ABSs. With reference to Fig. 15, Fig. 18, and Fig. 21, in certain embodiments, the C-terminus of the constant region domain amino acid sequence of a domain O and/or a domain S is connected to the N-terminus of the variable region domain amino acid sequence of a domain A and/or a domain H, respectively. In some preferred embodiments, the constant region domain is a CH3 amino acid sequence and the variable region domain is a VL amino acid sequence. In some preferred embodiments, the constant region domain is a CL amino acid sequence and the variable region domain is a VL amino acid sequence. In certain embodiments, the constant region domain is connected to the variable region domain through a peptide linker. In a preferred embodiment, the peptide linker is a 6 amino acid GSGSGS peptide sequence.

[00236] In a variety of embodiments, light chains of antibodies (e.g. the second and fourth polypeptide chains) are extended at their N-terminus to include additional variable domain-constant domain segments of an antibody. In certain embodiments, the constant region domain is a CH1 amino acid sequence and the variable region domain is a VH amino acid sequence.

6.5. Specific Bivalent antigen-binding CHI-substituted proteins

[00237] In a further aspect, bivalent antigen-binding CH1 -substituted proteins are provided.

[00238] With reference to FIG. 3, in a first series of embodiments the antigen-binding CH1- substituted proteins comprise a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B- D-E orientation, and domain A has a VL amino acid sequence, domain B has a CH3 amino acid sequence, domain D has a CH2 amino acid sequence, and domain E has a constant region domain amino acid sequence; (b) the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a VH amino acid sequence and domain G has a CH3 amino acid sequence; (c) the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C- terminus, in a H-I-J-K orientation, and wherein domain H has a variable region domain amino acid sequence, domain I has a constant region domain amino acid sequence, domain J has a CH2 amino acid sequence, and K has a constant region domain amino acid sequence; (d) the fourth polypeptide chain comprises a domain L and a domain M, wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and wherein domain L has a variable region domain amino acid sequence and domain M has a constant region domain amino acid sequence; (e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains; (f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains; and (g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen-binding CH1 -substituted protein.

[00239] In a preferred embodiment, domain E has a CH3 amino acid sequence, domain H has a VL amino acid sequence, domain I has a CL amino acid sequence, domain K has a CH3 amino acid sequence, domain L has a VH amino acid sequence, and domain M has a CH1 amino acid sequence.

[00240] In certain embodiments, the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, and the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the antigen-binding CH1 -substituted protein is a bispecific bivalent antigen-binding CH1- substituted protein. In certain embodiments, the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, and the interaction between the H domain and the L domain form a second antigen binding site specific for the first antigen, and the antigen-binding CH1- substituted protein is a monospecific bivalent antigen-binding CH1 -substituted protein.

6.5.1. Bivalent Bispecific B-Body“BCl”

[00241] With reference to FIG. 3 and FIG. 4, in a series of embodiments, the antigen binding CH1 -substituted protein has a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B- D-E orientation, and domain A has a first VL amino acid sequence, domain B has a human IgGl CH3 amino acid sequence with a T366K mutation and a C-terminal extension incorporating a KSC tripeptide sequence that is followed by the DKTHT motif of an IgGl hinge region, domain D has a human IgGl CH2 amino acid sequence, and domain E has human IgGl CH3 amino acid with a S354C and T366W mutation; (b) the second polypeptide chain has a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a first VH amino acid sequence and domain G has a human IgGl CH3 amino acid sequence with a L351D mutation and a C-terminal extension incorporating a GEC amino acid disulfide motif; (c) the third polypeptide chain has a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C-terminus, in a H-I-J-K orientation, and wherein domain H has a second VL amino acid sequence, domain I has a human CL kappa amino acid sequence, domain J has a human IgGl CH2 amino acid sequence, and K has a human IgGl CH3 amino acid sequence with a Y349C, a D356E, a L358M, a T366S, a L368A, and a Y407V mutation; (d) the fourth polypeptide chain has a domain L and a domain M, wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and wherein domain L has a second VH amino acid sequence and domain M has a human IgGl CH1 amino acid sequence; (e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains; (f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains; (g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen-binding CH1 -substituted protein; (h) domain A and domain F form a first antigen binding site specific for a first antigen; and (i) domain H and domain L form a second antigen binding site specific for a second antigen.

[00242] In preferred embodiments, the first polypeptide chain has the sequence SEQ ID NO: 8, the second polypeptide chain has the sequence SEQ ID NO: 9, the third polypeptide chain has the sequence SEQ ID NO: 10, and the fourth polypeptide chain has the sequence SEQ ID NO: 11.

6.5.2. Bivalent Bispecific B-Body“BC6”

[00243] With reference to FIG. 3 and FIG. 11, in a series of embodiments, the antigen-binding CH1- substituted protein has a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, and domain A has a first VL amino acid sequence, domain B has a human IgGl CH3 amino acid sequence with a C-terminal extension incorporating a KSC tripeptide sequence that is followed by the DKTHT motif of an IgGl hinge region, domain D has a human IgGl CH2 amino acid sequence, and domain E has human IgGl CH3 amino acid with a S354C and a T366W mutation; (b) the second polypeptide chain has a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F- G orientation, and wherein domain F has a first VH amino acid sequence and domain G has a human IgGl CH3 amino acid sequence with a C-terminal extension incorporating a GEC amino acid disulfide motif; (c) the third polypeptide chain has a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C- terminus, in a H-I-J-K orientation, and wherein domain H has a second VL amino acid sequence, domain I has a human CL kappa amino acid sequence, domain J has a human IgGl CH2 amino acid sequence, and K has a human IgGl CH3 amino acid sequence with a Y349C, a D356E, a L358M, a T366S, a L368A, and a Y407V mutation; (d) the fourth polypeptide chain has a domain L and a domain M, wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and wherein domain L has a second VH amino acid sequence and domain M has a human IgGl amino acid sequence; (e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains; (f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains; (g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen-binding CH1 -substituted protein; (h) domain A and domain F form a first antigen binding site specific for a first antigen; and (i) domain H and domain L form a second antigen binding site specific for a second antigen.

6.5.3. Bivalent Bispecific B-Body“BC28”

[00244] With reference to FIG. 3 and FIG. 13, in a series of embodiments, the antigen-binding CH1- substituted protein has a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, and domain A has a first VL amino acid sequence, domain B has a human IgGl CH3 amino acid sequence with a Y349C mutation and a C-terminal extension incorporating a PGK tripeptide sequence that is followed by the DKTHT motif of an IgGl hinge region, domain D has a human IgGl CH2 amino acid sequence, and domain E has a human IgGl CH3 amino acid with a S354C and a T366W mutation; (b) the second polypeptide chain has a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a first VH amino acid sequence and domain G has a human IgGl CH3 amino acid sequence with a S354C mutation and a C-terminal extension incorporating a PGK tripeptide sequence; (c) the third polypeptide chain has a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C-terminus, in a H-I-J-K orientation, and wherein domain H has a second VL amino acid sequence, domain I has a human CL kappa amino acid sequence, domain J has a human IgGl CH2 amino acid sequence, and K has a human IgGl CH3 amino acid sequence with a Y349C, a D356E, a L358M, a T366S, a L368A, and a Y407V; (d) the fourth polypeptide chain has a domain L and a domain M, wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and wherein domain L has a second VH amino acid sequence and domain M has a human IgGl CH1 amino acid sequence; (e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains; (f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains; (g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen-binding CH1 -substituted protein; (h) domain A and domain F form a first antigen binding site specific for a first antigen; and (i) domain H and domain L form a second antigen binding site specific for a second antigen.

[00245] In preferred embodiments, the first polypeptide chain has the sequence SEQ ID NO:24, the second polypeptide chain has the sequence SEQ ID NO:25, the third polypeptide chain has the sequence SEQ ID NO: 10, and the fourth polypeptide chain has the sequence SEQ ID NO: 11.

6.5.4. Bivalent Bispecific B-Body“BC44”

[00246] With reference to FIG. 3 and FIG. 19, in a series of embodiments, the antigen-binding CH1- substituted protein has a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, and domain A has a first VL amino acid sequence, domain B has a human IgGl CH3 amino acid sequence with a Y349C mutation, a P343 V mutation, and a C-terminal extension incorporating a PGK tripeptide sequence that is followed by the DKTHT motif of an IgGl hinge region, domain D has a human IgGl CH2 amino acid sequence, and domain E has human IgGl CH3 amino acid with a S354C mutation and a T366W mutation; (b) the second polypeptide chain has a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a first VH amino acid sequence and domain G has a human IgGl CH3 amino acid sequence with a S354C mutation and a C-terminal extension incorporating a PGK tripeptide sequence; (c) the third polypeptide chain has a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C- terminus, in a H-I-J-K orientation, and wherein domain H has a second VL amino acid sequence, domain I has a human CL kappa amino acid sequence, domain J has a human IgGl CH2 amino acid sequence, and K has a human IgGl CH3 amino acid sequence with a Y349C, T366S, L368A, and aY407V; (d) the fourth polypeptide chain has a domain L and a domain M, wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and wherein domain L has a second VH amino acid sequence and domain M has a human IgGl amino acid sequence; (e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains; (f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains; and (g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen-binding CH1 -substituted protein; (h) domain A and domain F form a first antigen binding site specific for a first antigen; and (i) domain H and domain L form a second antigen binding site specific for a second antigen.

[00247] In preferred embodiments, the first polypeptide chain has the sequence SEQ ID NO:32, the second polypeptide chain has the sequence SEQ ID NO:25, the third polypeptide chain has the sequence SEQ ID NO: 10, and the fourth polypeptide chain has the sequence SEQ ID NO: 11. 6.6. Specific Trivalent antigen-binding CHI-substituted proteins

6.6.1. Trivalent 1x2 Bispecific B-Body“BC28-lx2”

[00248] With reference to Section 6.5.3. and FIG. 18, in a series of embodiments, the antigen-binding CH1- substituted proteins further comprise a sixth polypeptide chain, wherein (a) the third polypeptide chain further comprises a domain R and a domain S, wherein the domains are arranged, from N-terminus to C-terminus, in a R-S-H-I-J-K orientation, and wherein domain R has the first VL amino acid sequence and domain S has a human IgGl CH3 amino acid sequence with a Y349C mutation and a C-terminal extension incorporating a PGK tripeptide sequence that is followed by GSGSGS linker peptide connecting domain S to domain H; (b) the antigen-binding CH1- substituted protein further comprises a sixth polypeptide chain, comprising: a domain T and a domain U, wherein the domains are arranged, from N-terminus to C-terminus, in a T-U orientation, and wherein domain T has the first VH amino acid sequence and domain U has a human IgGl CH3 amino acid sequence with a S354C mutation and a C-terminal extension incorporating a PGK tripeptide sequence; (c) the third and the sixth polypeptides are associated through an interaction between the R and the T domains and an interaction between the S and the U domains to form the antigen-binding CH1- substituted protein, and (d) domain R and domain T form a third antigen binding site specific for the first antigen.

[00249] In preferred embodiments, the first polypeptide chain has the sequence SEQ ID NO:24, the second polypeptide chain has the sequence SEQ ID NO:25, the third polypeptide chain has the sequence SEQ ID NO: 37, the fourth polypeptide chain has the sequence SEQ ID NO:l 1, and the sixth polypeptide chain has the sequence SEQ ID NO:25.

6.6.2. Trivalent 1x2 Trispecific B-Body“BC28-lxlxla”

[00250] With reference to Section 6.5.3. and FIG. 18 and FIG. 19, in a series of embodiments, the antigen-binding CH1 -substituted proteins further comprise a sixth polypeptide chain, wherein (a) the third polypeptide chain further comprises a domain R and a domain S, wherein the domains are arranged, from N-terminus to C-terminus, in a R- S-H-I-J-K orientation, and wherein domain R has a third VL amino acid sequence and domain S has a human IgGl CH3 amino acid sequence with a T366K mutation and a C- terminal extension incorporating a KSC tripeptide sequence that is followed by GSGSGS linker peptide connecting domain S to domain H; (b) the antigen-binding CH1- substituted protein further comprises a sixth polypeptide chain, comprising: a domain T and a domain U, wherein the domains are arranged, from N-terminus to C-terminus, in a T-U orientation, and wherein domain T has a third VH amino acid sequence and domain EG has a human IgGl CH3 amino acid sequence with a L351D mutation and a C-terminal extension incorporating a GEC amino acid disulfide motif; and (c) the third and the sixth polypeptides are associated through an interaction between the R and the T domains and an interaction between the S and the EG domains to form the antigen-binding CH1- substituted protein, and (d) domain R and domain T form a third antigen binding site specific for a third antigen.

[00251] In preferred embodiments, the first polypeptide chain has the sequence SEQ ID NO:24, the second polypeptide chain has the sequence SEQ ID NO:25, the third polypeptide chain has the sequence SEQ ID NO:45, the fourth polypeptide chain has the sequence SEQ ID NO:l 1, and the sixth polypeptide chain has the sequence SEQ ID NO:

53.

6.7. Additional CHI-substituted proteins

[00252] In some embodiments, the antigen-binding CH1 -substituted protein is a lxl MH2 bivalent bispecific platform, described in, e.g., W02017011342, which is hereby

incorporated by reference in its entirety.

[00253] In some embodiments, the antigen-binding CH1 -substituted protein is a CH3 Domain Substitution multispecific platform, described in WO2016087650, which is hereby incorporated by reference in its entirety.

6.8. Antigen specificities

[00254] The antigen binding sites of the antigen-binding CH1 -substituted proteins described herein may be chosen to specifically bind a wide variety of molecular targets.

For example, an antigen binding site or sites may specifically bind E-Cad, CLDN7, FGFR2b, N-Cad, Cad-l l, FGFR2c, ERBB2, ERBB3, FGFR1, FOLR1, IGF-Ira, GLP1R, PDGFRa, PDGFRb, EPHB6, ABCG2, CXCR4, CXCR7, Integrin-avb3, SPARC, VCAM, ICAM, Annexin, ROR1, ROR2, TNFa, CD 137, angiopoietin 2, angiopoietin 3, BAFF, beta amyloid, C5, CA-125, CD147, CD125, CD147, CD152, CD19, CD20, CD22, CD23,

CD24, CD25, CD274, CD28, CD3, CD30, CD33, CD37, CD4, CD40, CD44, CD44v4, CD44v6, CD44v7, CD50, CD51, CD52, CEA, CSF1R, CTLA-2, DLL4, EGFR, EPCAM, HER3, GD2 ganglioside, GDF-8, Her2/neu, CD2221, IL-17A, IL-12, IL-23, IL-13, IL-6, IL-23, an integrin, CDl la, MUC1, Notch, TAG-72, TGFp, TRAIL-R2, VEGF-A, VEGFR- 1, VEGFR2, VEGFc, hematopoietins (four-helix bundles) (such as EPO (erythropoietin), IL-2 (T-cell growth factor), IL-3 (multicolony CSF), IL-4 (BCGF-l, BSF-l), IL-5 (BCGF- 2), IL-6 IL-4 (IFN-D2, BSF-2, BCDF), IL-7, IL-8, IL-9, IL-l l, IL-13 (P600), G-CSF, IL- 15 (T-cell growth factor), GM-CSF (granulocyte macrophage colony stimulating factor), OSM (OM, oncostatin M), and LIF (leukemia inhibitory factor)); interferons (such as IFN- g, IFN-a, and IFN-b); immunoglobin superfamily (such as B7.1 (CD80), and B7.2 (B70, CD86)); TNF family (such as TNF-a (cachectin), TNF-b (lymphotoxin, LT, LT-a), LT-b, Fas, CD27, CD30, and 4-1BBL); and those unassigned to a particular family (such as TGF- b, IL la, IL- 1 b, IL-l RA, IL-10 (cytokine synthesis inhibitor F), IL-12 (NK cell stimulatory factor), MIF, IL-16, IL-17 (mCTLA-8), and/or IL-18 (IGIF, interferon-g inducing factor)); in embodiments relating to bispecific antibodies, the antibody may for example bind two of these targets. Furthermore, the Fc portion of the heavy chain of an antibody may be used to target Fc receptor-expressing cells such as the use of the Fc portion of an IgE antibody to target mast cells and basophils.

[00255] An antigen binding site or sites may be chosen that specifically binds the TNF family of receptors including, but not limited to, TNFR1 (also known as CD 120a and TNFRSF1A), TNFR2 (also known as CDl20b and TNFRSF1B), TNFRSF3 (also known as I^R), TNFRSF4 (also known as 0X40 and CD 134), TNFRSF5 (also known as CD40), TNFRSF6 (also known as FAS and CD95), TNFRSF6B (also known as DCR3), TNFRSF7 (also known as CD27), TNFRSF8 (also known as CD30), TNFRSF9 (also known as 4- 1BB), TNFRSF10A (also known as TRAILR1, DR4, and CD26), TNFRSF10B (also known as TRAILR2, DR5, and CD262), TNFRSF10C (also known as TRAILR3, DCR1, CD263), TNFRSF10D (also known as TRAILR4, DCR2, and CD264), TNFRSF11 A (also known as RANK and CD265), TNFRSF11B (also known as OPG), TNFRSF12A (also known as FN14, TWEAKR, and CD266), TNFRSF13B (also known as TACI and CD267), TNFRSF13C (also known as BAFFR, BR3, and CD268), TNFRSF14 (also known as HVEM and CD270), TNFRSF16 (also known as NGFR, p75NTR, and CD271), or

TNFRSF17 (also known as BCMA and CD269), TNFRSF18 (also known as GITR and CD357), TNFRSF19 (also known as TROY, TAJ, and TRADE), TNFRSF21 (also known as CD358), TNFRSF25 (also known as Apo-3, TRAMP, LARD, or WS-l), EDA2R (also known as XEDAR).

[00256] An antigen binding site or sites may be chosen that specifically binds immune- oncology targets including, but not limited to, checkpoint inhibitor targets such as PD1, PDL1, CTLA-4, PDL2, B7-H3, B7-H4, BTLA, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, BY55, and CGEN-15049.

[00257] In a series of embodiments, an antigen binding site or sites may be chosen that specifically target tumor-associated cells. In various embodiments, the antigen binding site or sites specifically target tumor associated immune cells. In certain embodiments, the antigen binding site or sites specifically target tumor associated regulatory T cells (Tregs). In specific embodiments, an antigen-binding CH1 -substituted protein has antigen binding sites specific for antigens selected from one or more of CD25, 0X40, CTLA-4, and NRP1 such that the antigen-binding CH1 -substituted protein specifically targets tumor associated regulatory T cells. In specific embodiments, an antigen-binding CH1- substituted protein has antigen binding sites that specifically bind CD25 and 0X40, CD25 and CTLA-4, CD25 and NRP1, 0X40 and CTLA-4, 0X40 and NRP1, or CTLA-4 and NRP1 such that the antigen-binding CH1 -substituted protein specifically targets tumor associated regulatory T cells. In preferred embodiments, a bispecific bivalent antigen-binding CH1 -substituted protein has antigen binding sites that specifically bind CD25 and 0X40, CD25 and CTLA- 4, CD25 and NRP1, 0X40 and CTLA-4, 0X40 and NRP1, or CTLA-4 and NRP1 such that the antigen-binding CH1- substituted protein specifically targets tumor associated regulatory T cells. In specific embodiments, the specific targeting of the tumor associated regulatory T cells results in depletion (e.g. killing) of the regulatory T cells. In preferred

embodiments, the depletion of the regulatory T cells is mediated by an antibody-drug conjugate (ADC) modification, such as an antibody conjugated to a toxin, as discussed in more detail below in Section 6.9.1.

[00258] In a series of embodiments, an antigen-binding CH1 -substituted protein has antigen binding sites selected from one or more of CD3, ROR1, and ROR2. In a specific embodiment, a bispecific bivalent has antigen binding sites that specifically bind CD3 and ROR1. In a specific embodiment, a bispecific bivalent has antigen binding sites that specifically bind CD3 and ROR2. In a specific embodiment, a trispecific trivalent has antigen binding sites that specifically bind CD3, ROR1, and ROR2.

6.9. Further modifications

[00259] In a further series of embodiments, the antigen-binding CH1- substituted protein has additional modifications.

6.9.1. Antigen-binding CHI-substituted protein-Drug

Conjugates

[00260] In various embodiments, the antigen-binding CH1- substituted protein is conjugated to a therapeutic agent (i.e. drug) to form an antigen-binding CH1- substituted protein-drug conjugate. Therapeutic agents include, but are not limited to,

chemotherapeutic agents, imaging agents (e.g. radioisotopes), immune modulators (e.g. cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g. cytotoxic agents). In certain embodiments, the therapeutic agents are attached to the antigen-binding CH1- substituted protein through a linker peptide, as discussed in more detail below in Section 6.9.3.

[00261] Methods of preparing antibody-drug conjugates (ADCs) that can be adapted to conjugate drugs to the antigen-binding CH1- substituted proteins disclosed herein are described, e.g., in US patent no. 8,624,003 (pot method), US patent no. 8,163,888 (one- step), US patent no. 5,208,020 (two-step method), US patent No. 8,337,856, US patent no. 5,773,001, US patent no. 7,829,531, US patent no. 5,208,020, US patent no. 7,745,394, WO 2017/136623, WO 2017/015502, WO 2017/015496, WO 2017/015495, WO 2004/010957, WO 2005/077090, WO 2005/082023, WO 2006/065533, WO 2007/030642, WO

2007/103288, WO 2013/173337, WO 2015/057699, WO 2015/095755, WO 2015/123679, WO 2015/157286, WO 2017/165851, WO 2009/073445, WO 2010/068759, WO

2010/138719 , WO 2012/171020, WO 2014/008375, WO 2014/093394, WO 2014/093640, WO 2014/160360, WO 2015/054659, WO 2015/195925, WO 2017/160754, Storz ( MAbs . 2015 Nov-Dec; 7(6): 989-1009), Lambert et al {Adv Ther, 2017 34: 1015), Di am antis et al. ( British Journal of Cancer , 2016, 114, 362-367), Carrico et al. ( Nat Chem Biol , 2007.

3: 321-2), We et al. {Proc Natl Acad Sci USA, 2009. 106: 3000-5), Rabuka et al. {Curr Opin Chem Biol., 2011 14: 790-6), Hudak et al. ( Angew Chem Int Ed Engl., 2012: 4161-5), Rabuka et al. {Nat Protoc., 2012 7: 1052-67), Agarwal et al. {Proc Natl Acad Sci USA., 2013, 110: 46-51), Agarwal et al. {Bioconjugate Chem., 2013, 24: 846-851), Barfield et al. {Drug Dev. andD., 2014, 14:34-41), Drake et al. {Bioconjugate Chem., 2014, 25: 1331-41), Liang et al. {J Am Chem Soc., 2014, 136:10850-3), Drake et al. {Curr Opin Chem Biol., 2015, 28: 174-80), and York et al. {BMC Biotechnology, 2016, l6(l):23), each of which is hereby incorporated by reference in its entirety for all that it teaches.

6.9.2. Additional Binding Moieties

[00262] In various embodiments, the antigen-binding CH1- substituted protein has modifications that comprise one or more additional binding moieties. In certain

embodiments the binding moieties are antibody fragments or antibody formats including, but not limited to, full-length antibodies, Fab fragments, Fvs, scFvs, tandem scFvs, Diabodies, scDiabodies, DARTs, tandAbs, minibodies, camelid VHH, and other antibody fragments or formats known to those skilled in the art. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), herein incorporated by reference for all that it teaches.

[00263] In particular embodiments, the one or more additional binding moieties are attached to the C-terminus of the first or third polypeptide chain. In particular

embodiments, the one or more additional binding moieties are attached to the C-terminus of both the first and third polypeptide chain. In particular embodiments, the one or more additional binding moieties are attached to the C-terminus of both the first and third polypeptide chains. In certain embodiments, individual portions of the one or more additional binding moieties are separately attached to the C-terminus of the first and third polypeptide chains such that the portions form the functional binding moiety.

[00264] In particular embodiments, the one or more additional binding moieties are attached to the N-terminus of any of the polypeptide chains (e.g. the first, second, third, fourth, fifth, or sixth polypeptide chains). In certain embodiments, individual portions of the additional binding moieties are separately attached to the N-terminus of different polypeptide chains such that the portions form the functional binding moiety.

[00265] In certain embodiments, the one or more additional binding moieties are specific for a different antigen or epitope of the ABSs within the antigen-binding CH1- substituted protein. In certain embodiments, the one or more additional binding moieties are specific for the same antigen or epitope of the ABSs within the antigen-binding CH1- substituted protein. In certain embodiments, wherein the modification is two or more additional binding moieties, the additional binding moieties are specific for the same antigen or epitope. In certain embodiments, wherein the modification is two or more additional binding moieties, the additional binding moieties are specific for different antigens or epitopes.

[00266] In certain embodiments, the one or more additional binding moieties are attached to the antigen-binding CH1 -substituted protein using in vitro methods including, but not limited to, reactive chemistry and affinity tagging systems, as discussed in more detail below in Section 6.9.3. In certain embodiments, the one or more additional binding moieties are attached to the antigen-binding CH1 -substituted protein through Fc-mediated binding (e.g. Protein A/G). In certain embodiments, the one or more additional binding moieties are attached to the antigen-binding CH1 -substituted protein using recombinant DNA techniques, such as encoding the nucleotide sequence of the fusion product between the antigen-binding CH1- substituted protein and the additional binding moieties on the same expression vector (e.g. plasmid). 6.9.3. Functional/Reactive Groups

[00267] In various embodiments, the antigen-binding CH1- substituted protein has modifications that comprise functional groups or chemically reactive groups that can be used in downstream processes, such as linking to additional moieties (e.g. drug conjugates and additional binding moieties, as discussed in more detail above in Sections 6.9.1. and 6.9.2.) and downstream purification processes.

[00268] In certain embodiments, the modifications are chemically reactive groups including, but not limited to, reactive thiols (e.g. maleimide based reactive groups), reactive amines (e.g. A-hydroxy sued ni mi de based reactive groups),“click chemistry” groups (e.g. reactive alkyne groups), and aldehydes bearing formylglycine (FGly). In certain

embodiments, the modifications are functional groups including, but not limited to, affinity peptide sequences (e.g. HA, HIS, FLAG, GST, MBP, and Strep systems etc.). In certain embodiments, the functional groups or chemically reactive groups have a cleavable peptide sequence. In particular embodiments, the cleavable peptide is cleaved by means including, but not limited to, photocleavage, chemical cleavage, protease cleavage, reducing conditions, and pH conditions. In particular embodiments, protease cleavage is carried out by intracellular proteases. In particular embodiments, protease cleavage is carried out by extracellular or membrane associated proteases. ADC therapies adopting protease cleavage are described in more detail in Choi et al. ( Theranostics , 2012; 2(2): 156-178.), the entirety of which is hereby incorporated by reference for all it teaches.

6.10. Pharmaceutical compositions

[00269] In another aspect, pharmaceutical compositions are provided that comprise an antigen-binding CH1- substituted protein as described herein and a pharmaceutically acceptable carrier or diluent. In typical embodiments, the pharmaceutical composition is sterile.

[00270] In various embodiments, the pharmaceutical composition comprises the antigen-binding CH1 -substituted protein at a concentration of 0.1 mg/ml - 100 mg/ml. In specific embodiments, the pharmaceutical composition comprises the antigen-binding CH1- substituted protein at a concentration of 0.5 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml, 5 mg/ml, 7.5 mg/ml, or 10 mg/ml. In some embodiments, the pharmaceutical composition comprises the antigen-binding CH1 -substituted protein at a concentration of more than 10 mg/ml. In certain embodiments, the antigen-binding CH1- substituted protein is present at a concentration of 20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, or even 50 mg/ml or higher. In particular embodiments, the antigen-binding CH1- substituted protein is present at a concentration of more than 50 mg/ml.

[00271] In various embodiments, the pharmaceutical compositions are described in more detail in U.S. Pat No. 8,961,964, U.S. Pat No. 8,945,865, U.S. Pat No. 8,420,081,

U.S. Pat No. 6,685,940, U.S. Pat No. 6,171,586, U.S. Pat No. 8,821,865, U.S. Pat No. 9,216,219, US application 10/813,483, WO 2014/066468, WO 2011/104381, and WO 2016/180941, each of which is incorporated herein in its entirety.

6.11. Methods of Manufacturing

[00272] The antigen-binding CH1 -substituted proteins described herein can readily be manufactured by expression using standard cell free translation, transient transfection, and stable transfection approaches currently used for antibody manufacture.

[00273] In some embodiments, Expi293 cells (ThermoFisher) can be used for production of the antigen-binding CH1 -substituted proteins using protocols and reagents from ThermoFisher, such as ExpiFectamine, or other reagents known to those skilled in the art, such as polyethylenimine as described in detail in Fang et al. {Biological Procedures Online , 2017, 19: 11), herein incorporated by reference for all it teaches.

[00274] In some embodiments, ExpiCHO (ThermoFisher) can be used for production of the antigen-binding CH1 -substituted proteins using protocols and reagents from

ThermoFisher, such as ExpiFectamine, or other reagents known to those skilled in the art, such as polyethylenimine as described in detail in Fang et al. {Biological Procedures Online , 2017, 19: 11).

[00275] As further described in the Examples below, the expressed proteins can be readily separated from undesired proteins and protein complexes using a CH1 affinity resin, such as the CaptureSelect CH1 resin and provided protocol from ThermoFisher. Further purification can be affected using ion exchange chromatography as is routinely used in the art.

6.12. Methods of Treatment

[00276] In another aspect, methods of treatment are provided, the methods comprising administering an antigen-binding CH1 -substituted protein as described herein to a patient in an amount effective to treat the patient.

[00277] In some embodiments, an antibody of the present disclosure may be used to treat a cancer. The cancer may be a cancer from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In some embodiments, the cancer may be a neoplasm, malignant; carcinoma; carcinoma,

undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma;

nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous

adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma;

cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant;

paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma;

alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;

hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma;

ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma;

primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[00278] An antibody of the present disclosure may be administered to a subject per se or in the form of a pharmaceutical composition for the treatment of, e.g., cancer, autoimmunity, transplantation rejection, post-traumatic immune responses, graft-versus- host disease, ischemia, stroke, and infectious diseases, for example by targeting viral antigens, such as gpl20 of HIV.

6.13. Examples

[00279] The following examples are provided by way of illustration, not limitation.

6.13.1. Methods

[00280] Non-limiting, illustrative methods for the purification of the various antigen binding proteins are described in more detail below.

6.13.1.1. Expi293 Expression

[00281] The various antigen-binding proteins tested were expressed using the Expi293 transient transfection system according to manufacturer’s instructions. Briefly, four plasmids coding for four individual chains were mixed at 1 : 1 : 1 : 1 mass ratio, unless otherwise stated, and transfected with ExpiFectamine 293 transfection kit into Expi293 cells. Cells were cultured at 37°C with 8% C02, 100% humidity and shaking at 125 rpm. Transfected cells were fed once after 16-18 hours of transfections. The cells were harvested at day 5 by centrifugation at 2000 g for 10 minutes. The supernatant was collected for affinity chromatography purification.

6.13.1.1. ExpiCHO Expression

[00282] The various antigen-binding proteins tested were expressed using the

ExpiCHO transient transfection system according to manufacturer’s instructions. Briefly, four plasmids coding for four individual chains were mixed at 1 : 1 : 1 : 1 mass ratio, unless otherwise stated, and transfected with ExpiFectamine CHO transfection kit into ExpiCHO. Cells were cultured at 37°C with 8% C02, 100% humidity and shaking at 125 rpm.

Transfected cells were fed once after 16-18 hours of transfections. The cells were harvested at day 5 by centrifugation at 2000 g for 10 munities. The supernatant was collected for affinity chromatography purification.

6.13.1.2. Protein A and Anti-CHI Purification

[00283] Cleared supernatants containing the various antigen-binding proteins were separated using either a Protein A (ProtA) resin or an anti-CHl resin on an AKTA Purifier FPLC. In examples where a head-to-head comparison was performed, supernatants containing the various antigen-binding proteins were split into two equal samples. For ProtA purification, a 1 mL Protein A column (GE Healthcare) was equilibrated with PBS (5 mM sodium potassium phosphate pH 7.4, 150 mM sodium chloride). The sample was loaded onto the column at 5 ml/min. The sample was eluted using 0.1 M acetic acid pH 4.0. The elution was monitored by absorbance at 280 nm and the elution peaks were pooled for analysis. For anti-CHl purification, a 1 mL CaptureSelect™ XL column (ThermoFisher) was equilibrated with PBS. The sample was loaded onto the column at 5 ml/min. The sample was eluted using 0.1 M acetic acid pH 4.0. The elution was monitored by absorbance at 280 nm and the elution peaks were pooled for analysis.

6.13.1.3. SDS-Page Analysis

[00284] Samples containing the various separated antigen-binding proteins were analyzed by reducing and non-reducing SDS-PAGE for the presence of complete product, incomplete product, and overall purity. 2 ug of each sample was added to 15 uL SDS loading buffer. Reducing samples were incubated in the presence of 10 mM reducing agent at 75°C for 10 minutes. Non-reducing samples were incubated at 95°C for 5 minutes without reducing agent. The reducing and non-reducing samples were loaded into a 4-15% gradient TGX gel (BioRad) with running buffer and run for 30 minutes at 250 volts. Upon completion of the run, the gel was washed with DI water and stained using GelCode Blue Safe Protein Stain (ThermoFisher). The gels were destained with DI water prior to analysis. Densitometry analysis of scanned images of the destained gels was performed using standard image analysis software to calculate the relative abundance of bands in each sample.

6.13.1.4. IEX Chromatography

[00285] Samples containing the various separated antigen-binding proteins were analyzed by cation exchange chromatography for the ratio of complete product to incomplete product and impurities. Cleared supernatants were analyzed with a 5-ml MonoS (GE Lifesciences) on an AKTA Purifier FPLC. The MonoS column was equilibrated with buffer A 10 mM MES pH 6.0. The samples were loaded onto the column at 2 ml/min. The sample was eluted using a 0-30% gradient with buffer B (10 mM MES pH 6.0, 1 M sodium chloride) over 6 CV. The elution was monitored by absorbance at 280 nm and the purity of the samples were calculated by peak integration to identify the abundance of the monomer peak and contaminants peaks. The monomer peak and contaminant peaks were separately pooled for analysis by SDS-PAGE as described above.

6.13.1.5. Analytical SEC Chromatography

[00286] Samples containing the various separated antigen-binding proteins were analyzed by analytical size exclusion chromatography for the ratio of monomer to high molecular weight product and impurities. Cleared supernatants were analyzed with an industry standard TSK G3000SWxl column (Tosoh Bioscience) on an Agilent 1100 HPLC. The TSK column was equilibrated with PBS. 25 uL of each sample at 1 mg/mL was loaded onto the column at 1 ml/min. The sample was eluted using an isocratic flow of PBS for 1.5 CV. The elution was monitored by absorbance at 280 nm and the elution peaks were analyzed by peak integration.

6.13.1.6. Mass Spec

[00287] Samples containing the various separated antigen-binding proteins were analyzed by mass spectrometry to confirm the correct species by molecular weight. All analysis was performed by a third-party research organization. Briefly, samples were treated with a cocktail of enzymes to remove glycosylation. Samples were both tested in the reduced format to specifically identify each chain by molecular weight. Samples were all tested under non-reducing conditions to identify the molecular weights of all complexes in the samples. Mass spec analysis was used to identify the number of unique products based on molecular weight.

6.13.2. Example 1: Anti-CHI purification of bispecifics with

standard CHI architectures contains significant incomplete background antibodies

[00288] We tested the anti-CHl purification efficiency of bispecific antibodies that have standard knob-hole orthogonal mutations introduced into CH3 domains found in their native positions within the Fc portion of the bispecific antibody. Therefore, the two antibodies tested, KL27-6 and KL27-7, each contained two CH1 domains, one on each arm of the antibody. As described in more detail in Section 6.13.1, each bispecific antibody was expressed using the Expi293 system, purified from undesired protein products on an anti- CHl column, and run on an SDS-PAGE gel. As shown in Fig. 1, a significant band at 75 kDa representing an incomplete bispecific antibody was present, interpreted as a complex containing only (i) a first and second or (ii) third and fourth polypeptide chains with reference to FIG. 3. Thus, methods using anti-CHl to purify complete bispecific molecules that have a CH1 domain in each arm resulted in significant background contamination of incomplete antibody complexes.

6.13.3. Example 2: Bivalent bispecific B-Body“BCl”

[00289] We constructed a bivalent bispecific construct, termed“BCl”, specific for PD1 and a second antigen,“Antigen A”. Salient features of the“BCl” architecture are illustrated in FIG. 4.

[00290] In greater detail, with domain and polypeptide chain references in accordance with FIG. 3 and modifications from native sequence indicated in parentheses, the architecture was:

I^st polypeptide chain (SEQ ID NO: 8)

Domain A = VL (“Antigen A”)

Domain B = CH3 (T366K; 445K, 446S, 447C tripeptide insertion)

Domain D = CH2

Domain E = CH3 (T366W, S354C)

2^nd polypeptide chain (SEQ ID NO: 9):

Domain F = VH (“Antigen A”)

Domain G = CH3 (L351D; 445G, 446E, 447C tripeptide insertion)

3^rd polypeptide chain (SEQ ID NO: 10):

Domain H = VL (“Nivo”)

Domain I = CL (Kappa)

Domain J = CH2

Domain K = CH3 (Y349C, D356E, L358M, T366S, L368A, Y407V)

4^th polypeptide chain (SEQ ID NO: 11):

Domain L = VH (“Nivo”)

Domain M = CH1_.

[00291] As described above, the bivalent bispecific construct has a single CH1 domain at Domain M with the sequence of a human IgGl CH1 region [SEQ ID NO: 23] With reference to a typical native antibody architecture, the CH1 domain typically found at domain B has been substituted with a CH3 amino acid sequence having the noted orthogonal mutations.

[00292] The A domain (SEQ ID NO: 12) and F domain (SEQ ID NO: 16) form an antigen binding site (A:F) specific for“Antigen A”. The H domain has the VH sequence from nivolumab and the L domain has the VL sequence from nivolumab; H and L associate to form an antigen binding site (H:L) specific for human PD1.

[00293] The B domain (SEQ ID NO: 13) has the sequence of human IgGl CH3 with several mutations: T366K, 445K, 446S, and 447C insertion. The T366K mutation is a charge pair cognate of the L351D residue in Domain G. The“447C” residue on domain B comes from the C-terminal KSC tripeptide insertion.

[00294] Domain D (SEQ ID NO: 14) has the sequence of human IgGl CH2 [00295] Domain E (SEQ ID NO: 15) has the sequence of human IgGl CH3 with the mutations T366W and S354C. The 366W is the“knob” mutation. The 354C introduces a cysteine that is able to form a disulfide bond with the cognate 349C mutation in Domain K.

[00296] Domain G (SEQ ID NO: 17) has the sequence of human IgGl CH3 with the following mutations: L351D, and 445G, 446E, 447C tripeptide insertion. The L351D mutation introduces a charge pair cognate to the Domain B T366K mutation. The“447C” residue on domain G comes from the C-terminal GEC tripeptide insertion.

[00297] Domain I (SEQ ID NO: 19) has the sequence of human C kappa light chain (CK)

[00298] Domain J (SEQ ID NO: 20) has the sequence of human IgGl CH2 domain, and is identical to the sequence of domain D.

[00299] Domain K (SEQ ID NO: 21) has the sequence of human IgGl CH3 with the following changes: Y349C, D356E, L358M, T366S, L368A, Y407V. The 349C mutation introduces a cysteine that is able to form a disulfide bond with the cognate 354C mutation in Domain E. The 356E and L358M introduce isoallotype amino acids that reduce immunogenicity. The 366S, 368A, and 407V are“hole” mutations.

[00300] “BC1” could readily be expressed at high levels using mammalian expression at concentrations greater than 100 pg/ml.

[00301] We found that the“BC1” protein - an antigen-binding CH1- substituted protein that is bivalent (and bispecific) and has a single CH1 domain - could easily be purified in a single step using a CH1 -specific CaptureSelect™ affinity resin from

ThermoFisher following expression using the Expi293 system.

[00302] As shown in FIG. 5A, SEC analysis demonstrates that a single-step CH1 affinity purification step yields a single, monodisperse peak via gel filtration in which >98% is monomer. FIG. 5B shows comparative literature data of SEC analysis of a CrossMab bivalent antibody construct having 2 CH1 domains.

[00303] FIG. 6A is a cation exchange chromatography (IEX) elution profile of“BC1” following one-step purification using the CaptureSelect™ CH1 affinity resin, showing a single tight peak. FIG. 6B is a cation exchange chromatography elution profile of“BC1” following purification using standard Protein A purification, showing additional elution peaks consistent with the co-purification of incomplete assembly products. As quantified by ion exchange chromatography (IEX) in Table 1, use of a CH1 binding reagent to purify the antigen-binding CH1- substituted protein resulted in over a 50% increase in the percentage of complete complexes present in the purified sample. [00304] FIG. 7 shows SDS-PAGE gels under non-reducing conditions. As seen in lane 3, single-step purification of“BC1” with CH1 affinity resin provided a nearly homogeneous single band. Lane 7, by comparison, shows less substantial purification when standard Protein A purification was performed. As quantified by SDS-PAGE in Table 1, comparing lane 3 to lane 7 reveals use of a CH1 binding reagent to purify the antigen binding CH1- substituted protein resulted in an 80% increase in the percentage of complete complexes present in the purified sample.

[00305] Additionally, lane 4 shows minimal additional purification of the anti- CHleluate with a subsequent cation exchange polishing step, while lanes 8-10

demonstrates further purification of the Protein A purified material using cation exchange chromatography.

[00306] FIG. 8 compares SDS-PAGE gels of “BC1” after single-step CHl-affmity purification, under both non-reducing and reducing conditions (Panel A) with SDS-PAGE gels of a CrossMab bispecific antibody under non-reducing and reducing conditions as published in the referenced literature (Panel B).

[00307] FIG. 9 shows mass spec analysis of“BC1”, demonstrating two distinct heavy chains (FIG. 9A) and two distinct light chains (FIG. 9B) under reducing conditions. The mass spectrometry data in FIG. 10 confirms the absence of incomplete pairing after purification.

[00308] Table 2 compares“BC1” to CrossMab in key developability characteristics:

*Data from Schaefer el a/. (Proc Natl Acad Sci USA. 2011 Jul 5; 108(27): 11187-92)

6.13.4. Example 3: Bivalent bispecific B-Body“BC6”

[00309] We constructed a bivalent bispecific B-Body, termed“BC6”, that is identical to“BC1” but for retaining wild type residues in Domain B at residue 366 and Domain G at residue 351.“BC6” thus lacks the charge-pair cognates T366K and L351D that had been designed to facilitate correct pairing of domain B and domain G in“BC1,” but still retains the single CH1 domain at Domain M. Salient features of the“BC6” architecture are illustrated in FIG. 11.

[00310] Notwithstanding the absence of the charge-pair residues present in“BC1”, we found that a single step purification of the“BC6” bivalent antigen-binding CH1- substituted protein using CH1 affinity resin following expression using the Expi293 system resulted in a highly homogeneous sample. FIG. 12A shows SEC analysis of“BC6” following one- step purification using the CaptureSelect™ CH1 affinity resin. The data demonstrate that the single step CH1 affinity purification yields a single monodisperse peak, similar to what we observed with“BC1”, demonstrating that the disulfide bonds between polypeptide chains 1 and 2 and between polypeptide chains 3 and 4 are intact. The chromatogram also shows the absence of non-covalent aggregates.

[00311] FIG. 12B shows a SDS-PAGE gel under non-reducing conditions, with lane 1 loaded with a first lot of“BC6” after a single-step CH1 affinity purification, lane 2 loaded with a second lot of“BC6” after a single-step CH1 affinity purification. Lanes 3 and 4 demonstrate further purification can be achieved with ion exchange chromatography subsequent to CH1 affinity purification. 6.13.5. Example 4: Bivalent bispecific B-Bodies“BC28” and“BC30”

[00312] We constructed bivalent lxl bispecific B-Bodies“BC28” and“BC30” having an engineered disulfide within the CH3 interface in Domains B and G as an alternative S-S linkage to the C-terminal disulfide present in“BC1” and“BC6”. Thus, each of the constructs still retain the single CH1 domain at Domain M, and are antigen-binding CH1- substituted proteins. Literature indicates that CH3 interface disulfide bonding is insufficient to enforce orthogonality in the context of Fc CH3 domains. The general architecture of these B-Body constructs is schematized in FIG. 13 with salient features of “BC28” summarized below:

Polypeptide chain 1 :“BC28” chain 1 (SEQ ID NO:24)

Domain A = VL (Antigen“A”)

Domain B = CH3 (Y349C; 445P, 446G, 447K insertion)

Domain D = CH2

Domain E= CH3 (S354C, T366W)

Polypeptide chain 2:“BC28” chain 2 (SEQ ID NO:25)

Domain F = VH (Antigen“A”)

Domain G = CH3 (S354C; 445P, 446G, 447K insertion)

Polypeptide chain 3:“BC1” chain 3 (SEQ ID NO: 10)

Domain H = VL (“Nivo”)

Domain I = CL (Kappa)

Domain J = CH2

Domain K = CH3 (Y349C, D356E, L358M, T366S, L368A, Y407V)

Polypeptide chain 4:“BC1” chain 4 (SEQ ID NO: 11)

Domain L = VH (“Nivo”)

Domain M = CH1_.

[00313] The“BC28” A:F antigen binding site is specific for“Antigen A”. The “BC28” H:L antigen binding site is specific for PD1 (nivolumab sequences).“BC28” domain B has the following changes as compared to wild type CH3: Y349C; 445P, 446G, 447K insertion. “BC28” domain E has the following changes as compared to wild type CH3: S354C, T366W. “BC28” domain G has the following changes as compared to wild type: S354C; 445P, 446G, 447K insertion. [00314] “BC28” thus has an engineered cysteine at residue 349C of Domain B and engineered cysteine at residue 354C of domain G (“349C-354C”).“BC30” has an engineered cysteine at residue 354C of Domain B and 349C of Domain G (“354C-349C”).

[00315] FIG. 14 shows SEC analysis of“BC28” and“BC30” following one-step purification using the CaptureSelect™ CH1 affinity resin following expression using the Expi293 system.

6.13.6. Example 5: Variable-CH3 junction engineering

[00316] We produced a series of variants in which we mutated the VL-CH3 junction between Domains A and B and the VH-CH3 junction between domains F and G to assess the expression level, assembly and stability of bivalent lxl B-Body constructs. Although there are likely many solutions, to reduce introduction of T cell epitopes we chose to only use residues found naturally within the VL, VH and CH3 domains. Structural assessment of the domain architecture further limits desirable sequence combinations. Table 3 and Table 4 below show junctions for several junctional variants based on“BC1” and other bivalent constructs.

6.13.7. Example 6: Trivalent 2x1 Bispecific B-Body construct (“BCl-2xl”)

[00317] We constructed a trivalent 2x1 bispecific B-Body“BCl-2xl” based on “BC1”. As designed,“BCl-2xl” retains a single CH1 domain at Domain M. This is a trivalent construct with a single CH1 domain, and is thus an antigen-binding CH1- substituted protein. Other salient features of the architecture are illustrated in FIG. 16.

[00318] In greater detail, using the domain and polypeptide chain references summarized in FIG. 15,

I^st polypeptide chain

Domain N = VL (“Antigen A”)

Domain O = CH3 (T366K, 447C)

Domain A = VL (“Antigen A”)

Domain B = CH3 (T366K, 447C)

Domain D = CH2

Domain E = CH3 (Knob, 354C)

5^th polypeptide chain (=“BC1” chain 2)

Domain P = VH (“Antigen A”)

Domain Q = CH3 (L351D, 447C)

2^nd polypeptide chain (=“BC1” chain 2)

Domain F = VH (“Antigen A”)

Domain G = CH3 (L351D, 447C)

3^rd polypeptide chain (=“BC1” chain 3)

Domain H = VL (“Nivo”)

Domain I = CL (Kappa)

Domain J = CH2

Domain K = CH3 (Hole, 349C)

4^th polypeptide chain (=“BC1” chain 4)

Domain L = VH (“Nivo”)

Domain M = CH1.

[00319] FIG. 17 shows non-reducing SDS-PAGE of protein expressed using the ThermoFisher Expi293 transient transfection system.

[00320] Lane 1 shows the eluate of the trivalent 2x1“BC1-2X1” protein following one-step purification using the CaptureSelect™ CH1 affinity resin. Lane 2 shows the lower molecular weight, faster migrating, bivalent“BC1” protein following one-step purification using the CaptureSelect™ CH1 affinity resin. Lanes 3-5 demonstrate purification of“BC1- 2x1” using protein A. Lanes 6 and 7 show purification of“BCl-2xl” using CH1 affinity resin. The abundance of lower bands representing incomplete complexes is decreased when purified with the CH1 affinity resin.

6.13.8. Example 7: Trivalent 1x2 Bispecific Construct (“BC28-lx2”)

[00321] We constructed a trivalent 1x2 bispecific B-Body having the following domain structure (domain nomenclature is set forth in FIG. 18). As designed,“BC28-lx2” retains a single CH1 domain at Domain M. This is a trivalent construct with a single CH1 domain, and is thus an antigen-binding CH1- substituted protein:

I^st polypeptide chain (=“BC28” chain 1) (SEQ ID NO:24)

Domain A = VL (Antigen“A”)

Domain B = CH3 (Y349C; 445P, 446G, 447K insertion)

Domain D = CH2

Domain E=CH3 (S354C, T366W)

2^nd polypeptide chain (=“BC28” chain 2) (SEQ ID NO:25)

Domain F = VH (Antigen“A”)

Domain G = CH3 (S354C; 445P, 446G, 447K insertion)

3^rd polypeptide chain (SEQ ID NO:37)

Domain R = VL (Antigen“A”)

Domain S = CH3 (Y349C; 445P, 446G, 447K insertion)

Linker = GSGSGS

Domain H = VL (“Nivo”)

Domain I = CL

Domain J = CH2

Domain K = CH3 (Y349C, D356E, L358M, T366S, L368A, Y407V)

4^th polypeptide chain (=“BC1” chain 4) (SEQ ID NO: 11):

Domain L = VH (“Nivo”)

Domain M = CH1_.

6^th polypeptide chain (=“BC28” chain 2) (SEQ ID NO:25)

Domain T = VH (Antigen“A”)

Domain U = CH3 (S354C; 445P, 446G, 447K insertion) [00322] The A:F antigen binding site is specific for“Antigen A”, as is the H:L binding antigen binding site. The R:T antigen binding site is specific for PD. The specificity of this construct is thus Antigen“A” x (PD 1 -Antigen“A”).

6.13.9. Example 8: Trivalent 1x2 trispecific construct“BC28- lxlxla”

[00323] We constructed a trivalent 1x2 trispecific molecule having the general structure schematized in FIG. 19. As designed,“BC28-lxlxla” has a single CH1 domain at Domain M. This is a trivalent construct with a single CH1 domain, and is thus an antigen-binding CH1- substituted protein. With reference to the domain nomenclature set forth in FIG. 18,

I^st polypeptide chain (=“BC28” chainl) [SEQ ID NO:24]

Domain A = VL (Antigen“A”)

Domain B = CH3 (Y349C; 445P, 446G, 447K insertion)

Domain D = CH2

Domain E = CH3 (S354C, T366W)

2^nd polypeptide chain (=“BC28” chain 2) (SEQ ID NO:25)

Domain F = VH (Antigen“A”)

Domain G = CH3 (S354C; 445P, 446G, 447K insertion)

3^rd polypeptide chain (SEQ ID NO:45)

Domain R = VL (CTLA4-4)

Domain S = CH3 (T366K; 445K, 446S, 447C insertion)

Linker = GSGSGS

Domain H = VL (“Nivo”)

Domain I = CL

Domain J = CH2

Domain K = CH3 (Y349C, D356E, L358M, T366S, L368A, Y407V)

4^th polypeptide chain (=“BCl” chain 4) (SEQ ID NO: 11)

Domain L = VH (“Nivo”)

Domain M = CH1_.

6^th polypeptide chain (=hCTLA4-4 chain2) (SEQ ID NO:53)

Domain T = VH (CTLA4)

Domain U = CH3 (L351D, 445G, 446E, 447C insertion) [00324] The antigen binding sites of this tri specific construct were:

Antigen binding site A:F was specific for’’Antigen A”

Antigen binding site H:L was specific for PD1 (nivolumab sequence)

Antigen binding site R:T was specific for CTLA4.

[00325] FIG. 20 shows size exclusion chromatography with“BC28-lxlxla” following expression using the Expi293 system and one-step purification using the

CaptureSelect™ CH1 affinity resin, demonstrating a single well-defined peak.

6.13.10. Example 9: Tetravalent constructs

[00326] FIG. 22 shows the overall architecture of a 2x2 tetravalent bispecific construct “BC22 -2x2”. The 2x2 tetravalent bispecific was constructed with“BC1” scaffold by duplicating each variable domain-constant domain segment. Domain nomenclature is schematized in FIG. 21.

[00327] FIG. 23 is a SDS-PAGE gel. Lanes 7-9 show the“BC22-2x2” tetravalent construct respectively following expression using the Expi293 system and one-step purification using the CaptureSelect™ CH1 affinity resin (“CH1 eluate”), and after an additional ion exchange chromatography purification (lane 8,“pk 1 after IEX”; lane 9,“pk 2 after IEX”). Lanes 1-3 are the trivalent 2x1 construct“BC2l-2xl” after CH1 affinity purification (lane 1) and, in lanes 2 and 3, subsequent ion exchange chromatography.

Lanes 4-6 are the 1x2 trivalent construct“BCl2-lx2”.

6.13.11. Example 10: SDS-PAGE analysis of bivalent and trivalent constructs

[00328] FIG. 24 shows a SDS-PAGE gel with various constructs, each following expression using the Expi293 system and one-step purification using the CaptureSelect™ CH1 affinity resin, under non-reducing and reducing conditions.

[00329] Lanes 1 (nonreducing conditions) and 2 (reducing conditions, + DTT) are the bivalent lxl bispecific construct“BC1”. Lanes 3 (nonreducing) and 4 (reducing) are the bivalent lxl bispecific construct“BC28” (see Example 4). Lanes 5 (nonreducing) and 6 (reducing) are the bivalent lxl bispecific construct“BC44” (see Example 5). Lanes 7 (nonreducing) and 8 (reducing) are the trivalent 1x2 bispecific“BC28-lx2” construct (see Example 9). Lanes 9 (nonreducing) and 10 (reducing) are the trivalent 1x2 trispecific “BC28-lxlxla” construct described in Example 11. [00330] The SDS-PAGE gel demonstrates the complete assembly of each construct, with the predominant band in the non-reducing gel appearing at the expected molecular weight for each construct.

6.13.12. Example 11: Control of CHI Expression

[00331] CH1 expression is considered the rate limiting step in antibody folding and secretion. Therefore, we tested controlling the expression ratio of the four chains, particularly the ratio of the chain having the CH1 domain. The Expi293 Expression system was used to test the assembly efficiency by varying the ratio of transfected expression vectors for each of the four polypeptide chains. In brief, lpg of total plasmid for all chains combined was transfected into 1 mL Expi293 cells. The expression of the CH1 domain was controlled by varying the relative ratio of the expression vector for the polypeptide chain containing the CH1 domain. The construct tested used the BC28 architecture. Thus, the ratio of the 4^th polypeptide chain containing the single CH1 (Domain M) was varied in the transfection mixture, as well as the ratio of the other chains in separate samples. The various ratios tested are shown in Table 5. Supernatant from the Expi293 Expression system was run directly on an SDS-PAGE gel. As shown in FIG. 25, the plasmid mix with ratio chain 1 : chain 2: chain 3 : chain 4 = 1 : 1 : 1 :3 (lane 9) increased the abundance and purity of complete antigen-binding CH1- substituted protein complexes (-150 kDa band) in comparison to increasing the relative ratio of other polypeptide chains. Thus, controlling the expression of the CH1 domain containing polypeptide chain was demonstrated to improve the efficiency of expressing and forming the desired complete antigen-binding CH1- substituted proteins.

6.13.13. Example 12: Anti-CHI purification of Constructs with

Reduced Numbers of CHI Domains using Expi293 System

[00332] Trivalent, bispecific constructs having the architecture shown in FIG. 26A were constructed. The domain architecture of the constructs is described below.

Chain 1 : VL (0X40:24) - CH3 (BC1) - GS linker - VL (0X40: 11) - CL -CH2-CH3 (Knob, 354C)

Chain 2: VH (0X40: 11) - CH1

Chain 3: VL (0X40: 11) - CL -CH2 -CH3 (Hole, 349C)

Chain 4: VH (0X40: 11) - CH1

Chain 5: VH (0X40:24) - CH3 (BC1)

[00333] The VH and VL antigen binding sites (ABSs) from 0X40 ABS clone numbers 24 and 11 (0X40:24 and 0X40: 11) are described in W02019/040791A1, which is hereby incorporated by reference in its entirety.

[00334] The constructs each contain at least one fewer CH1 than the valency of the construct. Each was expressed using the Expi293 system and subjected to one-step purification using the CaptureSelect™ CH1 affinity resin.

[00335] FIG. 26B shows an SDS-PAGE gel of the purification products of the constructs described in FIG. 26A (lanes A and B).

[00336] The SDS-PAGE gel demonstrates the complete assembly of each construct, with the predominant band in the non-reducing gel appearing at the expected molecular weight for each construct.

6.13.14. Example 14: Anti-CHI purification of Constructs using

ExpiCHO System

[00337] Trivalent, bispecific constructs having the architecture shown in FIG. 27A were constructed. The domain architecture of the constructs is described below.

Chain 1 : VL (I^st antigen binding site (ABS)) - CH3 (T366K, 477C) -CH2-CH3 (Hole, 349C) Chain 2: VH (I^st ABS) - CH3 (L351D, 447C)

Chain 3: VL (3^rd ABS) - CH3(T366K, 447C) -VL (2^nd ABS) -CH2 -CH3 (Knob,4C)

Chain 4: VH (2^nd ABS) - CH1

Chain 5: VH (3^rd ABS) - CH3 (L351D, 447C)

[00338] The construct was expressed using the ExpiCHO system and subjected to one- step purification using the CaptureSelect™ CH1 affinity resin. FIG. 27B shows an SDS- PAGE gel of the resulting purification products, demonstrating the complete assembly of each construct, with the predominant band in the non-reducing gel appearing at the expected molecular weight for each construct.

[00339] FIG. 27C shows size exclusion chromatography results following ExpiCHO expression and one-step purification described above, showing a single peak denoting purity of the sample comprising the correctly assembled product.

6.13.15. Example 15: Anti-CHI purification for various CHI- substituted architectures

[00340] To examine the benefits of anti-CHl purification, various antibody

architectures are compared using different purification strategies. Antibody architectures that are tested include the various B-body formats described above, other antibody platforms that have substituted CH1 for another domain leaving only a single CH1, and other similar antibody platforms but that still retain a number of CH1 domains equivalent to the valency of the antibody. Specific architecture platforms tested are: 1) bivalent bispecific B-Body formats, e.g ., those described above including“BC1,”“BC28,” and“BC44,” 2) Abbvie lxl MH2 bivalent bispecific platforms, described in, e.g., W02017011342, which is hereby incorporated by reference in its entirety; 3) Merck Kga CH3 Domain Substitution bivalent bispecific platforms, described in WO2016087650, which is hereby incorporated by reference in its entirety 4) and trivalent bispecific/trispecific B-Body formats described herein.

[00341] The different platforms are expressed and purified using the anti-CHl binding reagent, as described above. The different platforms are also purified using other standard techniques, such as Protein A purification. The purified antibodies are analyzed using various analytical tools and methods to assess purity and abundance of desired products, as described above. The different platforms are then compared against one another, and from the analysis it is determined that the anti-CHl purification strategy improves purifying antibody platforms having a single CH1 domain and/or antibody platforms having fewer CH1 domains than antibody valencies.

6.14. Sequences

> Example 1, bivalent monospecific construct CHAIN 1 [SEQ ID NO : l]

(VL) -VEIKRTPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV HEALHNHYTQKSLSLSPGKDKTHTCPP CPAPELLGGPSVFLFPPKPKDTL ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSV HEALHNHYTQKSLSLSPGK

>Example 1, bivalent monospecific construct CHAIN 2 [SEQ ID NO : 2 ]

(VH) -VTVSSASPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV HEALHNHYTQKSLSLSPGK

> Example 1, bivalent, bispecific construct CHAIN 1 [SEQ ID NO:3]

(VL) -VEIKRTPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV HEALHNHYTQKSLSLSPGKDRrjjrCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK

PREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

PCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

VL- CH3- Hinge- CH2- CH3 (knob)

> Example 1, bivalent, bispecific construct CHAIN 2 [SEQ ID NO:4]

(VH) -VTVSSASPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK VH- CH3

> Example 1, bivalent, bispecific construct CHAIN 3_ [SEQ ID NO: 5]

(VL) -VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDRrjjrCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK

PREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLP

PSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

VL- CL- Hinge- CH2- CH3 (hole)

> Example 1, bivalent, bispecific construct CHAIN 4 [SEQ ID NO: 6]

(VH) -VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSC

VH- CHI > Fc Fragment of Human IgGl [SEQ ID NO: 7]

GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

>BC1 chain 1 [SEQ ID NO: 8]

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTPREPQ VYTLPPSRDELTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCDiCTHTCPPCPAPEL

LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

YTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Domain arrangement :

A- B- Hinge- D- E

VL- CH3- Hinge- CH2- CH3 (knob)

Mutations in first CH3 (Domain B) :

T366K; 445K, 446S, 447C insertion

Mutations in second CH3 (Domain E) :

S354C , T366W

>BC1 chain 2 [SEQ ID NO: 9]

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIHWVRQAPGKGLEWVGDITPYDGT TNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARLVGEIATGFDYWGQGT LVTVSSASPREPQVYTDPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGEC

Domain arrangement :

F- G

VH- CH3

Mutations in CH3 (Domain G) :

L351D; 445G, 446E, 447C insertion >BC1 chain 3 [SEQ ID NO: 10]

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATG IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDiCmTCPPCPAPEL

LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

CTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Domain arrangement :

H- I- Hinge- J- K

VL- CL- Hinge- CH2- CH3 (hole)

Mutations in CH3 (domain K) :

Y349C, D356E, L358M, T366S, L368A, Y407V

>BC1 chain 4 [SEQ ID NO: 11]

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSK RYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSC

Domain arrangement :

L- M

VH- CHI

_> BC1 Domain A [SEQ ID NO: 12]

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT

_> BC1 Domain B [SEQ ID NO: 13]

PREPQVYTLPPSRDELTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSC

_> BC1 Domain D [SEQ ID NO: 14]

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

_> BC1 Domain E [SEQ ID NO: 15]

GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

_> BC1 Domain F [SEQ ID NO: 16]

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIHWVRQAPGKGLEWVGDITPYDGT TNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARLVGEIATGFDYWGQGT

LVTVSSAS

_> BC1 Domain G [SEQ ID NO: 17]

PREPQVYTDPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGEC

_> BC1 Domain H [SEQ ID NO: 18]

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATG IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK

_> BC1 Domain I [SEQ ID NO: 19]

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC _> BC1 Domain J [SEQ ID NO: 20]

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

_> BC1 Domain K [SEQ ID NO: 21]

GQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

_> BC1 Domain L [SEQ ID NO: 22]

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSK RYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

_> BC1 Domain M [SEQ ID NO: 23]

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSC

_>BC28 chain 1 [SEQ ID NO:24]

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTPREPQ VCTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG DKTfiTCPPCPAPEL

LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

Domain arrangement :

A- B- Hinge- D- E

VL- CH3- Hinge- CH2- CH3 (knob)

Mutations in domain B:

Y349C; 445P , 446G, 447K insertion

Mutations in domain E:

S354C, T366W

_>BC28 chain 2 [SEQ ID NO:25]

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIHWVRQAPGKGLEWVGDITPYDGT TNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARLVGEIATGFDYWGQGT LVTVSSASPREPQVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Domain arrangement :

F- G

VH- CH3

Mutations in domain G:

S354C; 445P , 446G, 447K insertion _>BC28 domain A [SEQ ID NO: 26]

_>BC28 domain B [SEQ ID NO: 27]

PREPQVCTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

_>BC28 domain D [SEQ ID NO: 28]

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

_>BC28 domain E [SEQ ID NO: 29]

GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

_>BC28 domain F [SEQ ID NO: 30]

LVTVSSAS

_>BC28 domain G [SEQ ID NO: 31]

PREPQVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>BC44 chain 1 [SEQ ID NO: 32]

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVREPQVCTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKDiCTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPGK

Domain arrangement :

A- B- Hinge- D- E

VL- CH3- Hinge- CH2- CH3 (knob)

Mutations in domain B:

P343V; Y349C; 445P, 446G, 447K insertion

Mutations in domain E:

S354C , T366W

>BC44 Domain A [SEQ ID NO: 33]

>BC44 Domain B [SEQ ID NO: 34]

VREPQVCTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>BC44 Domain D [SEQ ID NO: 35]

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

>BC44 Domain E [SEQ ID NO: 36]

GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>BC28 bivalent chain 3 equivalent to SEQ ID NO: 10 >BC28 bivalent chain 4 equivalent to SEQ ID NO: 11

_>BC28 1x2 chain 3 [SEQ ID NO: 37]

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTPREPQ VCTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKG5G5G5EXVLrQSP ATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGS GSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGOGTKVEIKRTVAAPSVFJFPPSO EOLKSGTASWCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTL

TLSKADYEKHKVYACEVTHOGLSSPVTKSFNRGECIIKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPgRE EMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Domain arrangement :

R- S- linker- H-I- Hinge- J- K-

VL- CH3- linker- VL- CL- Hinge- CH2- CH3 (hole)

Mutations in domain S:

Y349C; 445P , 446G, 447K insertion

Six amino acids linker insertion: GSGSGS

Mutations in domain K:

Y349C, D356E, L358M, T366S, L368A, Y407V

_>BC28 1x2 domain R [SEQ ID NO: 38]

_>BC28 1x2 domain S [SEQ ID NO: 39]

PREPQVCTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

_>BC28 1x2 linker [SEQ ID NO:40]

GSGSGS

_>BC28 1x2 domain H [SEQ ID NO:41]

_>BC28 1x2 domain I [SEQ ID NO:42]

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

_>BC28 1x2 domain J [SEQ ID NO:43]

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

_>BC28 1x2 domain K [SEQ ID NO:44]

GQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>BC28-lxlxla chain 3 [SEQ ID NO:45]

DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQRDSYLWTFGQGTKVEIKRTPREPQ VYTLPPSRDELTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSCGSGSGSEXVLrQSP ATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGS GSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGOGTKVEIKRTVAAPSVFJFPPSO EOLKSGTASWCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTL TLSKADYEKHKVYACEVTHOGLSSPVTKSFNRGECDiCTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRE EMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Domain arrangement :

R- S- linker- H-I- Hinge- J- K-

VL- CH3- linker- VL- CL- Hinge- CH2- CH3 (hole)

Mutations in domain S:

T366K; 445K, 446S, 447C insertion

Six amino acids linker insertion: GSGSGS

Mutations in domain K:

Y349C, D356E, L358M, T366S, L368A, Y407V

>BC28-lxlxla domain R [SEQ ID NO:46]

DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQRDSYLWTFGQGTKVEIKRT

_>BC28-lxlxla domain S [SEQ ID NO:47]

PREPQVYTLPPSRDELTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSKSC

>BC28-lxlxla linker [SEQ ID NO:48]

GSGSGS

_>BC28-lxlxla domain H [SEQ ID NO:49]

>BC28-lxlxla domain I [SEQ ID NO:50]

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

>BC28-lxlxla domain J [SEQ ID NO:51]

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

_>BC28-lxlxla domain K [SEQ ID NO:52]

GQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

>hCTLA4-4. chain 2 [SEQ ID NO:53]

EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYYIHWVRQAPGKGLEWVAVIYPYTGF TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGEYTVLDYWGQGTLVT VSSASPREPQVYTDPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGEC

Domain arrangement :

F- G

VH- CH3

Mutations in domain G

L351D, 445G, 446E, 447C insertion

>hCTLA4 -4 domain F [SEQ ID NO: 54]

EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYYIHWVRQAPGKGLEWVAVIYPYTGF TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGEYTVLDYWGQGTLVT VSSAS

>hCTLA4 -4 domain G [SEQ ID NO: 55]

PREPQVYTDPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGEC

Other Sequences :

>Hinge : DKTHTCPPCP [SEQ ID NO: 56]

>BC1-Polypeptide

Domain Junction: IKRTPREP [SEQ ID NO: 57] >BC15 -Polypeptide 1 Domain Junction: IKRTVREP [SEQ ID NO: 58] >BC16 -Polypeptide 1 Domain Junction: IKRTREP [SEQ ID NO: 59] >BC17 -Polypeptide 1 Domain Junction: IKRTVPREP [SEQ ID NO: 60] >BC26-Polypeptide 1 Domain Junction: IKRTVAEP [SEQ ID NO: 61] >BC27 -Polypeptide 1 Domain Junction: IKRTVAPREP [SEQ ID NO: 62] >BC1-Polypeptide

Domain Junction: SSASPREP [SEQ ID NO: 63] >BC13 -Polypeptide 2 Domain Junction: SSASTREP [SEQ ID NO: 64] >BC14 -Polypeptide 2 Domain Junction: SSASTPREP [SEQ ID NO: 65] >BC24 -Polypeptide 2 Domain Junction: SSASTKGEP [SEQ ID NO: 66] >BC25 -Polypeptide 2 Domain Junction: SSASTKGREP [SEQ ID NO: 67]

7. INCORPORATION BY REFERENCE

[00342] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

8. EQUIVALENTS

[00343] While various specific embodiments have been illustrated and described, the above specification is not restrictive. It will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s). Many variations will become apparent to those skilled in the art upon review of this specification.

Claims

WHAT IS CLAIMED IS:

1. A method of purifying an antigen-binding CH1 -substituted protein, the method

comprising the steps of:

i) contacting a sample comprising the antigen-binding CH1 -substituted protein with a CH1 binding reagent,

wherein the antigen-binding CH1- substituted protein comprises at least a first, a second, a third, and a fourth polypeptide chain associated in a complex,

wherein the complex comprises at least one CH1 domain, or portion thereof, and wherein the number of CH1 domains in the complex is at least one fewer than the valency of the complex, and

wherein the contacting is performed under conditions sufficient for the CH1 binding reagent to bind the CH1 domain, or portion thereof; and

ii) purifying the complex away from one or more incomplete complexes, wherein the incomplete complexes do not comprise the first, the second, the third, and the fourth polypeptide chain.

2. The method of claim 1, wherein the antigen-binding CH1 -substituted protein has a single

CH1 domain, or portion thereof.

3. The method of claim 2, wherein

(a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E,

wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation,

wherein domain A has a variable region domain amino acid sequence, and wherein domain B, domain D, and domain E have a constant region domain amino acid sequence;

(b) the second polypeptide chain comprises a domain F and a domain G,

wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence;

(c) the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K,

wherein the domains are arranged, from N-terminus to C-terminus, in a H-I-J-K orientation, and

wherein domain H has a variable region domain amino acid sequence, domain I has a CL amino acid sequence, and domains J and K have a constant region domain amino acid sequence;

(d) the fourth polypeptide chain comprises a domain L and a domain M,

wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and

wherein domain L has a variable region domain amino acid sequence, and

wherein domain M is the single CH1 domain, or portion thereof;

(e) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains;

(f) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains;

(g) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the antigen-binding CH1 -substituted protein.

4. The method of claim 2, wherein

wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and wherein domain B, domain D, and domain E have a constant region domain amino acid sequence; (b) the second polypeptide chain comprises a domain F and a domain G,

wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and

wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence;

wherein domain I is the single CH1 domain, or portion thereof, domain H has a variable region domain amino acid sequence, and domains J and K have a constant region domain amino acid sequence;

(d) the fourth polypeptide chain comprises a domain L and a domain M,

wherein domain L has a variable region domain amino acid sequence, and

wherein domain M has a CL amino acid sequence;

5. The method of claims 3 or 4, wherein domain B and domain G have a CH3 amino acid sequence.

6. The method of claim 5, wherein the amino acid sequences of the B and the G domains are identical, wherein the sequence is an endogenous CH3 sequence.

7. The method of claim 5, wherein the amino acid sequences of the B and the G domains are different and separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, wherein the B domain interacts with the G domain, and wherein neither the B domain nor the G domain significantly interacts with a CH3 domain lacking the orthogonal modification.

8. The method of claim 7, wherein the orthogonal modifications of the B and the G domains comprise mutations that generate engineered disulfide bridges between domain B and G.

9. The method of claim 8, wherein the mutations of the B and the G domains that generate engineered disulfide bridges are a S354C mutation in one of the B domain and G domains, and a 349C in the other domain.

10. The method of any one of claims 7-9, wherein the orthogonal modifications of the B and the G domains comprise knob-in-hole mutations.

11. The method of claim 10, wherein the knob-in hole mutations of the B and the G

domains are a T366W mutation in one of the B domain and G domain, and a T366S, L368A, and aY407V mutation in the other domain.

12. The method of any one of claims 7-11, wherein the orthogonal modifications of the B and the G domains comprise charge-pair mutations.

13. The binding molecule of claim 12, wherein the charge-pair mutations of the B and the G domains are a T366K mutation in one of the B domain and G domain, and a L351D mutation in the other domain.

14. The method of claims 3 or 4, wherein domain B and domain G have an IgM CH2 amino acid sequence or an IgE CH2 amino acid sequence.

15. The method of claim 14, wherein the IgM CH2 amino acid sequence or the IgE CH2 amino acid sequence comprise orthogonal modifications.

16. The method of any of claims 3-15, wherein domain E and domain K have a CH3 amino acid sequence.

17. The method of claim 16, wherein the amino acid sequences of the E and K domains are identical, wherein the sequence is an endogenous CH3 sequence.

18. The method of claim 16, wherein the amino acid sequences of the E and the K

domains are different.

19. The method of claim 18, wherein the different sequences of the E and the K domains separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, wherein the E domain interacts with the K domain, and wherein neither the E domain nor the K domain significantly interacts with a CH3 domain lacking the orthogonal modification.

20. The method of claim 19, wherein the orthogonal modifications of the E and the K

domains comprise mutations that generate engineered disulfide bridges between domain E and K.

21. The method of claim 20, wherein the mutations of the E and the K domains that

generate engineered disulfide bridges are a S354C mutation in one of the E domain and K domain, and a 349C in the other domain.

22. The method of any one of claims 19-21, wherein the orthogonal modifications in the E and K domains comprise knob-in-hole mutations.

23. The method of claim 22, wherein the knob-in hole mutations of the E and the K

domains are a T366W mutation in one of the E domain or K domain and a T366S, L368A, and aY407V mutation in the other domain.

24. The method of any one of claims 19-23, wherein the orthogonal modifications of the E and the K domains comprise charge-pair mutations.

25. The method of claim 24, wherein the charge-pair mutations of the E and the K

domains are a T366K mutation in one of the E domain or K domain and a

corresponding L351D mutation in the other domain.

26. The method of claim 2, wherein

wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence,

wherein domain B and domain D have a constant region domain amino acid sequence, and

wherein domain E is the single CH1 domain, or portion thereof;

-HO- (b) the second polypeptide chain comprises a domain F and a domain G,

wherein the domains are arranged, from N-terminus to C-terminus, in a H-I-J-K orientation, and wherein domain H has a variable region domain amino acid sequence, and domains I and J have a constant region domain amino acid sequence, and

wherein domain K has a CL amino acid sequence;

(d) the fourth polypeptide chain comprises a domain L and a domain M,

wherein domain L has a variable region domain amino acid sequence, and

wherein domain M has a constant region domain amino acid sequence;

27. The method of claim 2, wherein

wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and

wherein domain E has a CL amino acid sequence,

(b) the second polypeptide chain comprises a domain F and a domain G,

wherein domain K is the single CH1 domain, or portion thereof;

(d) the fourth polypeptide chain comprises a domain L and a domain M,

wherein domain L has a variable region domain amino acid sequence, and

wherein domain M has a constant region domain amino acid sequence;

28. The method of any of claims 3-27, wherein domain A has a VL amino acid sequence and domain F has a VH amino acid sequence.

29. The method of any of claims 3-27, wherein domain A has a VH amino acid sequence and domain F has a VL amino acid sequence.

30. The method of any of claims 3-29, wherein domain H has a VL amino acid sequence and domain L has a VH amino acid sequence.

31. The method of any of claims 3-29, wherein domain H has a VH amino acid sequence and domain L has a VL amino acid sequence.

32. The method of any of claims 3-31, wherein domain D and domain J have a CH2 amino acid sequence.

33. The method of any one of claims 3-32, wherein the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, and the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen.

34. The method of any one of the preceding claims, wherein:

(a) the first polypeptide chain or the third polypeptide chain further comprises a domain N and a domain O, wherein domain N has a variable region domain amino acid sequence, wherein domain O has a constant region amino acid sequence, wherein domains N and O are arranged, from N-terminus to C-terminus, in a N-0 orientation, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N- terminus of domain A of the first polypeptide chain or to the N-terminus of domain H of the third polypeptide chain;

(b) the binding molecule further comprises a fifth polypeptide chain, comprising: a domain P and a domain Q, wherein the domains are arranged, from N-terminus to C-terminus, in a P-Q orientation, and

wherein domain P has a variable region domain amino acid sequence and domain Q has a constant region amino acid sequence; and

(c) either the first or third polypeptide chain is associated with the fifth polypeptide chain through an interaction between the N and the P domains and an interaction between the O and the Q domains to form the binding molecule.

35. The method of claim 34, wherein the first polypeptide chain further comprises domain N and domain O, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain A of the first polypeptide chain.

36. The method of claim 34, wherein the third polypeptide chain further comprises

domain N and domain O, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain H of the third polypeptide chain.

37. The method of any one of claims 34-36, wherein:

(a) the amino acid sequences of domain N and domain A are identical,

the amino acid sequences of domain H is different from domains N and A, the amino acid sequences of domain O and domain B are identical,

the amino acid sequences of domain I is different from domains O and B,

the amino acid sequences of domain P and domain F are identical,

the amino acid sequences of domain L is different from domains P and F,

the amino acid sequences of domain Q and domain G are identical,

the amino acid sequences of domain M is different from domains Q and G; and

(b) wherein the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for the first antigen.

38. The method of claim 34-36, wherein:

(a) the amino acid sequences of domain N, domain A, and domain H are different, the amino acid sequences of domain O, domain B, and domain I are different, the amino acid sequences of domain P, domain F, and domain L are different, and the amino acid sequences of domain Q, domain G, and domain M are different; and

(b) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen,

the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for a third antigen.

39. The method of claim 35, wherein:

a. domain N, domain A, and domain H each comprise a VL amino acid sequence,

b. domain P, domain F, and domain L each comprise a VH amino acid sequence, c. domain O and domain Q each comprise a CH3 amino acid sequence, d. domain B and domain I each comprise a CL amino acid sequence, and e. domain G and domain M each comprise a CH1 amino acid sequence.

40. The method of claim 39, wherein:

a. domain N, domain A, and domain H are VL domains,

b. domain P, domain F, and domain L are VH domains,

c. domain O and domain Q are CH3 domains,

d. domain B and domain I are CL domains, and

e. domain G and domain M are CH1 domains.

41. The method of claim 36, wherein

a. domain N, domain A, and domain H each comprise a VL amino acid sequence,

b. domain P, domain F, and domain L each comprise a VH amino acid sequence, c. domain O and domain Q each comprise a CH3 amino acid sequence, d. domain B and domain G each comprise a CH3 amino acid sequence, e. domain I comprises a CL amino acid sequence, and

f. domain M comprises a CH1 amino acid sequence.

42. The method of claim 41, wherein

a. domain N, domain A, and domain H are VL domains,

b. domain P, domain F, and domain L are VH domains, c. domain O and domain Q are CH3 domains, d. domain B and domain G are CH3 domains,

e. domain I is a CL domain, and

f. domain M is a CH1 domain.

43. The method of any one of claims 39-42, wherein the amino acid sequences of the O and the Q domains are identical, and wherein the sequences of the O and the Q domains are endogenous CH3 sequences.

44. The method of any one of claims 39-42, wherein the amino acid sequences of the O and the Q domains are different and separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, wherein the O domain interacts with the Q domain, and wherein neither the O domain nor the Q domain significantly interacts with a CH3 domain lacking the orthogonal modification.

45. The method of claim 44, wherein the orthogonal modifications of the O and the Q domains comprise mutations that generate engineered disulfide bridges between domain O and G.

46. The method of claim 45, wherein the mutations of the O and the Q domains that generate engineered disulfide bridges are a S354C mutation in one of the O domain and Q domains, and a 349C in the other domain.

47. The method of any one of claims 44-46, wherein the orthogonal modifications of the O and the Q domains comprise knob-in-hole mutations.

48. The method of claim 47, wherein the knob-in hole mutations of the O and the Q

domains are a T366W mutation in one of the O domain and Q domain, and a T366S, L368A, and aY407V mutation in the other domain.

49. The method of any one of claims 44-48, wherein the orthogonal modifications of the O and the Q domains comprise charge-pair mutations.

50. The binding molecule of claim 49, wherein the charge-pair mutations of the O and the Q domains are a T366K mutation in one of the O domain and Q domain, and a L351D mutation in the other domain.

51. The method of any one of claims 3-38, wherein the antigen-binding CH1- substituted protein further comprises:

a sixth polypeptide chain, wherein: (a) the third polypeptide chain further comprises a domain R and a domain S, wherein the domains are arranged, from N-terminus to C-terminus, in a R-S-H-I-J-K orientation, and

wherein domain R has a variable region domain amino acid sequence and domain S has a constant domain amino acid sequence;

(b) the binding molecule further comprises a sixth polypeptide chain, comprising: a domain T and a domain U,

wherein the domains are arranged, from N-terminus to C-terminus, in a T-U orientation, and wherein domain T has a variable region domain amino acid sequence and domain U has a constant domain amino acid sequence; and

(c) the third and the sixth polypeptides are associated through an interaction between the R and the T domains and an interaction between the S and the U domains to form the binding molecule.

52. The method of claim 51, wherein

(a) the amino acid sequences of domain R and domain A are identical,

the amino acid sequences of domain H is different from domain R and A, the amino acid sequences of domain S and domain B are identical,

the amino acid sequences of domain I is different from domain S and B,

the amino acid sequences of domain T and domain F are identical,

the amino acid sequences of domain L is different from domain T and F, the amino acid sequences of domain U and domain G are identical,

the amino acid sequences of domain M is different from domain U and G and

the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for the first antigen.

53. The method of claim 51, wherein the antigen-binding CH1 -substituted protein further comprises a second CH1 domain, or portion thereof.

54. The method of claim 53, wherein

(a) the amino acid sequences of domain R and domain H are identical,

the amino acid sequences of domain A is different from domain R and H,

the amino acid sequences of domain S and domain I are identical,

the amino acid sequences of domain B is different from domain S and I,

the amino acid sequences of domain T and domain L are identical,

the amino acid sequences of domain F is different from domain T and L,

the amino acid sequences of domain U and domain M are identical,

the amino acid sequences of domain G is different from domain U and M and

the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and

the domain R and domain T form a third antigen binding site specific for the second antigen.

55. The method claim 54, wherein the amino acid sequences of domain S and domain I are CH1 sequences.

56. The method claim 54, wherein the amino acid sequences of domain U and domain M are CH1 sequences.

57. The method of claim 51, wherein

(a) the amino acid sequences of domain R, domain A, and domain H are different, the amino acid sequences of domain S, domain B, and domain I are different,

the amino acid sequences of domain T, domain F, and domain L are different, and the amino acid sequences of domain U, domain G, and domain M are different; and

the domain R and domain T form a third antigen binding site specific for a third antigen.

58. The method of claim 57, wherein the antigen-binding CH1 -substituted protein further comprises a second CH1 domain, or portion thereof.

59. The method claim 58, wherein the amino acid sequences of domain S and domain I are CH1 sequences.

60. The method claim 58, wherein the amino acid sequences of domain U and domain M are CH1 sequences.

61. The method of any one of claims 3-57, wherein the sequence that forms the junction between the A domain and the B domain is IKRTPREP or IKRTVREP.

62. The method of any one of claims 3-61, wherein the sequence that forms the junction between the F domain and the G domain is SSASPREP.

63. The method of any one of claims 3-62, wherein at least one CH3 amino acid sequence has a C-terminal tripeptide insertion connecting the CH3 amino acid sequence to a hinge amino acid sequence, wherein the tripeptide insertion is selected from the group consisting of PGK, KSC, and GEC.

64. The method of any one of claims 3-63, wherein the sequences are human sequences.

65. The method of any one of claims 3-64, wherein at least one CH3 amino acid sequence is an IgG sequence.

66. The method of any one of claims 3-65, wherein the IgG sequences are IgGl

sequences.

67. The method of any one of claims 3-66, wherein at least one CH3 amino acid sequence has one or more isoallotype mutations.

68. The method of any one of claims 3-67, wherein the isoallotype mutations are D356E and L358M.

69. The method of any one of the preceding claims, wherein at least one of the at least one CH1 domain comprises a human CH1 amino acid sequence, and wherein the CH1 binding reagent binds to a human CH1 epitope.

70. The method of any one of the preceding claims, wherein at least one of the at least one CH1 domain comprises an CH1 amino acid sequence selected from the group consisting of: an IgG CH1, an IgA CH1, an IgE CH1, an IgM CH1, and an IgD CH1.

71. The method of claim 70, wherein at least one of the at least one CH1 domain

comprises an IgG CH1 amino acid sequence.

72. The method of claim 71, wherein the IgG CH1 amino acid sequence comprises an IgGl CH1 amino acid sequence.

73. The method of claim 70, wherein at least one of the at least one CH1 domain

comprises an IgA CH1 amino acid sequence.

74. The method of any one of the preceding claims, wherein at least one of the at least one CH1 domain comprises SEQ ID NO: 23.

75. The method of any one of the preceding claims, wherein at least one of the at least one CH1 domain comprises one or more orthogonal modifications.

76. The method of claim 75, wherein the orthogonal modifications comprise mutations that generate engineered disulfide bridges between the at least one CH1 domain and a CL domain, the mutations selected from the group consisting of: an engineered cysteine at position 138 of the CH1 sequence and position 116 of the CL sequence; an engineered cysteine at position 128 of the CH1 sequence and position 119 of the CL sequence, and an engineered cysteine at position 129 of the CH1 sequence and position 210 of the CL sequence.

77. The method of claim 75, wherein the orthogonal modifications comprise mutations that generate engineered disulfide bridges between the at least one CH1 domain and a CL domain, wherein the mutations comprise and engineered cysteines at position 128 of the CH1 sequence and position 118 of a CL Kappa sequence.

78. The method of claim 75, wherein the orthogonal modifications comprise mutations that generate engineered disulfide bridges between the at least one CH1 domain and a CL domain, the mutations selected from the group consisting of: a Fl 18C mutation in the CL sequence with a corresponding A141C in the CH1 sequence; a Fl 18C mutation in the CL sequence with a corresponding L128C in the CH1 sequence; and a S162C mutations in the CL sequence with a corresponding P171C mutation in the CH1 sequence.

79. The method of any of claims 75-78, wherein the orthogonal modifications comprise charge-pair mutations between the at least one CH1 domain and a CL domain, the charge-pair mutations selected from the group consisting of: a Fl 18S mutation in the CL sequence with a corresponding A141L in the CH1 sequence; a Fl 18A mutation in the CL sequence with a corresponding A141L in the CH1 sequence; a Fl 18V mutation in the CL sequence with a corresponding A141L in the CH1 sequence; and a T129R mutation in the CL sequence with a corresponding K147D in the CH1 sequence.

80. The method of any of claims 75-78, wherein the orthogonal modifications comprise charge-pair mutations between the at least one CH1 domain and a CL domain, the charge-pair mutations selected from the group consisting of: a N138K mutation in the CL sequence with a corresponding G166D in the CH1 sequence,; and a N138D mutation in the CL sequence with a corresponding G166K in the CH1 sequence.

81. The method of any of the preceding claims, wherein at least one of the at least one CH1 domain comprises one or more orthogonal modifications.

82. The method of claim 1, wherein the antigen-binding CH1 -substituted protein is

selected from a lxl MH2 bivalent bispecific protein, and a CH3 domain substitution multispecific protein.

83. The method of any one of the preceding claims, wherein the CH1 binding reagent comprises an anti-CHl antigen binding site.

84. The method of any one of the preceding claims, wherein the CH1 binding reagent comprises an anti-CHl antibody.

85. The method of claim 84, wherein the anti-CHl antibody comprises a single-domain antibody.

86. The method of claim 85, wherein the single-domain antibody comprises a Camelid- derived antibody.

87. The method of any of claims 1-86, wherein the CH1 binding reagent is attached to a surface of a solid support.

88. The method of claim 87, wherein the solid support is selected from the group

consisting of: an agarose bead, a magnetic bead, and a resin.

89. The method of any of claims 87 or 88, wherein the CH1 binding reagent is attached to the surface prior to step (ii).

90. The method of any of claims 87 or 88, wherein the CH1 binding reagent is attached to the surface subsequent to step (ii).

91. The method of any of claims 87-90, wherein the purifying step is selected from the group consisting of: magnetic isolation, column purification, bead centrifugation, resin centrifugation, flow cytometry, and combinations thereof.

92. The method of any one of claims 1-91, further comprising an elution step following step (ii) generating an eluate comprising antigen-binding CH1- substituted protein.

93. The method of claim 92, wherein the elution step comprises contacting the antigen binding CH1 -substituted protein bound to the CH1 binding reagent with a low-pH solution.

94. The method of claim 92, wherein the low-pH solution comprises 0.1 M acetic acid pH 4.0.

95. The method of any one of claims 92-94, wherein the method further comprises an additional purification step following the elution step.

96. The method of claim 95, wherein the additional purification step comprises an ion exchange chromatography purification.

97. The method of claim 96, wherein the ion exchange chromatography purification

comprises cation exchange chromatography.

98. The method of any one of claims 92-97, wherein greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% (w/w) of the total protein in the eluate is the antigen-binding CH1 -substituted protein.

99. The method of claim 98, wherein the greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% (w/w) of the total protein in the eluate is obtained following a single iteration of steps (i)-(iii).

100. The method of claim 98or 99, wherein the greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% (w/w) of the total protein in the eluate is obtained using any of the above methods wherein the purifying step does not comprise use of a binding reagent other than the CH1 binding reagent of any of the above claims.

101. The method of any one of claims 92-97, wherein less than 5% of the first and the third polypeptides are unassociated in the eluate.

102. The method of any one of claims 92-97, wherein less than 5% of the first and the second polypeptides are unassociated in the eluate.

103. The method of any one of claims 92-97, wherein less than 5% of the third and the fourth polypeptides are unassociated in the eluate.

104. The method of any of claims 101-103, wherein the less than less than 5% of the first and the third polypeptides, the less than 5% of the first and the second polypeptides, or the less than 5% of the third and the fourth polypeptides unassociated in the eluate is obtained following a single iteration of steps (i)-(iii).

105. The method of claim 101-104, wherein the less than less than 5% of the first and the third polypeptides, the less than 5% of the first and the second polypeptides, or the less than 5% of the third and the fourth polypeptides unassociated in the eluate is obtained using any of the above methods wherein the purifying step does not comprise use of a binding reagent other than the CH1 binding reagent of any of the above claims.

106. The method of any of claims 1-105, wherein the sample is a supernatant or a lysate of an expression system.

107. The method of claim 106, wherein the expression system is selected from the group consisting of: a cell free expression system, a mammalian cell culture, a bacterial cell culture, a yeast cell culture.

108. The method of claim 107, wherein the mammalian cell culture comprises an

immortalized cell line.

109. The method of claim 108, wherein the immortalized cell line is a chine hamster ovary (CHO) or human 293 derived cell line.

110. The method of any of claims 106-109, wherein the expression system stably expresses the polypeptide chains of the antigen-binding CH1 -substituted protein.

111. The method of any of claims 106-110, wherein the expression system is a serum-free expression system.

112. An antigen-binding CH1 -substituted protein purified by the method of any one of claims 1-111.

113. A pharmaceutical composition comprising the antigen-binding CH1 -substituted

protein of claim 112.

114. A method of treatment, comprising:

administering to a subject in need of treatment the pharmaceutical composition of claim 113.