CN113880950A

CN113880950A - Coagulation Factor XI (FXI) binding proteins

Info

Publication number: CN113880950A
Application number: CN202010636876.0A
Authority: CN
Inventors: 徐霆; 汪皛皛; 逄敏洁; 郭康平; 金宇灏
Original assignee: Suzhou Alphamab Co Ltd
Current assignee: Suzhou Alphamab Co Ltd
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2022-01-04

Abstract

The invention relates to the field of medical biology, and discloses a single-domain antibody for coagulation Factor XI (FXI) and derived proteins thereof. In particular, the invention discloses a coagulation Factor XI (FXI) binding protein derived from a single domain antibody against coagulation Factor XI (FXI) and uses thereof.

Description

Coagulation Factor XI (FXI) binding proteins

Technical Field

Background

Coagulation factors are various protein components involved in the process of blood coagulation. Its physiological role is to be activated when a blood vessel bleeds, to bind platelets together and to fill leaks in the blood vessel. This process is called coagulation. They are produced in part by the liver. Can be inhibited by coumarin. For uniform nomenclature, the world health organization is numbered with Roman numerals in the order in which they were discovered, with coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII, and the like.

Coagulation factor xi (fxi) is a dimer composed of identical 80KDa subunits, each subunit consisting, starting from the N-terminus, of four Apple domains (a1, a2, A3 and a4) and a catalytic domain. FXI is a zymogen that circulates in complex with high molecular weight kininogen (HK). HK binds the a2 domain in FXI and is a physiological cofactor for FXIIa activation of FXI to FXIa. The remaining applet domain in FXI also mediates important physiological functions. For example, the FIX binding exosite is located in A3, whereas the FXIIa binding site is in a 4. Residues critical for FXI dimerization are also located in a 4.

Studies have shown that FXI plays a key role in the pathological process of thrombosis, contributes relatively little to hemostasis, and is therefore a promising target for thrombi. In the Ionis Pharmaceuticals Inc. FXI antisense oligonucleotide (ASO) phase II trial (Buller et al, N Engl J Med 2015,372: 232-. Human genetics and epidemiological studies (Duga et al, Semin Thromb Hemost 2013; Chen et al, Drug Discov Today 2014; Key, Hematology Am Soc Hematol Educ Program 2014,2014:66-70) indicate that severe FXI deficiency (hemophilia C) reduces the risk of ischemic stroke and deep vein embolism; conversely, increased FXI levels are associated with higher risk of VTE and ischemic stroke. Furthermore, several preclinical studies have shown that FXIa inhibition or loss of function mediates thrombus protection without compromising hemostasis (Chen et al, Drug Discov Today 2014). Notably, monoclonal antibodies 14E11 and 1A6 produced significant thrombus mitigation in the baboon AV branch thrombosis model (U.S. Pat. No. 8,388,959; Tucker et al, Blood 2009,113: 936-. Furthermore, 14E11 (which cross-reacts with mouse FXI) provides protection in an experimental model of mouse acute ischemic Stroke (Leung et al, Transl Stroke Res 2012,3: 381-389). Additional studies of mabs targeting FXI in preclinical models have also been reported, confirming FXI as an antithrombotic target with a minimal risk of hemorrhage (van Montfoort et al, Thromb Haemost 2013,110; Takahashi et al, Thromb Res 2010,125: 464-. Inhibition of FXI is therefore a promising strategy for novel antithrombotic therapy with an improved benefit-risk ratio compared to current standard anticoagulants.

Brief Description of Drawings

FIG. 1 shows the FXI blocking activity of FXI single domain antibody Fc fusion proteins (ATPP assay).

FIG. 2 shows the blocking activity of humanized FXI single domain antibody Fc fusion protein against FXI (ATPP assay).

Figure 3 shows the inhibitory effect of huFE bispecific antibody Fc fusion proteins on human FXI activity.

Figure 4 shows the inhibitory activity of huFE bispecific antibody Fc fusion proteins against human whole plasma APTT.

Figure 5 shows inhibitory activity of huFE bispecific antibody Fc fusion protein on monkey whole plasma APTT.

Figure 6 shows the inhibitory activity of huFE bispecific antibody Fc fusion protein on rabbit whole plasma APTT.

FIG. 7 shows the effect of FXI single domain antibody Fc fusion protein and bispecific antibody on rabbit venous thrombosis.

Detailed Description

Definition of

Unless otherwise indicated or defined, all terms used have the ordinary meaning in the art that will be understood by those skilled in the art. Reference is made, for example, to standard manuals, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2 nd edition), Vol.1-3, Cold Spring Harbor Laboratory Press (1989); lewis, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al, "Immunology" (2 nd edition), Gower Medical Publishing, London, New York (1989), and the general prior art cited herein; moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically recited may be and have been performed in a manner known per se to those of skill in the art. Reference is also made, for example, to standard manuals, the general prior art mentioned above and to other references cited therein.

Unless otherwise indicated, the terms "antibody" or "immunoglobulin" used interchangeably herein, whether referring to a heavy chain antibody or to a conventional 4 chain antibody, are used as general terms to include full-length antibodies, individual chains thereof, as well as all portions, domains or fragments thereof (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH/VL domains, respectively). Furthermore, the term "sequence" as used herein (e.g. in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence" etc.) should generally be understood to include both the relevant amino acid sequences and the nucleic acid or nucleotide sequences encoding the sequences, unless a more limited interpretation is required herein.

As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, domains are responsible for individual functional properties of proteins, and in many cases may be added, removed, or transferred to other proteins without loss of function of the rest of the protein and/or domain.

The term "immunoglobulin domain" as used herein refers to a globular region of an antibody chain (e.g., a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or to a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized in that they maintain the immunoglobulin folding characteristics of the antibody molecule.

The term "immunoglobulin variable domain" as used herein refers to an immunoglobulin domain consisting essentially of four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR 1", "framework region 2" or "FR 2", "framework region 3" or "FR 3", and "framework region 4" or "FR 4", respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR 1", "complementarity determining region 2" or "CDR 2", and "complementarity determining region 3" or "CDR 3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Immunoglobulin variable domains confer specificity for an antigen to an antibody by virtue of having an antigen binding site.

The term "immunoglobulin single variable domain" as used herein refers to an immunoglobulin variable domain that is capable of specifically binding an epitope of an antigen without pairing with other immunoglobulin variable domains. An example of an immunoglobulin single variable domain within the meaning of the present invention is a "domain antibody", e.g. immunoglobulin single variable domains VH and VL (VH and VL domains). Another example of an immunoglobulin single variable domain is a camelidae "VHH domain" (or simply "VHH") as defined below.

"VHH domains", also known as heavy chain single domain antibodies, VHHs, VHH domains, VHH antibody fragments and VHH antibodies, are variable domains of antigen-binding immunoglobulins (Hamers-Casterman C, Atarhouch T, Muydermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: Natural curing antibodies void of light chains "; Nature 363,446-448(1993)) known as" heavy chain antibodies "(i.e." antibodies lacking a light chain). The term "VHH domain" is used to distinguish the variable domain from a heavy chain variable domain (which is referred to herein as a "VH domain") present in conventional 4 chain antibodies, and a light chain variable domain (which is referred to herein as a "VL domain") present in conventional 4 chain antibodies. The VHH domain specifically binds to an epitope without the need for an additional antigen binding domain (as opposed to the VH or VL domain in conventional 4 chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed from a single immunoglobulin domain.

In the context of the present invention, the terms "heavy chain single domain antibody", "VHH domain", "VHH antibody fragment" and "VHH antibody" are used interchangeably.

For example, as shown in FIG. 2 of Riechmann and Muydermans, J.Immunol.methods 231,25-38(1999), amino acid residues employed for Camelidae VHH domains may be numbered according to the general numbering of VH domains given by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed.public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

Substitution methods for numbering amino acid residues of VH domains are known in the art and may also be applied analogously to VHH domains. For example, the Chothia CDR refers to the position of the structural loop (Chothia and Lesk, J.mol.biol.196:901-917 (1987)). AbM CDR representation of Kabat hypervariable region and Chothia structural loop compromise, and in Oxford Molecular's AbM antibody modeling software using. The "Contact" CDR is based on an analysis of the available complex crystal structure. The residues from the CDRs from each approach are described below:

the CDRs of the antibody may also be IMGT-CDRs, a form of CDR definition based on the IMGT antibody code, which is obtained by integrating the structural information of more than 5000 sequences. In VH CDR encoding of IMGT, CDR 1: 27-38; CDR 2: 56-65; CDR 3: 105-117.

It should be noted, however, that the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering, as is well known in the art for VH and VHH domains (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.

For example, the CDRs may include "extended (CDRs"), such as: 24-36 or 24-34(LCDR1), 46-56 or 50-56(LCDR2) and 89-97 or 89-96(LCDR3) in VL; 26-35(HCDR1), 50-65 or 49-65(HCDR2) and 93-102, 94-102 or 95-102(HCDR3) in the VH.

The total number of amino acid residues in the VHH domain will generally range from 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

Other structural and functional properties of VHH domains and polypeptides comprising the same may be summarized as follows:

the VHH domain, which has been naturally "designed" to functionally bind to an antigen in the absence and without interaction with a light chain variable domain, can be used as a single and relatively small functional antigen binding unit, domain or polypeptide. This distinguishes VHH domains from VH and VL domains of conventional 4 chain antibodies, which are themselves generally unsuitable for practical application as single antigen binding proteins or immunoglobulin single variable domains, but need to be combined in some form or another to provide a functional antigen binding unit (e.g. in the form of a conventional antibody fragment such as a Fab fragment; or in the form of a scFv consisting of a VH domain covalently linked to a VL domain).

Because of these unique properties, the use of VHH domains-alone or as part of a larger polypeptide-offers a number of significant advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g. Fab-or F (ab') 2-fragments): only a single domain is required to bind antigen with high affinity and high selectivity, so that neither two separate domains need be present, nor is it required to ensure that the two domains are present in the proper spatial conformation and configuration (e.g., scFv's typically require the use of specially designed linkers); the VHH domain may be expressed from a single gene and does not require post-translational folding or modification; VHH domains can be easily engineered into multivalent and multispecific formats (formatting); the VHH domain is highly soluble and has no tendency to aggregate; the VHH domain is highly stable to heat, pH, proteases and other denaturants or conditions, and therefore refrigeration equipment may not be used in preparation, storage or transport, thereby achieving cost, time and environmental savings; VHH domains are easy to prepare and relatively inexpensive, even on the scale required for production; the VHH domain is relatively small compared to conventional 4 chain antibodies and antigen binding fragments thereof (about 15kDa or 1/10 of conventional IgG in size), and therefore shows higher tissue permeability and can be administered at higher doses compared to conventional 4 chain antibodies and antigen binding fragments thereof; VHH domains may exhibit so-called cavity-binding properties (especially due to their extended CDR3 loops compared to conventional VH domains) allowing access to targets and epitopes not accessible by conventional 4-chain antibodies and antigen-binding fragments thereof.

Methods for obtaining VHHs that bind to a particular antigen or epitope have been previously disclosed in the following references: van der Linden et al, Journal of Immunological Methods,240(2000) 185-195; li et al, J Biol chem, 287(2012) 13713-13721; deffar et al, African Journal of Biotechnology Vol.8(12), pp.2645-2652,17June,2009 and WO 94/04678.

Camelid derived VHH domains may be "humanized" (also referred to herein as "sequence optimized", which in addition to humanization may also encompass other modifications to the sequence by one or more mutations providing VHH-modifying properties, such as removal of potential post-translational modification sites) by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in the VH domain of a human conventional 4 chain antibody. The humanized VHH domain may contain one or more fully human framework region sequences. Humanization can be accomplished using methods of protein surface amino acid humanization (resurfacing) and/or humanized universal framework CDR grafting (CDR grafting to a universal framework), for example, as exemplified in the examples.

As used herein, the term "epitope" or the interchangeably used term "antigenic determinant" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Antigenic determinants generally comprise chemically active surface groups of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically includes at least 3,4, 5, 6, 7, 8,9, 10,11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be a "linear" epitope or a "conformational" epitope. See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996). In a linear epitope, the points of all interactions between a protein and an interacting molecule (e.g., an antibody) are linearly present along the primary amino acid sequence of the protein. In conformational epitopes, the point of interaction exists across protein amino acid residues that are separated from each other.

Epitopes of a given antigen can be identified using a number of epitope mapping techniques well known in the art. See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996). For example, a linear epitope can be determined by, for example, the following methods: a plurality of peptides are simultaneously synthesized on a solid support, wherein the peptides correspond to portions of a protein molecule, and the peptides are reacted with an antibody while still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. nos. 4,708,871; geysen et al (1984) Proc.Natl.Acad.Sci.USA 81: 3998-; geysen et al (1986) molecular. Immunol.23: 709-715. Similarly, conformational epitopes can be identified by determining the spatial configuration of amino acids, such as by x-ray crystallography and 2-dimensional nuclear magnetic resonance, for example. See, e.g., Epitope Mapping Protocols (supra).

Antibodies can be screened for binding competition with the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to the antigen. A high throughput method for obtaining antibodies binding to the same epitope based on their cross-competition is described in International patent application WO 03/48731. Thus, antibodies and antigen-binding fragments thereof that compete with the antibody molecule of the invention for binding to the same epitope on FXI can be obtained using conventional techniques known to those skilled in the art.

In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein (e.g., an immunoglobulin single variable domain of the invention) can bind. Specificity of an antigen binding protein can be determined based on its affinity and/or avidity. The affinity, expressed by the dissociation equilibrium constant (KD) of an antigen to an antigen binding protein, is a measure of the strength of binding between an epitope and the antigen binding site on the antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen binding protein (alternatively, affinity can also be expressed as the association constant (KA), which is 1/KD). As will be appreciated by those skilled in the art, affinity can be determined in a known manner depending on the particular antigen of interest. Avidity is a measure of the strength of binding between an antigen binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and an associated antigen. Affinity is related to both: affinity to its antigen binding site on the antigen binding protein, and the number of relevant binding sites present on the antigen binding protein.

The term "factor xi (fxi) -binding protein" as used herein means any protein capable of specifically binding to factor xi (fxi). The FXI-binding protein may comprise an antibody as defined herein directed against FXI. FXI binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.

The "FXI-binding protein" of the invention may comprise at least one immunoglobulin single variable domain, such as VHH, that binds FXI. In some embodiments, an "FXI binding molecule" of the invention may comprise 2,3, 4 or more immunoglobulin single variable domains, such as VHH, that bind FXI. The FXI binding proteins of the invention may also comprise, in addition to binding to the immunoglobulin single variable domain of FXI, a linker and/or a moiety with effector function, e.g., a half-life extending moiety (such as an immunoglobulin single variable domain that binds serum albumin), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region. In some embodiments, the "FXI-binding proteins" of the invention also encompass bispecific antibodies that contain immunoglobulin single variable domains that bind to different antigens or different regions (e.g., different epitopes) of the same antigen.

Typically, the FXI binding proteins of the invention will be measured preferably 10 as measured in Biacore or KinExA or Fortibio assays^-7To 10^-10Mole/liter (M), more preferably 10^-8To 10^-10Mole/liter, even more preferably 10^-9To 10^-10Or a dissociation constant (KD) of at least 10, and/or⁷M^-1Preferably at least 10⁸M^-1More preferably at least 10⁹M^-1More preferably at least 10¹⁰M^-1Binds to the antigen to be bound (i.e., FXI). Any greater than 10^-4The KD value of M is generally considered to indicate non-specific binding. Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, Surface Plasmon Resonance (SPR) assays, Scatchard assays, and/or competitive binding assays (e.g., Radioimmunoassays (RIA), Enzyme Immunoassays (EIA), and sandwich competitive assays, as described herein.

Amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is well known and agreed upon in the art. In comparing two amino acid sequences, the term "amino acid difference" refers to the specified number of amino acid residues at a position in the reference sequence compared to the other sequence insertion, deletion or substitution. In the case of a substitution, the substitution will preferably be a conservative amino acid substitution, meaning that the amino acid residue is replaced with another amino acid residue that is chemically similar in structure and that has little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably made where one amino acid within the following groups (i) - (v) is replaced with another amino acid residue within the same group: (i) smaller aliphatic nonpolar or weakly polar residues: ala, Ser, Thr, Pro, and Gly; (ii) polar negatively charged residues and their (uncharged) amides: asp, Asn, Glu and Gln; (iii) polar positively charged residues: his, Arg and Lys; (iv) larger aliphatic non-polar residues: met, Leu, Ile, Val and Cys; and (v) aromatic residues: phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: ala substituted by Gly or Ser; arg is replaced by Lys; asn is replaced by Gln or His; asp substituted by Glu; cys is substituted with Ser; gln is substituted by Asn; glu is substituted with Asp; gly by Ala or Pro; his is substituted with Asn or Gln; ile is substituted by Leu or Val; leu is substituted by Ile or Val; lys is substituted with Arg, Gln, or Glu; met is substituted by Leu, Tyr or Ile; phe is substituted by Met, Leu or Tyr; ser substituted by Thr; thr is substituted by Ser; trp is substituted by Tyr; tyr is substituted with Trp or Phe; val is substituted by Ile or Leu.

"sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. "sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity. For the determination of sequence identity see, for example: computational Molecular Biology, Lesk, a.m., ed., Oxford University Press, New York, 1988; biocontrol, information and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; sequence Analysis in Molecular Biology, von Heinje, G., Academic Press,1987 and Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, New York, 1991.

A polypeptide or nucleic acid molecule is considered "isolated" when it has been separated from at least one other component with which it is normally associated in the source or medium (culture medium), such as another protein/polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or minor component, as compared to the reaction medium or culture medium from which it is naturally derived and/or from which it is obtained. In particular, a polypeptide or nucleic acid molecule is considered "isolated" when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold and up to 1000-fold or more than 1000-fold. The "isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous, as determined by suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).

By "effective amount" is meant an amount of an FXI-binding protein or pharmaceutical composition of the invention that results in a decrease in the severity of the disease symptoms, an increase in the frequency and duration of the asymptomatic phase of the disease, or prevention of injury or disability due to the affliction of the disease.

As used herein, "thrombosis" refers to the formation or presence of a clot (also referred to as "thrombus") within a blood vessel, thereby impeding the flow of blood through the circulatory system. Thrombosis is generally caused by abnormalities in the composition of the blood, the quality of the vessel walls and/or the nature of the blood flow. Clot formation is generally caused by injury to the vessel wall (such as from trauma or infected vessel wall damage) and the slowing or arresting of blood flow through the injury site. In some cases, the coagulation abnormality causes thrombosis.

As used herein, "does not impair hemostasis" means that little or no detectable bleeding is observed in a subject following administration of a subject FXI binding protein or pharmaceutical composition of the invention to the subject. In the case of targeted FXI, inhibition of conversion of FXI to FXIa or inhibition of FXIIa activation of FIX inhibits coagulation and associated thrombosis without bleeding.

The term "subject" as used herein means a mammal, particularly a primate, particularly a human.

FXI binding proteins of the invention

In one aspect, the invention provides an FXI binding protein comprising at least one immunoglobulin single variable domain capable of specifically binding FXI.

In some embodiments, the at least one immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in a VHH set forth in any one of SEQ ID NOs 1-23. The CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs, or IMGT CDRs.

In some embodiments, the at least one immunoglobulin single variable domain comprises a set of CDRs 1, CDR2, and CDR3 selected from:

in some embodiments, at least one immunoglobulin single variable domain in the FXI binding protein of the invention is a VHH. In some embodiments, the VHH comprises any one of the amino acid sequences of SEQ ID NOs 1-23.

In some embodiments, at least one immunoglobulin single variable domain in the FXI binding protein of the invention is a humanized VHH.

In some embodiments, at least one immunoglobulin single variable domain in an FXI binding protein of the invention is a humanized VHH comprising an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to any one of SEQ ID NOs 1-23. In some embodiments, the amino acid sequence of the humanized VHH comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NOs 1-23. For example, 1,2, 3,4, 5, 6, 7, 8,9, or 10 conservative amino acid substitutions are included.

In some embodiments, at least one immunoglobulin single variable domain in the FXI binding proteins of the invention is a humanized VHH, wherein said humanized VHH comprises any of the amino acid sequences of SEQ ID NO 300-335.

In some embodiments, the at least one immunoglobulin single variable domain binds an epitope within the Apple2 domain of FXI. An exemplary amino acid sequence of the Apple2 domain of FXI is shown in SEQ ID NO 338. For example, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in a VHH as set forth in any one of SEQ ID NO. 4, SEQ ID NO. 10 or SEQ ID NO. 14. In some embodiments, the immunoglobulin single variable domain comprises a set of CDRs 1, CDRs 2 and CDRs 3 selected from the group consisting of SEQ ID NOS 60-62, SEQ ID NOS 63-65, SEQ ID NOS 66-68, SEQ ID NOS 69-71, SEQ ID NOS 132-134, SEQ ID NOS 135-137, SEQ ID NOS 138-140, SEQ ID NOS 141-143, SEQ ID NOS 180-182, SEQ ID NOS 183-185, SEQ ID NOS 186-188, SEQ ID NOS 189-191. In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence set forth in any one of SEQ ID NO 4, SEQ ID NO 10, or SEQ ID NO 14. In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence set forth in any one of SEQ ID NO 306-323.

In some embodiments, the at least one immunoglobulin single variable domain does not bind to an epitope within the Apple2 domain of FXI. In some embodiments, the at least one immunoglobulin single variable domain does not bind the Apple2 domain of FXI. In some embodiments, the at least one immunoglobulin single variable domain does not bind to the Apple2 domain polypeptide of isolated FXI.

In some embodiments, the at least one immunoglobulin single variable domain binds an epitope within the Apple3 domain of FXI. An exemplary amino acid sequence of the Apple3 domain of FXI is shown in SEQ ID NO: 339. For example, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 of VHH shown in SEQ ID NO. 17. In some embodiments, the immunoglobulin single variable domain comprises a set of CDRs 1, CDRs 2 and CDRs 3 selected from the group consisting of SEQ ID NO 216-218, SEQ ID NO 219-221, SEQ ID NO 222-224, and SEQ ID NO 225-227. In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO 17. In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence set forth in any one of SEQ ID NO 324-329.

In some embodiments, the at least one immunoglobulin single variable domain binds an epitope within the Apple4 domain of FXI. An exemplary amino acid sequence of the Apple4 domain of FXI is shown in SEQ ID NO: 340. For example, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 of VHH shown in SEQ ID NO. 1. In some embodiments, the immunoglobulin single variable domain comprises a set of CDRs 1, CDRs 2, and CDRs 3 selected from the group consisting of SEQ ID NOS 24-26, SEQ ID NOS 27-29, SEQ ID NOS 30-32, SEQ ID NOS 33-35. In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO 1. In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence set forth in any one of SEQ ID NO 300-305.

In some embodiments, the at least one immunoglobulin single variable domain binds an epitope within the Apple1-2 region of FXI (the region between the Apple1 domain and the Apple2 domain). An exemplary amino acid sequence of the Apple1-2 region of FXI is shown in SEQ ID NO: 341.

In some embodiments, the at least one immunoglobulin single variable domain binds an epitope within the Apple2-3 region of FXI (the region between the Apple2 domain and the Apple3 domain). An exemplary amino acid sequence of the Apple2-3 region of FXI is shown in SEQ ID NO: 342. For example, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 of VHH represented by SEQ ID NO. 20. In some embodiments, the immunoglobulin single variable domain comprises a set of CDRs 1, CDRs 2 and CDRs 3 selected from the group consisting of SEQ ID NO:252-254, SEQ ID NO:255-257, SEQ ID NO:258-260, SEQ ID NO: 261-263. In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO 20. In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence set forth in any one of SEQ ID NO 330-335.

In some embodiments, the at least one immunoglobulin single variable domain binds an epitope within the Apple3-4 region of FXI (the region between the Apple3 domain and the Apple4 domain). An exemplary amino acid sequence of the Apple3-4 region of FXI is shown in SEQ ID NO: 343.

In some embodiments, the FXI-binding protein comprises an immunoglobulin single variable domain that specifically binds FXI.

In some embodiments, the FXI-binding protein comprises at least two, e.g., 2,3, 4, or more immunoglobulin single variable domains that specifically bind FXI.

In some embodiments, the at least two immunoglobulin single variable domains bind to the same region or epitope of FXI, or compete for binding or partially compete for binding to the same region or epitope of FXI, e.g., the at least two immunoglobulin single variable domains are identical.

In some embodiments, the at least two immunoglobulin single variable domains bind different regions or epitopes of FXI, or do not compete for binding to the same region or epitope of FXI.

Whether two antibodies or immunoglobulin single variable domains bind or compete for binding to the same region or epitope can be determined by epitope binding by the biofilm interference technique BLI, as exemplified in the examples of the present application.

In some embodiments, the at least two immunoglobulin single variable domains that specifically bind FXI are directly linked to each other.

In some embodiments, the at least two immunoglobulin single variable domains that specifically bind FXI are linked to each other by a linker. The linker may be a non-functional amino acid sequence of 1-20 or more amino acids in length without more than secondary structure. For example, the linker is a flexible linker such as GGGGS, GS, GAP, (GGGGS) x 3, and the like.

In some embodiments, the FXI-binding protein comprises a first immunoglobulin single variable domain capable of specifically binding FXI and a second immunoglobulin single variable domain capable of specifically binding FXI, wherein the first immunoglobulin single variable domain and the second immunoglobulin single variable domain bind different epitopes on FXI.

In some embodiments, the first immunoglobulin single variable domain binds an epitope within the Apple2 domain of FXI and the second immunoglobulin single variable domain binds an epitope within the Apple3 domain of FXI; or

The first immunoglobulin single variable domain binds to an epitope within the Apple2 domain of FXI and the second immunoglobulin single variable domain binds to an epitope within the Apple4 domain of FXI; or

The first immunoglobulin single variable domain binds to an epitope within the Apple2 domain of FXI and the second immunoglobulin single variable domain binds to an epitope within the Apple2-3 region of FXI; or

The first immunoglobulin single variable domain binds to an epitope within the Apple3 domain of FXI and the second immunoglobulin single variable domain binds to an epitope within the Apple4 domain of FXI; or

The first immunoglobulin single variable domain binds to an epitope within the Apple3 domain of FXI and the second immunoglobulin single variable domain binds to an epitope within the Apple2-3 region of FXI; or

The first immunoglobulin single variable domain binds to an epitope within the Apple4 domain of FXI and the second immunoglobulin single variable domain binds to an epitope within the Apple2-3 region of FXI.

In some embodiments, the FXI binding protein comprises a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, wherein,

the first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 1, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 4; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 1, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 9; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 1, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 10; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 1, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 14; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 1, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 17; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 1, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 20; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 4, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 9; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 4, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 10; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 4, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 14; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 4, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 17; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 4, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 20; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 9, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 10; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 9, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 14; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 9, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 17; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 9 and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 20; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 10 and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 14; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 10 and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 17; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 10 and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 20; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH represented by SEQ ID NO. 14 and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH represented by SEQ ID NO. 17; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 14, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH set forth in SEQ ID No. 20; or

The first immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH shown in SEQ ID NO. 17, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in VHH shown in SEQ ID NO. 20.

In some embodiments, wherein the CDR1, CDR2, and CDR3 in the VHH set forth in

SEQ ID NO

1, 4, 9, 10, 14, 17, or 20 are set forth in the following table:

the first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 1, 300-305 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 4, 306-311; or

The first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO 1, 300-305 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO 9; or

The first immunoglobulin single variable domain comprises an amino acid sequence as set forth in one of SEQ ID NO 1, 300-305 and the second immunoglobulin single variable domain comprises an amino acid sequence as set forth in one of SEQ ID NO 10, 312-317; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 1, 300-305 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 14, 318-323; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 1, 300-305 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 17, 324-329; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 1, 300-305 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 20, 330-335; or

The first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO 4, 306-311 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO 9; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 4, 306-311 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in VHH as set forth in one of SEQ ID NO 10, 312-317; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 4, 306-311 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 14, 318-323; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 4, 306-311 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 17, 324-329; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 4, 306-311 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 20, 330-335; or

The first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO 9 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO 10, 312-317; or

The first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO 9 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO 14, 318-323; or

The first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO 9 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO 17, 324-329; or

The first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO 9 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO 20, 330-335; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 10, 312-317 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 14, 318-323; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 10, 312-317 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 17, 324-329; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NOs 10, 312-317 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NOs 20, 330-335; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 14, 318-323 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 17, 324-329; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 14, 318-323 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 20, 330-335; or

The first immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 17, 324-329 and the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NO 20, 330-335.

In some embodiments, wherein the first immunoglobulin single variable domain is N-terminal to the second immunoglobulin single variable domain. In other embodiments, wherein the second immunoglobulin single variable domain is N-terminal to the first immunoglobulin single variable domain.

In some embodiments, the FXI binding protein of the invention comprises an immunoglobulin Fc region in addition to at least one immunoglobulin single variable domain capable of specifically binding FXI. The inclusion of an immunoglobulin Fc region in the FXI binding proteins of the invention allows the binding molecules to form dimers. Fc regions useful in the present invention may be from different subtypes of immunoglobulin, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.

In some embodiments, mutations can be introduced on the wild-type Fc sequence for altering the relevant Fc-mediated activity. Such mutations include, but are not limited to: a) a mutation that alters Fc-mediated CDC activity; b) a mutation that alters Fc-mediated ADCC activity; a mutation that alters FcRn-mediated half-life in vivo. Such mutations are described in the following documents: leonard G Presta, Current Opinion in Immunology 2008,20: 460-; esohe E.Idusogene et al, J Immunol 2000,164: 4178-; RAPHAEL A.CLYNES et al, Nature Medicine,2000, Volume 6, Number 4: 443-; paul R.Hinton et al, J Immunol,2006,176: 346-. For example, mutations of 1,2, 3,4, 5, 6, 7, 8,9 or 10 amino acids in the CH2 region may be used to increase or remove Fc-mediated ADCC or CDC activity or to enhance or reduce the affinity of FcRn. In addition, the stability of the protein can be increased by mutating 1,2, 3,4, 5, 6, 7, 8,9 or 10 amino acids of the hinge region.

In some embodiments, mutations can be introduced on the Fc sequence, thereby making the mutated Fc more susceptible to the formation of homodimers or heterodimers. The knob-hole model, which exploits steric effects of amino acid side chain groups at the Fc contact interface, as mentioned in Ridgway, Presta et al 1996 and Carter 2001, makes heterodimerization between different Fc mutations easier; for example, in CN 102558355A or CN 103388013a, the ionic interaction force between Fc contact interfaces is changed by changing the charges of the amino acids on the Fc contact interfaces, so that heterodimers (CN 102558355A) are more easily formed between different Fc mutation pairs or homodimers (CN 103388013a) are more easily formed between Fc with the same mutations.

The immunoglobulin Fc region is preferably a human immunoglobulin Fc region, for example, an Fc region of human IgG1, IgG2, IgG3, or IgG 4. In some embodiments, the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO 336.

In some embodiments, in the FXI-binding proteins of the invention, the immunoglobulin Fc region (e.g., the Fc region of human IgG 1) is linked directly or indirectly through a linker (e.g., a peptide linker) to the C-terminus of the immunoglobulin single variable domain (e.g., VHH).

In some embodiments, the FXI-binding protein of the invention comprises one immunoglobulin single variable domain that specifically binds FXI, linked directly or through a linker to an immunoglobulin Fc region that allows the FXI-binding protein to form a dimeric molecule comprising two FXI-binding domains. Such FXI binding proteins are also referred to as bivalent FXI binding proteins. In some embodiments, the dimer is a homodimer.

In some embodiments, the FXI-binding protein of the invention comprises two immunoglobulin single variable domains that specifically bind FXI and one immunoglobulin Fc region that allows the FXI-binding protein to form a dimeric molecule comprising four FXI-binding domains, linked to each other directly or through a linker. Such FXI binding proteins are also known as tetravalent FXI binding proteins. In some embodiments, the dimer is a homodimer. In some embodiments, two immunoglobulin single variable domains in the FXI binding protein that specifically bind FXI bind different regions or different epitopes of FXI, respectively.

In some embodiments, the FXI binding proteins of the invention are capable of inhibiting the activity of FXI. In some embodiments, the FXI binding proteins of the invention are capable of inhibiting the coagulation function of FXI.

Nucleic acids, vectors, host cells

In another aspect, the invention relates to a nucleic acid molecule encoding the FXI binding protein of the invention. The nucleic acid of the present invention may be RNA, DNA or cDNA. According to one embodiment of the invention, the nucleic acid of the invention is a substantially isolated nucleic acid.

The nucleic acid of the invention may also be in the form of a vector, may be present in and/or may be part of a vector, such as a plasmid, cosmid or YAC. The vector may especially be an expression vector, i.e. a vector providing for the expression of the FXI-binding protein in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression system). The expression vector typically comprises at least one nucleic acid of the invention operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, and the like). The selection of the elements and their sequences for expression in a particular host is within the knowledge of one skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for expression of the FXI binding proteins of the invention, such as promoters, enhancers, terminators, integration factors, selection markers, leaders, reporters.

The nucleic acids of the invention may be prepared or obtained in a known manner (e.g., by automated DNA synthesis and/or recombinant DNA techniques) based on information regarding the amino acid sequence of the polypeptides of the invention given herein, and/or may be isolated from a suitable natural source.

In another aspect, the invention relates to a recombinant host cell expressing or capable of expressing one or more FXI-binding proteins of the invention and/or comprising a nucleic acid or vector of the invention. Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.

Suitable bacterial cells include cells of gram-negative bacterial strains, such as Escherichia coli, Proteus and Pseudomonas strains, and gram-positive bacterial strains, such as Bacillus (Bacillus), Streptomyces, Staphylococcus and Lactococcus strains.

Suitable fungal cells include cells of species of the genera Trichoderma (Trichoderma), Neurospora (Neurospora) and Aspergillus (Aspergillus); or cells of species including Saccharomyces (Saccharomyces) such as Saccharomyces cerevisiae, Schizosaccharomyces (Schizosaccharomyces pombe), Pichia (Pichia) such as Pichia pastoris and Pichia methanolica, and Hansenula.

Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expression of heterologous proteins may also be used in the present invention.

The invention also provides a method of producing an FXI-binding protein of the invention, the method generally comprising the steps of:

-culturing a host cell of the invention under conditions allowing the expression of the FXI-binding protein of the invention; and

-recovering from the culture the FXI-binding protein expressed by the host cell; and

-optionally further purifying and/or modifying the FXI-binding protein of the invention.

The FXI-binding proteins of the invention can be produced intracellularly (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies) in cells as described above, followed by isolation from the host cell and optionally further purification; or it may be produced extracellularly (e.g. in the medium in which the host cell is cultured), followed by isolation from the medium and optionally further purification.

Methods and reagents for recombinant production of polypeptides, such as specifically adapted expression vectors, transformation or transfection methods, selection markers, methods of inducing protein expression, culture conditions, and the like, are known in the art. Similarly, protein isolation and purification techniques suitable for use in the methods of making FXI-binding proteins of the invention are well known to those skilled in the art.

However, the FXI-binding proteins of the invention may also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or solution phase synthesis.

Pharmaceutical composition

In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising one or a combination of FXI binding proteins of the invention formulated together with a pharmaceutically acceptable carrier. Such compositions may comprise one or a combination (e.g. two or more different) of the FXI binding proteins of the invention. For example, the pharmaceutical composition of the invention may contain a combination of antibody molecules that bind to different epitopes on the target antigen (FXI).

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody molecule, may be encapsulated in a material to protect the compound from acids and other natural conditions that may inactivate the compound.

The pharmaceutical compositions of the present invention may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

These compositions may also contain, for example, preservatives, wetting agents, emulsifying agents and dispersing agents.

Prevention of the presence of microorganisms can be ensured by sterilization procedures or by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium oxide in the composition. Prolonged absorption of the injectable drug can be achieved by incorporating into the composition a delayed absorption agent, such as monostearate salts and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Conventional media or agents, except insofar as any is incompatible with the active compound, may be present in the pharmaceutical compositions of the invention. Supplementary active compounds may also be incorporated into the composition.

Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form is generally that amount of the composition which produces a therapeutic effect. Typically, this amount ranges from about 0.01% to about 99% of the active ingredient, e.g., from about 0.1% to about 70%, or from about 1% to about 30%, by 100%, in combination with a pharmaceutically acceptable carrier.

Dosage regimens may be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be scaled down or up as required by the exigencies of the therapeutic condition. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specifics of the dosage unit forms of the invention are defined and directly dependent upon (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such active compounds for use in the treatment of sensitivity in an individual.

For administration of the antibody molecule, the dosage range is about 0.0001 to 100mg/kg, more usually 0.01 to 30mg/kg of the recipient's body weight. For example, the dose may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, 10mg/kg body weight, 20mg/kg body weight, or 30mg/kg body weight, or in the range of 1-30mg/kg body weight. Exemplary treatment regimens require weekly dosing, biweekly dosing, every three weeks, every four weeks, monthly dosing, every 3 months, every 3-6 months, or slightly shorter initial dosing intervals (e.g., weekly to every three weeks) followed by longer post dosing intervals (e.g., monthly to every 3-6 months).

Alternatively, the antibody molecule may be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency will vary depending on the half-life of the antibody molecule in the patient. Typically, human antibodies exhibit the longest half-life, followed by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at less frequent intervals over an extended period of time. Some patients continue to receive treatment for the remainder of their lives. In therapeutic applications, it is sometimes desirable to administer higher doses at shorter intervals until progression of the disease is reduced or halted, preferably until the patient exhibits partial or complete improvement in disease symptoms. Thereafter, the administration to the patient may be carried out in a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain amounts of the active ingredients effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without toxicity to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the invention or ester, salt or amide thereof employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in conjunction with the particular composition employed, the age, sex, weight, condition, general health and medical history of the patient being treated, and like factors well known in the medical arts

The compositions of the present invention may be administered by one or more routes of administration using one or more methods well known in the art. It will be appreciated by those skilled in the art that the route and/or manner of administration will vary depending on the desired result. Preferred routes of administration of the FXI-binding proteins of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically injections, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

Alternatively, the FXI binding proteins of the invention may be administered by a non-parenteral route, such as topical, epidermal or mucosal route, e.g., intranasal, oral, vaginal, rectal, sublingual or topical.

Treatment and/or prevention of diseases

In one aspect, the invention also provides a method of treating and/or preventing a thromboembolic disorder or disease in a subject, comprising administering to the subject a therapeutically effective amount of an FXI binding protein of the invention or a pharmaceutical composition of the invention.

In some embodiments, the subject has or is at risk of having: myocardial infarction, ischemic stroke, pulmonary thromboembolism, Venous Thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, thromboembolic disorders associated with medical devices, severe systemic inflammatory response syndrome, thromboembolism formed in extracorporeal circulation (e.g., cardiopulmonary bypass, hemodialysis and ECMO), arterial thrombosis, end stage renal disease, antiphospholipid syndrome, stroke, metastatic cancer or infectious disease.

In some embodiments, the subject has pathological activation of FXI.

In one aspect, the invention further provides the use of an FXI-binding protein of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment and/or prevention of a thromboembolic disorder or disease.

In some embodiments, the thromboembolic disorder or disease is myocardial infarction, ischemic stroke, pulmonary thromboembolism, Venous Thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, medical device-related thromboembolic disorder, severe systemic inflammatory response syndrome, thromboembolism formed in extracorporeal circulation (e.g., cardiopulmonary bypass, hemodialysis, and ECMO), arterial thrombosis, end stage renal disease, antiphospholipid syndrome, stroke, metastatic cancer, or an infectious disease.

In one aspect, the invention provides methods of inhibiting FXI activation (e.g., by factor xiia (fxiia)) in a subject, comprising: (a) selecting a subject in need of treatment, wherein the subject in need of treatment has or is at risk of thrombosis; and (b) administering to the subject an effective amount of an FXI-binding protein of the invention or a pharmaceutical composition of the invention, thereby inhibiting activation of FXI.

In some embodiments, the subject in need of treatment is a subject suffering from or at risk of suffering from: myocardial infarction, ischemic stroke, pulmonary thromboembolism, Venous Thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, thromboembolic disorders associated with medical devices, severe systemic inflammatory response syndrome, thromboembolism formed in extracorporeal circulation (e.g., cardiopulmonary bypass, hemodialysis and ECMO), arterial thrombosis, end stage renal disease, antiphospholipid syndrome, stroke, metastatic cancer or infectious disease.

In some embodiments, the subject in need of treatment is a subject with pathological activation of FXI.

In some embodiments, an effective amount of an FXI binding protein of the invention or a pharmaceutical composition of the invention is an amount sufficient to inhibit activation of FXI by at least 10%, 20%, 30%, 40%, 50%.

In another aspect, the invention provides a method of inhibiting blood coagulation and associated thrombosis in a subject in need thereof without compromising hemostasis, comprising administering to the subject a therapeutically effective amount of an FXI binding protein of the invention or a pharmaceutical composition of the invention, thereby inhibiting blood coagulation and associated thrombosis in the subject without compromising hemostasis.

In some embodiments, the subject is a subject with pathological activation of FXI.

In another aspect, the invention also provides the use of an FXI-binding protein of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for inhibiting blood coagulation and associated thrombosis without compromising hemostasis.

In some embodiments of the various aspects of the invention, the FXI binding protein of the invention or the pharmaceutical composition of the invention is administered to the subject by parenteral administration.

Detection of

In another aspect the invention also provides a method for detecting the presence and/or amount of FXI in a biological sample comprising contacting said biological sample and a control sample with an FXI-binding protein of the invention under conditions such that a complex can form between the FXI-binding protein of the invention and FXI. And detecting the formation of a complex, wherein a difference in complex formation between the biological sample and the control sample is indicative of the presence and/or amount of FXI in the sample.

In some embodiments, FXI binding proteins of the invention are also conjugated with fluorescent dyes, chemicals, polypeptides, enzymes, isotopes, tags or the like that can be used for detection or can be detected by other reagents.

Reagent kit

Also within the scope of the invention are kits for use in the methods of the invention, comprising an FXI binding protein of the invention, and instructions for use. The kit typically includes a label indicating the intended use of the kit contents. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

Examples

The invention will now be further illustrated by way of the following examples, without thereby limiting the invention to the scope of the examples described.

Example 1 screening of Single-Domain antibodies against FXI heavy chain

1.1 construction of the library

Before immunization, 5mL bactrian camel arterial blood was collected in a vacuum blood collection tube, and the supernatant was collected as preimmune serum. Performing primary immunization, namely selecting a healthy 2-year-old Xinjiang bactrian camel, taking 300 mu g of recombinant human factor XI (hFXI, prepared by self, with sequence reference to Uniprot database, accession number P03951) as an antigen, uniformly mixing the antigen with a complete Freund adjuvant in equal volume, completely emulsifying protein, and performing neck muscle multi-point injection on the bactrian camel; later stage immunization, namely uniformly mixing the same amount of antigen and incomplete Freund adjuvant in equal volume each time; the immunization is carried out once a week, and the bactrian camel is immunized six times in the later period. And (3) when the last immunization is finished, collecting 5mL bactrian camel artery blood in a vacuum blood collection tube, and collecting supernatant serving as post-immunization serum.

Lymphocytes were separated by density gradient centrifugation, and total RNA was extracted using an RNA extraction kit provided by QIAGEN corporation. The extracted RNA was all reverse transcribed into cDNA using the Super-Script III FIRST STRANDSUPERMIX kit as per the instructions, and the nucleic acid fragment encoding the variable region of the heavy chain antibody was amplified by nested PCR.

The heavy chain single domain antibody nucleic acid fragment of interest was recovered and cloned into the phage display vector pMECS using restriction enzymes (purchased from NEB) PstI and NotI. The product was then electro-transformed into E.coli electro-competent cells TG1, and immune single domain antibody phage display libraries against recombinant human factor XI were constructed and assayed. The size of the reservoir was calculated to be 1.5X 10 by gradient dilution plating⁸. To examine the insertion rate of the library, 50 clones were randomly selected for sequencing examination, and 50 clones with correct insertion of the foreign fragment were found with a 100% accuracy. The DNA and amino acid sequences cloned by sequencing are respectively analyzed and compared, all the sequences are verified to be camel VHH sequences, and the diversity of the camel VHH sequences can be estimated to be more than 95%.

1.2 heavy chain Single Domain antibody panning against FXI

For the first screen, the protein hApple-chs was used (fromFor preparation, the sequence refers to the Uniprot database, accession number P03951, the amino acid sequence at the front 387 is selected, His-tag is added at the C end of the sequence for purification), 5 ug/well is coated on a plate, and the plate is placed at 4 ℃ overnight. The next day, after 2 hours of blocking with 2% skim milk at room temperature, 100. mu.L of phage (about 10) was added⁸-10⁹pfu, from a 1.1hFXI-Chis single domain antibody display library), at room temperature for 2 hours. After that, the phage were washed 25 times with PBST (0.05% Tween 20 in PBS) to wash away unbound phage. Finally, the phage specifically bound to hApple-chs were dissociated with Glycine (100mM, pH 2.0).

Second screening, using protein mApple-chs (self-prepared, sequence reference Uniprot database, accession number Q91Y47, selecting the pre-389 amino acid sequence, and adding His-tag at the sequence C end for purification) 3 ug/hole coated plate, 4 ℃ standing overnight. The next day, after 2 hours of blocking with 2% skim milk at room temperature, 100. mu.L phage (about 10) was added⁸-10⁹pfu, hFXI-Chis single domain antibody display library from 1.1), at room temperature for 2 hours. After that, the phage were washed 25 times with PBST (0.05% Tween 20 in PBS) to wash away unbound phage. Finally, phage specifically binding to mApple-chs were dissociated with Glycine (100mM, pH 2.0) and infected with E.coli TG1, which was grown in log phase, and phages were generated and purified for the next round of screening. The 2 nd round plates were coated with 10. mu.g/well of the protein hApple-chs and the rest was as above.

Therefore, positive clones are enriched, and the aim of screening FXI specific antibodies in an antibody library by using a phage display technology is fulfilled.

1.3 screening of specific Single Positive clones by enzyme-linked immunosorbent assay (ELISA)

FXI-binding positive phages obtained after the above panning were infected with blank e.coli and plated. Subsequently, 190 single colonies were randomly selected, named iFE 1-iFE 190, and inoculated to 2TY-AG, respectively, and when the OD600 was about 0.8, IPTG was added to a final concentration of about 1mM, and overnight induction expression was performed at 25 ℃, a single domain antibody was expressed in E.coli periplasm, and the next day, the cells were lysed, and the supernatant was used for ELISA detection. Plates were coated with hFXI, hApple and mApple, respectively, overnight at 4 ℃ and the obtained sample lysis supernatant (control group was blank E.coli lysis supernatant) was added and reacted at room temperature for 2 hours. After washing, a secondary antibody, Goat anti-HA tag HRP (from abcam) was added and reacted at room temperature for 2 hours. Adding TMB color developing solution after washing, reading the absorption values at the wavelengths of 450nm and 650nm, and subtracting the absorption value at the wavelength of 650nm from the absorption value at the wavelength of 450nm to obtain the final absorption value. And when the OD value of the sample well is more than 2 times larger than that of the control well, judging the sample well to be a positive clone well. Positive clones were sent to the King for only intelligent sequencing.

Protein sequences of individual clones were analyzed according to the sequence alignment software BioEdit. Clones with > 90% sequence homology of CDR1, CDR2, and CDR3 were considered as identical antibody strains. Finally 23 different antibodies were obtained altogether. The results of the binding assays are shown in table 1 below, and it is known that iFE96, iFE97, iFE148, iFE163, iFE166, iFE168 bind to hFXI, hApple and mApple simultaneously, and iFE29, iFE30, iFE5, iFE7, iFE13, iFE15, iFE35, iFE56, iFE11, iFE17, iFE22, iFE43, iFE49, iFE50, iFE70, iFE108, iFE128 bind to hFXI and hApple and do not bind to mApple.

Binding characteristics of the antibodies of strains 23 of Table 1

1.4 prokaryotic expression and purification of Positive clones

The single colony of positive clone obtained by screening in 1.3 (Escherichia coli TG1 expression system, vector pMECS, HA and HIS label) was cultured in 2TY-AG overnight, the seed solution was transferred to 50mL of 2TY-AG medium, cultured at 37 ℃ to OD600 of about 0.8, IPTG was added to a final concentration of about 1mM, and expression was induced overnight at 25 ℃. And (3) harvesting thalli the next day, resuspending the thalli by using a Tris buffer solution, carrying out ultrasonic bacteria breaking, and purifying the obtained supernatant by using a His label on the single-domain antibody by using a Ni column affinity chromatography to obtain the corresponding target protein.

1.5 affinity detection of prokaryotic expression protein to hApple and mApple

Plates were coated with hApple and mApple proteins, respectively, at 0.5 μ g/well and overnight at 4 ℃. A gradient dilution series of the His-and HA-tagged candidate single domain antibody obtained at 1.4 was added and reacted at room temperature for 2 hours. After washing, a horseradish peroxidase-labeled chicken anti-HA-labeled secondary antibody (streptavidin-HRP, abcam) was added and reacted at room temperature for 2 hours. And adding a color development liquid after washing, reading the absorbance values of the wavelengths of 450nm and 650nm, and subtracting the absorbance value of the wavelength of 650nm from the absorbance value of the wavelength of 450nm to obtain the final absorbance value. Data processing and mapping analysis were performed using the software SotfMax Pro v5.4, and binding curves and EC50 values of candidate single domain antibodies against FXI to hApple and mApple were obtained by four-parameter fitting to reflect the affinity of these candidate antibodies to hApple and mApple.

The results are shown in table 2, and it is known that these candidate single domain antibodies all bind to hApple, wherein iFE43, iFE50, iFE96 have weak affinity with protein hApple; and iFE96, iFE97, iFE148, iFE163, iFE166, iFE168 bind to mApplet simultaneously.

TABLE 2 binding affinities of candidate antibodies to hApple and mApple

Sample	EC₅₀(ng/mL): and hApple	EC₅₀(ng/mL): and mApple
			iFE5	32.9	——
iFE7	18.7	——
			iFE11	43.6	——
iFE13	19.5	——
			iFE15	19.7	——
iFE17	31.4	——
			iFE22	116.0	——
iFE29	23.8	——
			iFE30	24.0	——
iFE35	24.1	——
			iFE43	Very weak binding, highest OD<0.5	——
iFE49	1160	——
			iFE50	Very weak binding, highest OD<0.5	——
iFE56	323.0	——
			iFE70	60.3	——
iFE96	1250 (OD 1.3 at 20 ug/mL)	OD0.8 at 20ug/mL
			iFE97	735.0	1230 (OD 1.4 at 20 ug/mL)
iFE107	43.3	——
			iFE128	54.7	——
iFE148	263	255
			iFE163	16.2	12.9
iFE166	49.8	46.8
			iFE168	20.5	58.4

Example 2 preparation of Fc fusion protein of FXI Single-Domain antibody Using mammalian cells

2.1 preparation of Fc fusion plasmid of FXI Single-Domain antibody

Primers were designed to PCR amplify an FXI single domain antibody VHH fragment (amino acid sequence shown in SEQ ID NO: 1-23; wherein individual amino acids in the FR2 region of the partial sequence were mutated in order to improve protein stability), fused with a DNA fragment encoding human IgG1-Fc (amino acid sequence SEQ ID:336), and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing the FXI single domain antibody-Fc fusion protein in mammals. Wherein different VHH fragments were amplified using universal primers fused to a DNA fragment of human IgG 1-Fc. The universal primers are as follows:

upstream primer cccACCGGTCAGGTGCAGCTGCAGGAGTC

Downstream primer cccGGATCCTGAGGAGACGGTGACCTGG

2.2 preparation of Fc fusion protein of FXI Single-Domain antibody

The 2.1 construction vector was transfected into HEK293 cells for transient expression of the antibody. Diluting the recombinant expression plasmid with Freestyle293 medium and adding PEI (polyethyleneimine) solution required for transformation, adding each group of plasmid/PEI mixture into HEK293 cell suspension respectively, and placing at 37 ℃ and 5% CO₂And (4) suspension culture. And after culturing for 5-6 days, collecting transient expression culture supernatant, and purifying by Protein A affinity chromatography to obtain the target FXI single-domain antibody-Fc fusion Protein. The purity of the protein obtained was checked initially by SDS-PAGE and SEC-HPLC. The expression and purity of each protein was analyzed as shown in Table 3 below.

Table 3, results of one-step purification after transient transformation of the obtained anti-FXI single-domain antibody-FC fusion protein

Antibodies	Amount of expression (mg/L)	SDS-PAGE purity%	SEC purity%
				iFE5-Fc	413	>95％	99.1
iFE7m-Fc	3.8	——	——
				iFE13-Fc	367	>95％	99.4
iFE15m-Fc	16	——	——
				iFE17EREG-Fc	42.9	>95％	——
iFE29m-Fc	25	——	——
				iFE30m-Fc	50	——	——
iFE35-Fc	397	>95％	98.4
				iFE49-Fc	495	>95％	99.3
iFE56-Fc	412	>95％	97.5
				iFE96-Fc	412	>95％	97.9
iFE97-Fc	378	>95％	99
				iFE148-Fc	418	>95％	97.4
iFE163-Fc	292	>95％	93.2
				iFE166-Fc	486	>95％	98.3
iFE168-Fc	368	>95％	98.3

Therefore, the expression levels of the FXI single-domain antibody-Fc fusion Protein iFE7m-Fc, iFE15m-Fc, iFE17EREG-Fc, iFE29m-Fc and iFE30m-Fc are very low, the rest expression levels are all more than 290mg/L, and the target Protein with stable concentration and high purity is obtained after one-step purification by a Protein A affinity chromatography column.

Example 3 identification of Functions of FXI Single Domain antibody-Fc fusion proteins

3.1 binding curves of FXI Single-Domain antibody-Fc fusion protein to mApple and hApple

Plates were coated with proteins mApple and hApple, respectively, 0.5. mu.g/well with a blank set at 4 ℃ overnight. A gradient dilution series of the FXI single-domain antibody-Fc fusion protein obtained in example 2.2 was added and reacted at room temperature for 2 hours. After washing, Goat anti-human IgG-HRP (from SIGMA) was added and the reaction was carried out at room temperature for 2 hours. And adding a color development liquid after washing, reading the absorption values at the wavelengths of 450nm and 650nm, and subtracting the absorption value at the wavelength of 650nm from the absorption value at the wavelength of 450nm to obtain the final absorption value. Data processing and mapping analysis were performed using the software SotfMax Pro v5.4, and the binding curves of the antibody to mApplel and hApplel and the EC50 value were obtained by four-parameter fitting to reflect the affinity of the antibody to mApplel and hApplel.

The results are shown in table 4, and the antibodies all have good binding with protein hApple, wherein the antibodies iFE96, iFE97, iFE148, iFE163, iFE166, iFE168 also have good binding with protein mApple.

TABLE 4 binding of FXI Single-Domain antibody-Fc fusion proteins to mApple and hApple

3.2 detection of affinity of FXI Single-Domain antibody-Fc fusion protein (biofilm interference technique BLI)

The binding kinetics of the FXI single domain antibody-Fc fusion protein obtained in the above examples against the recombinant proteins hApple and mApple were measured by biofilm interference (BLI) technique using a molecular interactor. FXI single-domain antibody-Fc fusion protein iFE5-Fc, iFE13-Fc, iFE35-Fc, iFE56-Fc, iFE97-Fc and iFE5-Fc are diluted to a final concentration of 10 μ g/mL and directly solidified on a ProteinAbiossor, for kinetic measurement, hApple is diluted to 5 concentrations of 200nM, 100nM, 50nM, 25nM and 12.5nM with 0.02% PBST20, mApple is diluted to 5 concentrations of 50nM, 25nM, 12.5nM, 6.25nM and 3.13nM, 150s is injected, the dissociation time is 900s, and 10mM glycine-HCl (pH1.7) is regenerated for 5 s. The association rate (kon) and dissociation rate (kdis) were calculated using a simple one-to-one Languir association model (Octet K2 Data analysis software version 9.0 (Data analysis 9.0)). The equilibrium dissociation constant (kD) is calculated as the ratio kdis/kon.

The results are shown in tables 5 and 6, where table 5 shows that the FXI single domain antibody-Fc fusion protein binds hApple equally well and table 6 shows that the antibodies iFE97-Fc and iFE148-Fc have better affinity for mApple.

The positive control 14E11 was prepared by transient expression from 293 cells according to the method described above after gene synthesis with reference to the sequence in WO 2010080623.

TABLE 5 affinity to hApple

Antibodies	KD(M)	kon(1/Ms)	kdis(1/s)
				Positive control 14E11	2.10E-09	9.55E+04	2.01E-04
iFE5-Fc	1.77E-09	6.41E+04	1.14E-04
				iFE13-Fc	6.59E-09	9.86E+04	6.50E-04
iFE35-Fc	2.02E-08	3.95E+04	7.98E-04
				iFE56-Fc	2.27E-08	3.33E+04	7.55E-04
iFE97-Fc	1.61E-09	1.50E+05	2.43E-04
				iFE148-Fc	9.52E-09	1.09E+05	1.03E-03

TABLE 6 affinity to mApple

Antibodies	KD(M)	kon(1/Ms)	kdis(1/s)
				iFE97-Fc	<1.0E-12	5.17E+05	<1.0E-07
iFE148-Fc	3.14E-11	3.60E+05	1.13E-05

3.3 detection of different epitopes of FXI Single-Domain antibody-Fc fusion protein binding to Apple protein (biofilm interference technique BLI: epitope binding)

The happy-chs-biotin and the mapper-chs-biotin were respectively diluted to 10ug/mL with 0.02% PBST20 using an in-tandem method and cured on a SAbiosensor for 100s and a height of about 1 nm. The FXI single-domain antibody-Fc fusion protein is diluted to 200nM by 0.02% PBST20, and divided into two groups, wherein the antibody binding time is 300s, and the regeneration solution is 10mM glycine-HCl (pH1.7); the first antibody (saturating antibody) binds to the sensor to saturation, after which the second antibody (competing antibody) competes with the first antibody at the same concentration, and the percentage is calculated. The percentage is calculated as Ab2 with Ab1/Ab2 with Ab 1.

The measurement results are shown in tables 7, 8 and 9. From the above results, it is clear from Table 7 that iFE13-Fc, iFE35-Fc and 14E11 compete, the iFE13-Fc epitope overlaps with 14E11, and the iFE35-Fc epitope partially overlaps with 14E 11; iFE56-Fc and iFE35-Fc compete, and the iFE56-Fc epitope is partially hindered from 14E 11; iFE5-Fc and iFE30-Fc compete, the epitopes are the same, and do not overlap with 14E 11; as can be seen from tables 8 and 9, iFE97-Fc and iFE163-Fc compete, have the same epitope, and do not overlap with 14E 11; iFE148-Fc recognizes both human and mouse, and the epitope does not overlap with other single domain antigens and 14E 11.

TABLE 7 hApple

TABLE 8 happle

TABLE 9 mapper

3.4 detection of non-specific binding of FXI Single-Domain antibody-Fc fusion protein to null cells

The CHOK1 empty cells and 293F empty cells were resuspended in 3% BSA-PBS and the cell number was adjusted to 6X 10⁶cell/mL, the final concentration of FXI single-domain antibody-Fc fusion protein is selected to be 100 mu g/mL according to the result of example 3.1, and a negative control and a blank control are arranged at the same time and are subjected to ice bath for 60 min. After washing, Biolegend secondary antibody FITC anti-human IgG FC was added and ice-cooled for 30 min. After washing, the cells were resuspended in 300. mu.L of 1% PBS-BSA Buffer and examined by flow cytometry.

The results are shown in tables 10 and 11, and neither candidate antibody specifically binds to the blank cells.

Watch 10

3.5 identification of FXI blocking Activity of FXI Single-Domain antibody Fc fusion protein (ATPP assay)

The method comprises the steps of quickly mixing and incubating pre-warmed blood plasma and a reagent by using an optical detection method, detecting a light absorption value at a wavelength of 660nm, changing fibrinogen into fibrin along with the extension of incubation time to increase the turbidity of a mixture so as to cause the change of scattered light intensity, and detecting the scattered light intensity changed due to the increase of the turbidity of a sample by using an instrument so as to determine the solidification time. The commercial blood coagulation factor XI was used as a standard, a standard curve was drawn by a coagulation method using a standard curve with the coagulation time as the Y-axis and the activity percentage of reference plasma as the X-axis, and the activity of the sample was calculated from the standard curve.

Antibody FXI plasma is respectively diluted to 1 mu g/mL, meanwhile, Buffer is used as negative control, 50 mu L of processed samples are respectively taken and incubated for 2h at 37 ℃, then the samples are loaded to a full-automatic coagulator, and after the samples are automatically mixed and incubated with Dade Actin Activated cytoloplastin Reagent, Calcium Chloride Solution and FACTOR IX DEICIENT Reagent, the absorbance value of the samples is detected at the position of 660 nm. The results are shown in table 11 below, iFE15, iFE29, iFE96, iFE166, iFE168 without blocking activity.

TABLE 11

Sample name	Clotting time (S)	Activity of
			Non-added antibody	60.9	88.6％
iFE13-Fc	69.9	48.1％
			iFE15m-Fc	61	88.0％
iFE29m-Fc	62.2	80.7％
			iFE30m-Fc	67.2	57.2％
iFE56-Fc	70.4	46.6％
			iFE5-Fc	64.9	66.8％
iFE35-Fc	70	47.7％
			iFE96-Fc	62.5	79.0％
iFE97-Fc	67.9	54.7％
			iFE148-Fc	64.2	70.1％
iFE163-Fc	68.4	52.9％
			iFE166-Fc	61.9	82.5％
iFE168-Fc	61.7	83.7％

According to the results in table 11, antibodies iFE13, iFE35, iFE97, iFE5, iFE148, iFE56 and iFE30 were serially diluted with standard human plasma, activity curves were detected respectively, and at the same time, a positive control 14E11 was set, and as shown in fig. 1, it was found that the inhibitor effects of iFE5, iFE13, iFE35, iFE56 and iFE97 were significantly better than those of the positive control 14E 11; the remaining antibodies iFE148, iFE30 inhibited equally well as the positive control.

3.6 binding of FXI Single Domain antibodies to different Apple domains

The FXI single-domain anti-Fc fusion protein obtained in the above example and the positive control 14E11 were selected and their binding kinetics to different human Apple domain proteins were examined by biofilm interference (BLI) technique. The sequences of the different Apple domain proteins were from Uniprot (accession number P03951), and all Apple domains had His-tag added to their C-terminus for purification (with the chs in the name) and mouse Fc fragments added to their C-terminus for purification (with the muFc in the name). The protein was obtained by transient transfection of 293 cells followed by purification by IMAC. The specific procedures were similar to those described above, and the assays used were self-prepared hApple1-2muFc, hApple2-3muFc, hApple3-4muFc, hApple 2-kis, hApple 4-kis, hApple1-muFc, and hApple3-muFc, respectively.

Binding of candidate FXI single domain antibody Fc fusion proteins to different Apple domains is shown in tables 12 and 13 below.

TABLE 12

	hApple1-2muFc	hApple2-3muFc	hApple3-4muFc	hApple1-muFc	hApple4-chis
						14E11	Bonding of	Bonding of	Is not combined with	Is not combined with	Is not combined with
iFE5-Fc	Is not combined with	Is not combined with	Bonding of	Is not combined with	Bonding of
						iFE13-Fc	Bonding of	Bonding of	Is not combined with	Is not combined with	Is not combined with
iFE35-Fc	Bonding of	Is not combined with	Is not combined with	Is not combined with	Is not combined with
						iFE56-Fc	Bonding of	Bonding of	Is not combined with	Is not combined with	Is not combined with
iFE97-Fc	Is not combined with	Bonding of	Bonding of	Is not combined with	Is not combined with
						iFE148-Fc	Is not combined with	Bonding of	Is not combined with	Is not combined with	Is not combined with

Watch 13

	hApple2-chis(KD，M)	hApple4-chis(KD，M)	hApple3-muFc(KD，M)
				14E11	2.15E-09	Without knottingCombination of Chinese herbs	Is not combined with
iFE5-Fc	Is not combined with	4.29E-10	Is not combined with
				iFE13-Fc	7.71E-10	Is not combined with	Is not combined with
iFE35-Fc	8.20E-07	Is not combined with	Is not combined with
				iFE56-Fc	3.02E-09	Is not combined with	Is not combined with
iFE97-Fc	Is not combined with	Is not combined with	<1.0E-12
				iFE148-Fc	Is not combined with	Is not combined with	Is not combined with

Example 4 humanization of FXI Single Domain antibodies

The humanization method is completed by adopting a protein surface amino acid humanization (resurfacing) method and a VHH (very high human) humanization universal framework grafting method (CDR grafting to a universal framework).

The humanization procedure was as follows: antibody strains iFE5, iFE13, iFE35, iFE56, iFE97 and iFE148 were homologously modeled with modeler 9. The reference homology sequence was NbBcII10 antibody (PDB accession number: 3DWT), and the relative solvent accessibility (relative solvent access) of amino acids was calculated from the three-dimensional structure of the protein. If an amino acid of the antibody strains iFE5, iFE13, iFE35, iFE56, iFE97 and iFE148 is exposed to a solvent, the amino acid is replaced with an amino acid at the same position of the reference human antibody 10HQ sequence, and finally, all the substitutions are completed.

The specific steps of the VHH humanized universal framework transplantation method are as follows: firstly, a universal humanized VHH framework hNbBcII10FGLA (PDB No. 3EAK) designed by Cicle Vincke et al according to sequence homology is obtained, the framework design is based on a nano antibody NbBcII10 antibody (PDB No. 3DWT), protein surface amino acid humanization is carried out by referring to the humanized antibody 10HQ, and the method is completed by modifying partial amino acid VLP of VHH sequence framework 1(frame 1), partial amino acid GL of VHH sequence framework 2(frame 2), partial amino acid RSKRAAV of VHH sequence framework 3(frame 3) and amino acid L of VHH sequence framework 4(frame 4). We used hnnbbcii 10FGLA directly as framework to replace the CDRs with the CDR regions of antibody strains iFE5, iFE13, iFE35, iFE56, iFE97 and iFE148, completing humanization of the antibody.

Antibody strains iFE5, iFE13, iFE35, iFE56, iFE97 and iFE148 were humanized to obtain humanized variants of huFE for the 6 antibody strains, respectively. The sequences of these humanized variants are suitable for SEQ ID NO 300-335, respectively.

Example 5 preparation of Fc fusion protein of humanized Single Domain antibody

5.1 preparation of Fc fusion plasmid of humanized Single Domain antibody

The huFE humanized sequence of example 4 was subjected to gene synthesis by Suzhou Hongyo Biotech Co., Ltd, and enzyme cleavage sites were added to both ends.

The huFE single domain antibody VHH fragment is subjected to double enzyme digestion, is fused with a DNA fragment encoding human IgG1FC, and is cloned to a conventional mammal expression vector, so that a recombinant plasmid for expressing the huFE single domain antibody Fc fusion protein in a mammal is obtained.

5.2 preparation of Fc fusion proteins of humanized Single Domain antibodies

The 4.1 construction vector was transfected into HEK293 cells for transient expression of the antibody. Diluting the recombinant expression plasmid with Freestyle293 medium and adding PEI (polyethyleneimine) solution required for transformation, adding each group of plasmid/PEI mixture into HEK293 cell suspension respectively, and placing at 37 ℃ and 5% CO₂Cultivation at 130 rpm. Four hours later, the cells were supplemented with EXCELL293 medium, 2mM glutamine, and cultured at 130 rpm. After 24 hours 3.8mM VPA was added and after 72 hours 4g/L glucose was added. And after culturing for 5-6 days, collecting transient expression culture supernatant, and purifying by Protein A affinity chromatography to obtain the target huFE single domain antibody Fc fusion Protein. The protein was initially checked for purity by SDSPAGE and SECHPC. The expression quantity of each protein exceeds 350mg/L, and the SEC purity after one-step purification is more than 95%.

Example 6 identification of the function of huFE Single Domain antibody-Fc fusion proteins

6.1 affinity of huFE Single Domain antibody Fc fusion proteins

The binding kinetics of the huFE single domain antibody Fc fusion protein obtained in the above examples against hApple-chs protein were measured by a biofilm interference (BLI) technique using a molecular interactor. The huFE single domain antibody Fc fusion protein obtained in example 5.2 was diluted to a final concentration of 10. mu.g/mL and directly immobilized on AHC biosensor, and for kinetic measurements, the hApple-CHis protein was diluted to 5 concentrations of 200nM, 100nM, 50nM, 25nM, and 12.5nM, respectively, at baseline 60s, association 120s, and dissociation 900 s. The diluent is Kinetic buffer, the regeneration solution is glycine-HCl (pH1.7), and the neutralization solution is diluent. The biosensor is ProteinA. The association rate (kon) and dissociation rate (kdis) were calculated using a simple one-to-one Languir association model (Octet K2 Data analysis software version 9.0 (Data analysis 9.0)). The equilibrium dissociation constant (kD) is calculated as the ratio kdis/kon.

The measured affinity of the huFE single domain antibody Fc fusion protein for the hApple-Chis protein is shown in Table 14.

TABLE 14

The binding kinetics of the huFE single domain antibody Fc fusion protein obtained in the above examples against the hFXI-chs protein were measured by a biofilm interference (BLI) technique using a molecular interactor. The specific operation process is the same as that described above, and the hFXI-Chis protein is used as the detection object. 4E11 (as before) and B1213790-F11a (prepared by itself according to the sequence in WO 2018134184) were used as controls.

The measured affinity of the huFE single domain antibody Fc fusion protein for the hFXI-CHis protein is shown in Table 15.

Watch 15

Sample	KD(M)	Sample	KD(M)
				huFE35huv1-Ld-Fc	1.02E-08	huFE35huv2-Ld-Fc	2.55E-08
huFE13huv1-Ld-Fc	8.84E-09	huFE13huv2-Ld-Fc	1.05E-08
				huFE56huv1-Ld-Fc	3.97E-09	huFE56huv2-Ld-Fc	4.21E-09
huFE97huv1-Ld-Fc	2.67E-09	huFE97huv2-Ld-Fc	2.51E-09
				huFE148huv1-Ld-Fc	2.64E-09	huFE148huv2-Ld-Fc	4.56E-09
huFE5huv1-Ld-Fc	6.56E-10	huFE5huv2-Ld-Fc	6.12E-10
				huFE35huv3-Ld-Fc	9.65E-09	huFE35huv4-Ld-Fc	3.13E-08
huFE13huv3-Ld-Fc	6.84E-09	huFE13huv4-Ld-Fc	8.84E-09
				huFE56huv3-Ld-Fc	5.97E-09	huFE56huv4-Ld-Fc	3.97E-09
huFE97huv3-Ld-Fc	1.98E-09	huFE97huv4-Ld-Fc	2.67E-09
				huFE148huv3-Ld-Fc	3.45E-09	huFE148huv4-Ld-Fc	2.64E-09
huFE5huv3-Ld-Fc	4.56E-10	huFE5huv4-Ld-Fc	6.56E-10
				huFE35huv5-Ld-Fc	1.02E-08	huFE35huv6-Ld-Fc	1.02E-08
huFE13huv5-Ld-Fc	8.84E-09	huFE13huv6-Ld-Fc	8.84E-09
				huFE56huv5-Ld-Fc	3.97E-09	huFE56huv6-Ld-Fc	3.97E-09
huFE97huv5-Ld-Fc	2.67E-09	huFE97huv6-Ld-Fc	2.67E-09
				huFE148huv5-Ld-Fc	2.64E-09	huFE148huv6-Ld-Fc	2.64E-09
huFE5huv5-Ld-Fc	6.56E-10	huFE5huv6-Ld-Fc	6.56E-10
				14E11	3.02E-09	B1213790-F11a	4.12E-09

The results show that the humanized single-domain antibody-Fc fusion protein binds well to hApple-CHis and to hFXI-CHis, and there is no significant difference between the humanized versions. And comparable binding compared to the two control antibodies.

Part of the huFE single-domain anti-Fc fusion protein obtained in the above example was selected, and the binding kinetics of the huFE single-domain anti-Fc fusion protein to FXI protein of New Zealand rabbit was examined by a biofilm interference (BLI) technique. The specific procedures are the same as those described above, and the detection substance is prepared from FXI protein of New Zealand rabbit (reference protein sequence to Uniprot database accession No. Q95ME7)

The measured affinity of the huFE single domain antibody Fc fusion protein for the RabFXI-Apple-CHis shown in Table 16 below.

TABLE 16

Sample	KD(M)	Sample	KD(M)
				huFE35huv1-Ld-Fc	/	huFE35huv2-Ld-Fc	/
huFE13huv1-Ld-Fc	2.95E-08	huFE13huv2-Ld-Fc	3.15E-08
				huFE56huv1-Ld-Fc	/	huFE56huv2-Ld-Fc	/
huFE97huv1-Ld-Fc	8.78E-10	huFE97huv2-Ld-Fc	9.60E-10
				huFE148huv1-Ld-Fc	7.83E-09	huFE148huv2-Ld-Fc	7.21E-09
huFE5huv1-Ld-Fc	2.87E-07	huFE5huv2-Ld-Fc	4.51E-07

Part of the huFE single-domain anti-Fc fusion protein obtained in the above example was selected, and the binding kinetics of the huFE single-domain anti-Fc fusion protein to the FXI protein of cynomolgus monkeys was examined by a biofilm interference (BLI) technique. The specific procedures were the same as those described above, and the assay used a self-prepared cynomolgus monkey FXI protein (reference to the related protein sequence in Uniprot database accession No. A0A2K5VVK2)

The measured affinity of the huFE single domain antibody Fc fusion protein for the cynomolgus monkey FXI-CHis protein is shown in Table 17.

TABLE 17

Sample	KD(M)	Sample	KD(M)
				huFE35huv1-Ld-Fc	<1.0E-12	huFE35huv2-Ld-Fc	1.24E-12
huFE13huv1-Ld-Fc	1.27E-08	huFE13huv2-Ld-Fc	2.06E-08
				huFE56huv1-Ld-Fc	5.93E-09	huFE56huv2-Ld-Fc	7.23E-09
huFE97huv1-Ld-Fc	<1.0E-12	huFE97huv2-Ld-Fc	<1.0E-12
				huFE148huv1-Ld-Fc	9.87E-10	huFE148huv2-Ld-Fc	8.25E-10
huFE5huv1-Ld-Fc	/	huFE5huv2-Ld-Fc	/

The above experimental results show that most candidate antibodies can simultaneously recognize coagulation F11 factors of human, New Zealand rabbit and cynomolgus monkey. But the binding was relatively reduced for new zealand rabbits. And antibodies 35 and 56 were substantially inactive in New Zealand rabbits, while antibody 5 was substantially inactive in cynomolgus monkeys. There was no significant difference in activity between the various humanized versions of the protein.

A part of the huFE single-domain anti-Fc fusion protein obtained in the above example was selected and its binding kinetics to activated human factor 11 protein (hfxa) was examined by a biofilm interference (BLI) technique. The procedure was the same as described above, and commercial hFXIa was used as the analyte. 14E11 (as before) and B1213790-F11a (as before) were used as controls.

The measured affinity of the huFE single domain antibody Fc fusion protein for hfxa protein is shown in table 18.

Watch 18

Sample	KD(M)
		huFE35huv1-Ld-Fc	4.55E-08
huFE13huv1-Ld-Fc	6.93E-09
		huFE56huv1-Ld-Fc	2.88E-08
huFE97huv1-Ld-Fc	1.42E-09
		huFE148huv1-Ld-Fc	5.05E-09
14E11	5.73E-10
		B1213790-F11a	6.85E-11

From the above results, it was found that the humanized FXI single domain antibody Fc fusion protein can efficiently bind to activated FXI factor. The binding force is lower than that of B1213790-F11a which directly binds to the FXI factor activation site.

At the same time, the results of binding of previously inactivated FXI factor were also found to be 14E11, also binding to Apple domain (Apple2), with improved binding to activated FXIa. Whereas the binding of the candidate antibodies of the invention to activated FXIa remains comparable to the binding activity of non-activated FXI or the binding to activated FXIa is reduced. This result demonstrates, on the other hand, that the binding patterns of the candidate antibodies of the invention are significantly different even though there is some crossover with 14E11 at the binding epitope.

6.2 inhibitory Effect of huFE Single-Domain antibody Fc fusion protein on human FXI factor Activity

The activity of human FXI in standard human plasma (purchased from WHO) is 92 percent, and the inhibition effect of the activity of FXI factor in the plasma after adding different single-domain antibody Fc fusion proteins and a reference substance (14E11) is detected by matching with FXI-poor plasma. The results of the activity of FXI factor mixed with different antibodies are shown in table 19 and figure 2. These humanized FXI antibodies all had better results for FXI factor inhibitory activity. 14E11 protein served as a positive control.

Watch 19

Example 7 preparation of Fc fusion protein of huFE bispecific antibody Using mammalian cells

7.1 preparation of Fc fusion plasmid of huFE bispecific antibody

The gene of the huFE single-domain antibody Fc fusion protein obtained in example 4 was subjected to molecular cloning to obtain a recombinant plasmid for expressing the huFE bispecific antibody Fc fusion protein in mammals, for preparing bispecific antibody proteins: huFE97n13-Ld-Fc, huFE13n97-Ld-Fc, huFE97n56-Ld-Fc, huFE56n97-Ld-Fc, huFE97di-Ld-Fc, huFE56n13-Ld-Fc, huFE13n56-Ld-Fc, huFE97n148-Ld-Fc, huFE148n97-Ld-Fc, huFE5n97-Ld-Fc, huFE97n5-Ld-Fc, huFE148n5-Ld-Fc, huFE5n148-Ld-Fc, huFE148n56-Ld-Fc, huFE56n148-Ld-Fc, huFE56n5-Ld-Fc, huFE5n56-Ld-Fc, huFE5n13-Ld-Fc, and huFE13n 5-Ld-Fc.

Meanwhile, the tetravalent monospecific antibody of HuFE97di-Ld-Fc is also used as a control antibody by the method or the control antibody.

7.2 preparation of Fc fusion proteins for huFE bispecific antibodies

The 7.1 construction vector was transfected into HEK293 cells for transient expression of the antibody. Diluting the recombinant expression plasmid with Freestyle293 medium and adding PEI (polyethyleneimine) solution required for transformation, adding each group of plasmid/PEI mixture into HEK293 cell suspension respectively, and placing at 37 ℃ and 5% CO₂And (5) medium suspension culture. And after culturing for 5-6 days, collecting transient expression culture supernatant, and purifying by Protein A affinity chromatography to obtain the target huFE bispecific antibody Fc fusion Protein. The protein was initially checked for purity by SDSPAGE and SECHPC.

The expression level of all proteins is between 250mg/L and 400mg/L, and the SEC purity after one-step purification is above 95%. The results preliminarily prove that the bispecific antibodies have excellent solubility stability and are suitable for being used as drug candidate molecules.

Example 8 high temperature acceleration of huFE bispecific antibody-Fc fusion proteins

The huFE bispecific antibody Fc fusion proteins obtained in the above examples were each 12mg, concentrated to 10mg/ml by ultrafiltration, and dissolved in PBS. Then, the mixture is placed at 40 ℃, and samples are periodically taken to detect the changes of protein content, SEC purity and the like so as to investigate the stability of the mixture. The results are shown in tables 20 and 21 below.

TABLE 20 detection of protein content by 7 proteins OD280nm after acceleration

TABLE 21 SE-HPLC detection

And (4) conclusion: after 20 days at elevated temperature, the purity decreased, but was within acceptable limits.

Example 9 identification of the function of huFE bispecific antibody-Fc fusion protein

9.1 affinity of huFE bispecific antibody Fc fusion protein

The binding kinetics of the huFE bispecific antibody Fc fusion protein obtained in the above examples against the hApple-Chis and hFXI-Chis proteins were measured by biofilm interference (BLI) technique using a molecular interactor. The huFE bispecific antibody Fc fusion protein obtained in example 7.2 was diluted to a final concentration of 10. mu.g/mL and directly immobilized on an AHC biosensor, and for kinetic measurements, the protein hApple-Chis or hFXI-Chis was diluted 5 concentrations, respectively, at a baseline of 60s, for binding 120s, and for dissociation of 900 s. The diluent is Kinetic buffer, the regeneration solution is glycine-HCl (pH1.7), and the neutralization solution is diluent. The biosensor is ProteinA. The association rate (kon) and dissociation rate (kdis) were calculated using a simple one-to-one Languir association model (Octet K2 Data analysis software version 9.0 (Data analysis 9.0)). The equilibrium dissociation constant (kD) is calculated as the ratio kdis/kon.

The measured affinity of the huFE bispecific antibody Fc fusion protein for the hFXI-Chis protein is shown in Table 22 below.

TABLE 22

	KD(M)
		huEF13n97-Ld-Fc	6.70E-10
huEF56n97-Ld-Fc	6.74E-10
		huEF97n13-Ld-Fc	1.31E-09
huEF97n56-Ld-Fc	5.51E-10
		huEF97n148-Ld-Fc	4.48E-10
huEF148n13-Ld-Fc	1.81E-09
		huEF13n148-Ld-Fc	1.93E-09
huEF148n56-Ld-Fc	1.66E-09
		huEF56n148-Ld-Fc	2.31E-09
huEF148n97-Ld-Fc	4.64E-10
		huEF5n13-Ld-Fc	1.25E-09
huEF13n5-Ld-Fc	9.52E-10
		huEF5n97-Ld-Fc	4.50E-10
huEF97n5-Ld-Fc	2.16E-10
		huEF56n5-Ld-Fc	5.77E-10
huEF5n56-Ld-Fc	6.11E-10
		huEF148n5-Ld-Fc	1.71E-09
huEF5n148-Ld-Fc	2.01E-09

The measured affinity of the huFE bispecific antibody Fc fusion protein for the hApple-Chis protein is shown in Table 23 below.

TABLE 23

	KD(M)
		huEF13n97-Ld-Fc	<1.0E-12
huEF56n97-Ld-Fc	<1.0E-12
		huEF97n13-Ld-Fc	<1.0E-12
huEF97n56-Ld-Fc	<1.0E-12
		huEF97n148-Ld-Fc	<1.0E-12
huEF148n13-Ld-Fc	9.04E-11
		huEF13n148-Ld-Fc	<1.0E-12
huEF148n56-Ld-Fc	1.59E-10
		huEF56n148-Ld-Fc	2.31E-10
huEF148n97-Ld-Fc	<1.0E-12
		huEF5n13-Ld-Fc	<1.0E-12
huEF13n5-Ld-Fc	<1.0E-12
		huEF5n97-Ld-Fc	<1.0E-12
huEF97n5-Ld-Fc	<1.0E-12
		huEF56n5-Ld-Fc	2.46E-10
huEF5n56-Ld-Fc	1.11E-10
		huEF148n5-Ld-Fc	4.59E-10
huEF5n148-Ld-Fc	2.01E-10

The above results indicate that all candidate FXI bispecific antibodies show good affinity for human FXI factor protein as well as for the human Apple domain and are higher than the maternal monospecific antibody.

9.2 specificity of the huFE bispecific antibody Fc fusion protein

The FXI bispecific antibody obtained in the above example was selected and its nonspecific binding to other coagulation-related proteins was examined by biofilm interference (BLI) technique. The antibody proteins were immobilized on AHC chips and the proteins examined were commercially available FVII, FIX, FV, FXII, pro-Thrombin, α -kallikrein, FVIIa, FIXa, FVa, FXIIa, Thrombin. The results of the experiments show that all bispecific antibodies do not bind to FVII, FIX, FV, FXII, pro-Thrombin, alpha-kallikrein, FVIIa, FIXa, FVa, FXIIa, Thrombin.

9.3 inhibitory Effect of huFE bispecific antibody Fc fusion protein

9.3.1 inhibitory Effect on human FXI Activity

The activity of standard human FXI (WHO) was 92%, and the activity of FXI in standard human plasma (purchased from sigma) was 87.5%. The effect of inhibiting the activity of FXI factor in plasma after adding different single-domain antibody Fc fusion proteins and a control (14E11, MAA868-F11) is detected by matching with FXI-lacking plasma. The results of the FXI factor activity in combination with different antibodies are shown in FIG. 3. The FXI antibodies have better inhibition activity results on FXI factors and are superior to control positive antibodies. Of these, 14E11 protein (prepared as above) was used as a positive control with MAA868-F11 (prepared according to patent application US 15/739414).

9.3.2 inhibitory Activity on human Whole plasma APTT

Whole blood APTT times were measured after incubation for 3min at 37 degrees with FXI antibody diluted to different concentrations with standard human plasma (purchased from Sigma). 14E11 and B1213790-F11a were positive controls.

Figure 4 shows the variation of APTT time with antibody concentration. The result shows that the candidate FXI antibody protein can effectively prolong the whole blood APTT blood coagulation time; and the bispecific antibody shows better effect of inhibiting blood coagulation.

9.3.3 inhibitory Activity on monkey Whole plasma APTT

Whole blood APTT time was measured after 3min incubation at 37 degrees with FXI antibody diluted to different concentrations in monkey plasma (purchased from Sigma). 14E11 and B1213790-F11a were positive controls.

The results are shown in FIG. 5.

9.3.4 inhibitory Activity on APTT in Rabbit Whole plasma

Whole blood APTT time was measured by diluting the example FXI antibody Fc fusion protein and standards to different concentrations with rabbit plasma (purchased from Sigma). 14E11 and B1213790-F11a were positive controls.

The results are shown in FIG. 6.

The above experimental results indicate that most FXI candidate antibodies, whether bispecific or not, show better APTT inhibitory activity in human or monkey plasma. Some antibodies were not active in rabbits.

The activity of the candidate antibody in human plasma is basically better than that of the control positive antibody. Bispecific antibodies tend to be more active than monospecific antibodies.

9.4 Effect of FXI Single-Domain antibody Fc fusion protein and bispecific antibody on Rabbit venous thrombosis

After the rabbits are fasted at night, blood is collected through the ear vein, anesthesia is carried out by injecting anesthetic into the ear vein, the far end and the near end of the jugular vein are ligated, and the interval between two ligatures is uniformly kept at about 3.0 cm. 15min before molding, 1mg/kg of the test sample (huFE single domain antibody Fc fusion protein obtained in example 5.2) or PBS negative control (see Table 24) was injected into the ear margin intravenously. The artery clamp clamps the proximal end and the distal end of the jugular vein respectively, the injector is used for exhausting the blood in the blood vessel from the facial vein, 0.3ml of agonist with the concentration of 5mg/ml is injected into the closed section of the blood vessel, the agonist is extracted by the injector after being incubated for 5 minutes, the normal saline is used for flushing for 2 times, the artery clamp is loosened, the blood is restored to flow, the diameter of the blood vessel is controlled to be 0.8mm, and the thrombosis is induced. And (3) after the blood returns to circulate for 25 minutes, clamping the vein distal end and proximal end of the closed section by using an artery clamp, tying the vein proximal end and distal end surgical lines tied before the closing section, cutting the closed vein, taking out the thrombus, immediately weighing the wet weight of the thrombus and recording the wet weight of the thrombus. After drying the thrombus in a 60 ℃ oven for 20h, the dried thrombus weight was weighed and the data recorded (see table 25).

The observation was for thrombus weight, the experimental data were expressed as X ± SD, and the significance test was performed using GraphPad Prism 51 way ANOVA (see fig. 7).

And only part of the antibodies show better thrombus inhibition effect under the influence of the cross of different antibodies and rabbit species. Monkeys with better species cross property are selected to be subjected to corresponding in vivo activity detection.

Watch 24

TABLE 25

Sequence information

Amino acid sequences and corresponding CDR sequences of 23 heavy chain single domain antibodies

>iFE5

>iFE7m

>iFE11m

>iFE13

>iFE15m

>iFE17EREG

>iFE22m

>iFE29m

>iFE30m

>iFE35

>iFE43

>iFE49

>iFE50

>iFE56

>iFE70m

>iFE96

>iFE97

>iFE107m

>iFE128m

>iFE148

>iFE163

>iFE166

>iFE168

>huFE5huv1

>huFE5huv2

>huFE5huv3

>huFE5huv4

>huFE5huv5

>huFE5huv6

>huFE13huv1

>huFE13huv2

>huFE13huv3

>huFE13huv4

>huFE13huv5

>huFE13huv6

>huFE35huv1

>huFE35huv2

>huFE35huv3

>huFE35huv4

>huFE35huv5

>huFE35huv6

>huFE56huv1

>huFE56huv2

>huFE56huv3

>huFE56huv4

>huFE56huv5

>huFE56huv6

>huFE97huv1

>huFE97huv2

>huFE97huv3

>huFE97huv4

>huFE97huv5

>huFE97huv6

>huFE148huv1

>huFE148huv2

>huFE148huv3

>huFE148huv4

>huFE148huv5

>huFE148huv4

>IgG1-FC

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:336)

>Apple1

ECVTQLLKDTCFEGGDITTVFTPSAKYCQVVCTYHPRCLLFTFTAESPSEDPTRWFTCVLKDSVTETLPRVNRTAAISGYSFKQCSHQISA(SEQ ID NO:337)

>Apple2

HQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLA(SEQ ID NO:338)

>Apple3

LSNLACIRDIFPNTVFADSNIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIPVF(SEQ ID NO:339)

>Apple4

IPVFCHSSFYHDTDFLGEELDIVAAKSHEACQKLCTNAVRCQFFTYTPAQASCNEGKGKCYLKLSSNGSPTKILHGRGGISGYTLRLCKMDNESTTK(SEQ ID NO:340)

>Apple1-2

ECVTQLLKDTCFEGGDITTVFTPSAKYCQVVCTYHPRCLLFTFTAESPSEDPTRWFTCVLKDSVTETLPRVNRTAAISGYSFKQCSHQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLA(SEQ ID NO:341)

>Apple2-3

HQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLACIRDIFPNTVFADSNIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIPVF(SEQ ID NO:342)

>Apple 3-4

HQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLACIRDIFPNTVFADSNIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIPVF(SEQ ID NO:343)

Claims

1. A coagulation Factor Xi (FXI) binding protein comprising at least one immunoglobulin single variable domain capable of specifically binding FXI comprising CDR1, CDR2 and CDR3 of VHH set forth in any one of SEQ ID NOs 1, 17 and 20.

2. The FXI binding protein of claim 1, wherein the CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs, or IMGT CDRs.

3. The FXI binding protein of claim 1, wherein CDR1, CDR2 and CDR3 in the VHH represented by SEQ ID NO:1 are selected from any one of the group consisting of: 24-26 of SEQ ID NO, 27-29 of SEQ ID NO, 30-32 of SEQ ID NO and 33-35 of SEQ ID NO.

4. The FXI binding protein of claim 3, wherein the at least one immunoglobulin single variable domain comprises an amino acid sequence as set forth in one of SEQ ID NO 1, 300-305.

5. The FXI binding protein of claim 1, wherein CDR1, CDR2 and CDR3 in the VHH represented by SEQ ID NO 17 are selected from any one of the group consisting of: 216-218, 219-221, 222-224 and 225-227.

6. The FXI binding protein of claim 5, wherein the at least one immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO 17, 324-329.

7. The FXI binding protein of claim 1, wherein CDR1, CDR2 and CDR3 in the VHH represented by SEQ ID NO:20 are selected from any one of the group consisting of: 254 for SEQ ID NO:252, 257 for SEQ ID NO:255, 260 for SEQ ID NO:258 and 263 for SEQ ID NO: 261.

8. The FXI binding protein of claim 7, wherein the at least one immunoglobulin single variable domain comprises the amino acid sequence as set forth in one of SEQ ID NOS 20, 330 and 335.

9. The FXI binding protein of any of claims 1-8, which does not bind to the Apple2 domain polypeptide of isolated FXI.

10. The FXI binding protein of any of claims 1-9, further comprising an immunoglobulin Fc-region, preferably a human immunoglobulin Fc-region, such as that of human IgG1, IgG2, IgG3 or IgG 4.

11. The FXI binding protein of claim 10, wherein the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO 336.

12. A nucleic acid molecule encoding the FXI binding protein of any of claims 1-11.

13. An expression vector comprising the nucleic acid molecule of claim 12 operably linked to an expression control element.

14. A recombinant cell comprising the nucleic acid molecule of claim 12 or transformed with the expression vector of claim 13 and capable of expressing said FXI binding protein.

15. A method of producing the FXI binding protein of any of claims 1-11, comprising:

a) culturing the recombinant cell of claim 14 under conditions that allow expression of said FXI-binding protein;

b) recovering the FXI binding protein expressed by the recombinant cells from the culture from step a); and

c) optionally further purifying and/or modifying the FXI-binding protein resulting from step b).

16. A pharmaceutical composition comprising the FXI binding protein of any of claims 1-11 and a pharmaceutically acceptable carrier.

17. A method of treating and/or preventing a thromboembolic disorder or disease in a subject, comprising administering to the subject a therapeutically effective amount of the FXI binding protein of any of claims 1-11 or the pharmaceutical composition of claim 16.

18. The method of claim 17, wherein the subject has or is at risk of having: myocardial infarction, ischemic stroke, pulmonary thromboembolism, Venous Thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, medical device-related thromboembolic disorder, severe systemic inflammatory response syndrome, arterial thrombosis, end stage renal disease, antiphospholipid syndrome, stroke, metastatic cancer, or infectious disease.

19. Use of an FXI binding protein according to any one of claims 1-11 or a pharmaceutical composition according to claim 16 for the manufacture of a medicament for the treatment and/or prevention of a thromboembolic disorder or disease.

20. The use of claim 19, wherein the thromboembolic disorder or disease is myocardial infarction, ischemic stroke, pulmonary thromboembolism, Venous Thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, medical device-related thromboembolic disorder, severe systemic inflammatory response syndrome, thromboembolism formed in extracorporeal circulation (e.g., cardiopulmonary bypass, hemodialysis, and ECMO), arterial thrombosis, end stage renal disease, antiphospholipid syndrome, stroke, metastatic cancer, or infectious disease.

21. A method of inhibiting activation of FXI in a subject, comprising: (a) selecting a subject in need of treatment, wherein the subject in need of treatment has or is at risk of thrombosis; and (b) administering to the subject an effective amount of the FXI binding protein of any of claims 1-11 or the pharmaceutical composition of claim 16, thereby inhibiting activation of FXI.

22. The method of claim 21, wherein the subject in need of treatment is a subject suffering from or at risk of suffering from: myocardial infarction, ischemic stroke, pulmonary thromboembolism, Venous Thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, thromboembolic disorders associated with medical devices, severe systemic inflammatory response syndrome, thromboembolism formed in extracorporeal circulation (e.g., cardiopulmonary bypass, hemodialysis and ECMO), arterial thrombosis, end stage renal disease, antiphospholipid syndrome, stroke, metastatic cancer or infectious disease.

23. A method of inhibiting blood coagulation and associated thrombosis in a subject in need thereof without compromising hemostasis, comprising administering to the subject a therapeutically effective amount of the FXI binding protein of any of claims 1-11 or the pharmaceutical composition of claim 16, thereby inhibiting blood coagulation and associated thrombosis in the subject without compromising hemostasis.

24. The method of claim 23, wherein the subject has or is at risk of having: myocardial infarction, ischemic stroke, pulmonary thromboembolism, Venous Thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, thromboembolic disorders associated with medical devices, severe systemic inflammatory response syndrome, thromboembolism formed in extracorporeal circulation (e.g., cardiopulmonary bypass, hemodialysis and ECMO), arterial thrombosis, end stage renal disease, antiphospholipid syndrome, stroke, metastatic cancer or infectious disease.

25. Use of an FXI binding protein according to any of claims 1-11 or a pharmaceutical composition according to claim 16 for the manufacture of a medicament for inhibiting blood coagulation and associated thrombosis without compromising hemostasis.