TGFβRII extracellular region variants and uses thereof

Abstract

Provided herein are polypeptides comprising variant human TGFβRII extracellular regions, and fusions of the same with antibody molecules. Also provided are polynucleotides, vectors, and host cells encoding these polypeptides and fusion molecules, and methods of making and using these polypeptides and fusion molecules.

Description

CROSS REFERENCE

[0001] This application claims the priority of U.S. Provisional Application No. 63 / 587,852, filed October 4, 2023, which is hereby incorporated by reference in its entirety. RFERENCE TO SEQUENCE LISTING

[0002] The content of the electronically submitted sequence listing in .XML file (Name: 213071_Seqlisting_ST26.XML; Size: 35,046 bytes; and Date Created: October 2, 2024), filed with the application, is incorporated herein by reference in its entirety. FIELD

[0003] extracellular regions, and fusions of the same with antigen-binding molecules. Also provided are polynucleotides, vectors, and host cells encoding these polypeptides and fusion molecules, and methods of making and using these polypeptides and fusion molecules. BACKGROUND

[0004] cell proliferation, differentiation, apoptosis, angiogenesis, fibrosis, and immune activation. crosstalk and a key factor in inducing the transdifferentiation of fibroblasts into cancer- BUSINESS.32102741.1resistance to chemotherapy, targeted therapy, and immunotherapy. Thus, there is a need for

[0005] During the production of biotherapeutics, recombinant proteins are often subject to undesirable product fragmentation, commonly referred to as “clipping.” Clipping of proteins occurs through the enzymatic or chemical disruption of a peptide bond. The peptide bond is generally resistant to nonenzymatic hydrolysis; although, cleavage has been described of specific side chain residues, such as aspartic acid, glycine, serine, threonine, cysteine, or asparagine, especially when solvent-exposed residues are under acidic or basic conditions and at high temperature. However, the clipping observed during biotherapeutic production is mainly due to the action of either intracellular or extracellular host cell proteases, such as metalloproteases, serine proteases, and cathepsin D. This clipping can result in a heterogeneous product, leading to negative impacts on yield, downstream processing requirements, immunogenicity risk, pharmacokinetics, and ultimately a higher cost of goods for the more difficult to produce than classical IgG molecules, with reduced yield compared to a traditional IgG and increased susceptibility to clipping during production.

[0006] exhibit lower levels of protease-mediated clipping when fused to antibodies. SUMMARY

[0007] extracellular regions, and fusions of the same with antigen-binding molecules. Also provided are polynucleotides, vectors, and host cells encoding these polypeptides and fusion molecules, and methods of making and using these polypeptides and fusion molecules. The variant human which, in turn, leads to improved final product yield, simpler downstream processing, a more

[0008] Accordingly, in one aspect, the present disclosure provides a polypeptide (a) lacks an amino acid sequence corresponding to amino acids 1-15 of SEQ ID NO: 1; (b) lacks an amino acid sequence corresponding to amino acids 1-22 of SEQ ID NO: 1; BUSINESS.32102741.1(c) comprises Threonine and Proline at the amino acid positions corresponding to amino acids 5 and 9, respectively, of SEQ ID NO: 1; (d) comprises Isoleucine and Alanine at the amino acid positions corresponding to amino acids 33 and 35, respectively, of SEQ ID NO: 1; (e) comprises Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 33, 35, and 36, respectively, of SEQ ID NO: 1; (f) comprises Threonine, Proline, Isoleucine, and Alanine at the amino acid positions corresponding to amino acids 5, 9, 33, and 35, respectively, of SEQ ID NO: 1; (g) comprises Threonine, Proline, Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 5, 9, 33, 35, and 36, respectively, of SEQ ID NO: 1; (h) comprises Isoleucine and Alanine at the amino acid positions corresponding to amino acids 33 and 35, respectively, of SEQ ID NO:1 and lacks an amino acid sequence corresponding to amino acids 1-15 of SEQ ID NO: 1; (i) comprises Isoleucine and Alanine at the amino acid positions corresponding to amino acids 33 and 35, respectively, of SEQ ID NO:1 and lacks an amino acid sequence corresponding to amino acids 1-22 of SEQ ID NO: 1; (j) comprises Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 33, 35, and 36, respectively, of SEQ ID NO: 1 and lacks the amino acid sequence corresponding to amino acids 1-15 of SEQ ID NO: 1; or (k) comprises Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 33, 35, and 36, respectively, of SEQ ID NO:1 and lacks the amino acid sequence corresponding to amino acids 1-22 of SEQ ID NO: 1.

[0009] 75% identical (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to SEQ ID NO: 1 over the full length of the human

[0010] extracellular region consists of the amino acid sequence set forth in any one of SEQ ID NOs: 2-12.

[0011] In an embodiment, the polypeptide comprises an antigen-binding region. In an embodiment, the antigen-binding region comprises an scFv, a VH, a VL, a VHH, an antibody heavy chain, or an antibody light chain.

[0012] In an embodiment, the polypeptide comprises an antibody constant region. BUSINESS.32102741.1

[0013] the antigen-binding region or antibody constant region via a linker sequence.

[0014] In an embodiment, the polypeptide comprises, from N-terminal to C-terminal: extracellular region.

[0015] In an embodiment, the polypeptide comprises, from N-terminal to C-terminal: an antibody heavy chain, or an Fc fragment thereof; a linker sequence; and the variant human

[0016] In an embodiment, the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 13. In an embodiment, the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0017] In an embodiment, the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-25.

[0018] In one aspect, the present disclosure provides an antibody molecule comprising two heavy chain polypeptides, wherein each heavy chain polypeptide comprises, from N- terminal to C-terminal: a variable region; a constant region; a linker sequence; and a variant of SEQ ID NOs: 2-12. In an embodiment, the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 13. In an embodiment, the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 14. In an embodiment, the antibody molecule comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-25. In an embodiment, the antibody molecule is a full-length heterotetrameric antibody molecule comprising two heavy chain polypeptides and two light chain polypeptides. In an embodiment, the antibody molecule is a full-length heavy chain antibody molecule, comprising two heavy chain polypeptides and no light chain polypeptides. In an embodiment, the constant region is a human heavy chain constant region. In an embodiment, the human heavy chain constant region is of the IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, or IgM isotype.

[0019] In one aspect, the present disclosure provides a polynucleotide encoding a polypeptide or antibody molecule disclosed herein.

[0020] In one aspect, the present disclosure provides a vector comprising a polynucleotide disclosed herein.

[0021] In one aspect, the present disclosure provides an engineered cell comprising a polynucleotide or vector disclosed herein. BUSINESS.32102741.1

[0022] In one aspect, the present disclosure provides a method of producing a polypeptide, the method comprising culturing an engineered cell disclosed herein under conditions such that the polynucleotide is expressed, and the polypeptide is produced.

[0023] In one aspect, the present disclosure provides a method of inhibiting the activity antibody molecule disclosed herein.

[0024] In one aspect, the present disclosure provides a method of inhibiting the activity molecule disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1A-FIG. 1B bispecific variants on SMAD-driven luciferase reporter gene activity, which is activated upon variant concentration (x-axis).

[0026] FIG.2 concentration (x-axis).

[0027] FIG.3 binding events that are consistent with the antibody binding simultaneously to human FAP and DETAILED DESCRIPTION

[0028] This disclosure provides polypeptides comprising improved variant human provided are polynucleotides, vectors, and host cells encoding these polypeptides and fusion molecules, and methods of making and using these polypeptides and fusion molecules. The proteolytic cleavage during protein production compared to the corresponding wild-type (WT) BUSINESS.32102741.1processing, a more homogeneous product, and a lower cost of goods, compared to WT human

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0030] As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of 5% to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., up to 5% to 10% below) the value or range remain within the intended meaning of the recited value or range.

[0031] As used herein, the term “FAP,” refers to fibroblast activation protein alpha. As used herein, the term “human FAP” refers to a FAP protein encoded by a wild-type human FAP gene, e.g., RefSeq accession number NG_027991.1. Exemplary immature wild-type human FAP amino acid sequences are provided as in RefSeq accession numbers NP_004451.2 and NP_001278736.1.

[0032] TGFB1, TGFB2, and TGFB3 by a human TGFB1 gene (e.g., a wild-type human TGFB1 gene). An exemplary wild-type TGFB2 gene (e.g., a wild-type human TGFB2 provided as in RefSeq accession numbers NP_001129071.1 and NP_003229.1. As used herein, TGFB3 gene (e.g., a wild-type human TGFB3 in RefSeq accession numbers NP_003230.1, NP_001316868.1, and NP_001316867.1. BUSINESS.32102741.1

[0033] As used herein, the terms “transforming growth factor beta receptor” and by the TGFBR1, TGFBR2, and TGFBR3 TGFBR1 gene (e.g., a wild-type human TGFBR1 by GenBank accession numbers NP_004603.1, NP_001124388.1, and NP_001293139.1. As encoded by a human TGFB2 gene (e.g., a wild-type human TGFB2 gene). Exemplary wild- TGFB3 gene (e.g., a wild-type human TGFB3 GenBank accession numbers NP_003230.1, NP_001316868.1, and NP_001316867.1.

[0034] to a polypeptide comprising one or more insertions, deletions, or substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein the polypeptide is capable of binding to human 75% identical (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to SEQ ID NO: 1 over the full length of the variant human

[0035] As used herein, the terms “antibody” and “antibodies” include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, without limitation, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’)2fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the above, and conjugates or fusion proteins comprising any of the above. In certain embodiments, BUSINESS.32102741.1antibodies described herein refer to polyclonal antibody populations. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, antibodies described herein are IgG antibodies, or a class (e.g., human IgG1or IgG4) or subclass thereof. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody. In certain embodiments, an antibody, as described herein, comprises a full-length antibody, or an antigen-binding fragment thereof, linked to a ligand-binding moiety that specifically binds to chimeric antigen receptor comprising a VH and / or a VL disclosed herein linked to a transmembrane domain and one or more intracellular domains that are capable of providing signals for T-cell activation.

[0036] “Multispecific antibodies” are antibodies (e.g., bispecific antibodies) that specifically bind to two or more different antigens or two or more different regions of the same antigen. Multispecific antibodies include bispecific antibodies that contain two different antigen-binding sites (exclusive of the Fc region). Multispecific antibodies can include, for example, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, heteroconjugate antibodies, linked single-chain antibodies or linked single-chain Fvs (scFv), camelized antibodies, affybodies, linked Fab fragments, F(ab’)2fragments, chemically-linked Fvs, and disulfide-linked Fvs (sdFv). Multispecific antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, multispecific antibodies described herein are IgG antibodies, or a class (e.g., human IgG1, IgG2, or IgG4) or subclass thereof.

[0037] As used herein, “antigen-binding region” refers to the portion of a polypeptide disclosed herein which comprises amino acid residues that confer specificity for an antigen. Examples of antigen-binding regions include antibody complementarity determining regions (CDR), heavy chain variable regions, light chain variable regions, heavy chains, light chains, and any fragment thereof. The antigen-binding region can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans.

[0038] As used herein, the term “CDR” or “complementarity determining region” means the noncontiguous antigen combining sites found within the variable region of both BUSINESS.32102741.1heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are herein incorporated by reference in their entireties, where the definitions include overlapping or subsets of amino acid residues when compared against each other. In certain embodiments, the term “CDR” is a CDR as defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term “CDR” is a CDR as defined by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In certain embodiments, heavy chain CDRs and light chain CDRs of an antibody are defined using different conventions. In certain embodiments, heavy chain CDRs and / or light chain CDRs are defined by performing structural analysis of an antibody and identifying residues in the variable region(s) predicted to make contact with an epitope region of a target molecule. CDRH1, CDRH2, and CDRH3 denote the heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 denote the light chain CDRs.

[0039] As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs). BUSINESS.32102741.1

[0040] As used herein, the terms “VH” and “VL” refer to antibody heavy and light chain variable regions, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is herein incorporated by reference in its entirety.

[0041] As used herein, the terms “constant region” and “constant domain” are interchangeable and are common in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and / or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with an Fc receptor (e.g., Fc gamma receptor).

[0042] As used herein, the term “heavy chain” when used in reference to an antibody can refer to any distinct type, e.g. based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0043] As used herein, the term “light chain” when used in reference to an antibody can refer to any distinct type, e.g. of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.

[0044] As used herein, the terms “specifically binds,” “specifically recognizes,” “immunospecifically binds,” and “immunospecifically recognizes” are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope or immune complex) as such binding is understood by one skilled in the art. For example, a molecule that specifically binds to an antigen can bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a specific embodiment, molecules that specifically bind to the antigen with a KA that is at least 2 logs (e.g., factors of 10), 2.5 logs, 3 logs, 4 logs, or greater than the KA when the molecules bind non-specifically to another antigen.

[0045] As used herein, the term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can BUSINESS.32102741.1generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0046] As used herein, the term “EU numbering system” refers to the EU numbering convention for the constant regions of an antibody, as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al, Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 5th edition, 1991, each of which is herein incorporated by reference in its entirety.

[0047] As used herein, the term “linked to” refers to covalent or noncovalent binding between two molecules or moieties. The skilled worker will appreciate that when a first molecule or moiety is linked to a second molecule or moiety, the linkage need not be direct, but instead, can be via an intervening molecule or moiety. For example, when a heavy chain variable region of a full-length antibody is linked to a ligand-binding moiety, the ligand- binding moiety can bind a constant region (e.g., a heavy chain constant region) of the full- length antibody (e.g., via a peptide bond), rather than bind directly to the heavy chain variable region.

[0048] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays (e.g., constraining peptides using CLIPS (Chemical Linkage of Peptides onto Scaffolds) to map discontinuous or conformational epitopes), and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, each of which is herein incorporated by reference in its entirety). Antibody:antigen crystals may be studied using well known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.; U.S.2004 / 0014194), and BUSTER (Bricogne G BUSINESS.32102741.1(1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323), each of which is herein incorporated by reference in its entirety. Mutagenesis mapping studies may be accomplished using any method known to one of skill in the art. See, e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085, each of which is herein incorporated by reference in its entirety, for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0049] As used herein, the term “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration of an antibody to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0050] As used herein, the term “effective amount” in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect.

[0051] As used herein, the term “subject” includes any human or non-human animal. In one embodiment, the subject is a human or non-human mammal. In one embodiment, the subject is a human.

[0052] As used herein with respect to a polypeptide, antibody, or polynucleotide, the term “isolated” refers to a polypeptide, antibody, or polynucleotide that is separated from one or more contaminants (e.g., polypeptides, polynucleotides, lipids, or carbohydrates, etc.) which are present in a natural source of the polypeptide, antibody, or polynucleotide. All instances of “isolated polypeptides” described herein are additionally contemplated as polypeptides that may be, but need not be, isolated. All instances of “isolated antibodies” described herein are additionally contemplated as antibodies that may be, but need not be, isolated. All instances of “isolated polynucleotides” described herein are additionally contemplated as polynucleotides that may be, but need not be, isolated. All instances of “polypeptides” described herein are additionally contemplated as antibodies that may be, but need not be, isolated. All instances of “antibodies” described herein are additionally contemplated as antibodies that may be, but need not be, isolated. All instances of “polynucleotides” described herein are additionally contemplated as polynucleotides that may be, but need not be, isolated. BUSINESS.32102741.1

[0053] The determination of “percent identity” between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0054] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0055] BUSINESS.32102741.1region and variants thereof.BUSINESS.32102741.1BUSINESS.32102741.1

[0056] In some embodiments, the disclosure provides a polypeptide comprising a acids 1-15 of SEQ ID NO: 1; lacks an amino acid sequence corresponding to amino acids 1-22 of SEQ ID NO: 1; comprises Threonine and Proline at the amino acid positions corresponding to amino acids 5 and 9, respectively, of SEQ ID NO: 1; comprises Isoleucine and Alanine at the amino acid positions corresponding to amino acids 33 and 35, respectively, of SEQ ID NO: 1; comprises Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 33, 35, and 36, respectively, of SEQ ID NO: 1; comprises Threonine, Proline, Isoleucine, and Alanine at the amino acid positions corresponding to amino acids 5, 9, 33, and 35, respectively, of SEQ ID NO: 1; comprises Threonine, Proline, Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 5, 9, 33, 35, and 36, respectively, of SEQ ID NO: 1; comprises Isoleucine and Alanine at the amino acid positions corresponding to amino acids 33 and 35, respectively, of SEQ ID NO: 1 and lacks an amino acid sequence corresponding to amino acids 1-15 of SEQ ID NO: 1; comprises Isoleucine and Alanine at the amino acid positions corresponding to amino acids 33 and 35, respectively, of SEQ ID NO: 1 and lacks an amino acid sequence corresponding to amino acids 1-22 of SEQ ID NO: 1; comprises Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 33, 35, and 36, respectively, of SEQ ID NO: 1 and lacks the amino acid sequence corresponding to amino acids 1-15 of SEQ ID NO: 1; or comprises Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 33, 35, and 36, respectively, of SEQ ID NO: 1 and lacks the amino acid sequence corresponding to amino acids 1-22 of SEQ ID NO: 1.

[0057] In some embodiments, the disclosure provides a polypeptide comprising a region is at least 75% identical (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to SEQ ID NO: 1 over the full length extracellular region is at least 75% identical (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, BUSINESS.32102741.186, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to SEQ ID NO: 1 over the

[0058] In some embodiments, the disclosure provides a polypeptide comprising a ID NOs: 2-12. In some embodiments, the disclosure provides a polypeptide comprising a ID NOs: 2-12.

[0059] In some embodiments, the disclosure provides a polypeptide comprising a binding region. In some embodiments, the antigen-binding region comprises an scFv, a VH, a VL, a VHH, an antibody heavy chain, or an antibody light chain.

[0060] In some embodiments, the disclosure provides a polypeptide comprising a constant region. Any antibody constant region can be used in the fusion molecules disclosed herein. In certain embodiments, the antibody constant region is a human immunoglobulin (Ig) of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) of immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. In an embodiment, the antibody constant region is an IgG constant region (e.g., a human IgG constant region). In an embodiment, the antibody constant region is an IgG1 constant region (e.g., a human IgG1 constant region). In an embodiment, the antibody constant region is a chimeric constant region comprising portions of several different constant regions. Suitable examples of chimeric constant regions are set forth in US 2011 / 0243966A1, which is incorporated herein by reference in its entirety. A variety of constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits.

[0061] directly linked to the antigen-binding region or antibody constant region. In some region or antibody constant region via a linker sequence. In some embodiments, the linker is a peptide linker. Examples of peptide linkers are well-known and those of skill in the art could BUSINESS.32102741.1to an antigen-binding region or antibody constant region. Exemplary linker sequences are described herein.

[0062] Linker sequence may be of any length. In some embodiments, the length and amino acid composition of the linker sequence can be optimized to vary the orientation and / or proximity of the moieties to one another to achieve a desired activity. In some embodiments, the linker sequence is between about 1 and about 100 amino acids in length, between about 8 and about 40 amino acids in length, or between about 15 amino acids and about 25 amino acids in length. In some embodiments, the linker sequence is between 1 and 100 amino acids in length, between 8 and 40 amino acids in length, or between 15 and 25 amino acids in length. In some embodiments, the linker sequence is about 8 amino acids in length, about 9 amino acids in length, about 10 amino acids in length, about 11 amino acids in length, about 12 amino acids in length, about 13 amino acids in length, about 14 amino acids in length, about 15 amino acids in length, about 16 amino acids in length, about 17 amino acids in length, about 18 amino acids in length, about 19 amino acids in length, about 20 amino acids in length, about 21 amino acids in length, about 22 amino acids in length, about 23 amino acids in length, about 24 amino acids in length, about 25 amino acids in length, about 26 amino acids in length, about 27 amino acids in length, about 28 amino acids in length, about 29 amino acids in length, about 30 amino acids in length, about 31 amino acids in length, about 32 amino acids in length, about 33 amino acids in length, about 34 amino acids in length, about 35 amino acids in length, about 36 amino acids in length, about 37 amino acids in length, about 38 amino acids in length, about 39 amino acids in length, or about 40 amino acids in length. In some embodiments, the linker sequence is 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, 20 amino acids in length, 21 amino acids in length, 22 amino acids in length, 23 amino acids in length, 24 amino acids in length, 25 amino acids in length, 26 amino acids in length, 27 amino acids in length, 28 amino acids in length, 29 amino acids in length, 30 amino acids in length, 31 amino acids in length, 32 amino acids in length, 33 amino acids in length, 34 amino acids in length, 35 amino acids in length, 36 amino acids in length, 37 amino acids in length, 38 amino acids in length, 39 amino acids in length, or 40 amino acids in length.

[0063] In some embodiments, the linker sequence contains only glycine and / or serine residues (e.g., glycine-serine linker or GS linker). In some embodiments, the linker sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and BUSINESS.32102741.114, or a variant thereof comprising 1-5 amino acid changes. In some embodiments, the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the linker sequence consists of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the linker sequence consists of the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the linker sequence is a GS linker of about 21 amino acids in length. In some embodiments, the linker sequence is a GS linker of 21 amino acids in length.

[0064] In some embodiments, the disclosure provides a polypeptide comprising a terminal to C-terminal: an antibody heavy chain, or an Fc fragment thereof; and the variant N-terminal to C-terminal: an antibody heavy chain, or an Fc fragment thereof; and the variant N-terminal to C-terminal: an antibody heavy chain, or an Fc fragment thereof; a linker polypeptide consists of, from N-terminal to C-terminal: an antibody heavy chain, or an Fc

[0065] In some embodiments, the disclosure provides a polypeptide comprising a acid sequence set forth in any one of SEQ ID NOs: 15-25. In some embodiments, the polypeptide consists of the amino acid sequence set forth in any one of SEQ ID NOs: 15-25.

[0066] In an aspect, the disclosure provides an antibody molecule comprising a variant molecule comprises two heavy chain polypeptides, wherein each heavy chain polypeptide comprises, from N-terminal to C-terminal: a variable region; a constant region; a linker sequence set forth in any one of SEQ ID NOs: 2-12. In some embodiments, the antibody molecule comprises two heavy chain polypeptides, wherein each heavy chain polypeptide comprises, from N-terminal to C-terminal: a variable region; a constant region; a linker sequence set forth in any one of SEQ ID NOs: 2-12. In some embodiments, the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the linker sequence consists of the amino acid sequence set forth in SEQ ID NO: 13. In some BUSINESS.32102741.1embodiments, the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the linker sequence consists of the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the antibody molecule comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-25.

[0067] In some embodiments, the antibody molecule is a full-length heterotetrameric antibody molecule comprising two heavy chain polypeptides and two light chain polypeptides. In some embodiments, the antibody molecule is a full-length heavy chain antibody molecule, comprising two heavy chain polypeptides and no light chain polypeptides. In some region consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2-12. In some embodiments, each heavy chain polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-25.

[0068] In some embodiments, the disclosure provides an antibody molecule comprising the antibody molecule is a human heavy chain constant region. In some embodiments, the antibody molecule comprises two heavy chain polypeptides, wherein each heavy chain polypeptide comprises a constant region. In some embodiments, each constant region of the antibody molecule is a human heavy chain constant region. In some embodiments, the human heavy chain constant region is of the IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, or IgM isotype. In some embodiments, each constant region of the antibody molecule is a human heavy chain constant region of the IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, or IgM isotype. Polynucleotides, Vectors, and Methods of Production

[0069] The disclosure also provides polynucleotides that encode any of the polypeptides, antibody molecules, or portions thereof described herein. In some embodiments, region of the disclosure. In some embodiments, the polynucleotide encodes an antibody molecule of the disclosure. In some embodiments, the polynucleotide encodes a polypeptide antibody constant region, and optionally a linker. In some embodiments, the polynucleotide antibody heavy chain, or an Fc fragment thereof, and optionally a linker. In some embodiments, the polynucleotides described herein are DNA molecules. In some embodiments, the polynucleotides described herein are RNA molecules. BUSINESS.32102741.1

[0070] In some embodiments, the polynucleotide comprises a nucleotide sequence that region at least 75% identical (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to SEQ ID NO: 1 over the full length of the comprises a nucleotide sequence that encodes a polypeptide comprising or consisting of a any one of SEQ ID NOs: 2-12. In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes a polypeptide comprising or consisting of a variant human SEQ ID NOs: 2-12.

[0071] In some embodiments, the polynucleotide comprises a nucleotide sequence that region and a linker sequence. In some embodiments, the polynucleotide comprises a nucleotide extracellular region at least 75% identical (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to SEQ ID NO: 1 over the the amino acid sequence set forth in SEQ ID NO: 13 or 14. In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes a polypeptide comprising or sequence set forth in any one of SEQ ID NOs: 2-12 and a linker sequence comprising the amino acid sequence set forth in SEQ ID NO: 13 or 14. In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes a polypeptide comprising or consisting of a any one of SEQ ID NOs: 2-12 and a linker sequence comprising the amino acid sequence set forth in SEQ ID NO: 13 or 14. In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes a polypeptide comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 15-25.

[0072] In some embodiments, the polynucleotide comprises a first nucleotide sequence region and a second nucleotide sequence that encodes a polypeptide comprising or consisting of an antigen-binding region. In some embodiments, the polynucleotide comprises a first nucleotide sequence that encodes a polypeptide comprising or consisting of a variant human BUSINESS.32102741.1comprising or consisting of an antibody constant region. In some embodiments, the first nucleotide sequence encodes a polypeptide comprising or consisting of a variant human e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to the amino acid sequence of any one of SEQ ID NOs: 2-12. In some region comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 2-12.

[0073] In some embodiments, the first or second nucleotide sequence also encodes a extracellular region and a linker and the second nucleotide sequence encodes an antigen- binding region. In some embodiments, the first nucleotide sequence encodes a variant human region and a linker. In some embodiments, the first nucleotide sequence encodes a variant an antibody constant region. In some embodiments, the first nucleotide sequence encodes a antibody constant region and a linker. The linker can be any linker described herein, including a peptide linker, or a linker comprising the amino acid sequence set forth in SEQ ID NO: 13 or 14. In some embodiments, the linker consists of the amino acid sequence set forth in SEQ ID NO: 13 or 14.

[0074] In some embodiments, the first nucleotide sequence encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 15-25, and the second nucleotide sequence encodes an antigen-binding region. In some embodiments, the first ID NOs: 2-12, and the second nucleotide sequence encodes an antigen-binding region and a linker. In some embodiments, the first nucleotide sequence encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 15-25, and the second nucleotide sequence encodes an antibody constant region. In some embodiments, the first nucleotide 2-12, and the second nucleotide sequence encodes an antibody constant region and a linker.

[0075] Also provided herein are polynucleotides encoding a polypeptide or an antibody molecule as provided above that are optimized, e.g., by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Methods BUSINESS.32102741.1to generate optimized nucleic acids for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly, all of which are herein incorporated by reference in their entireties. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequences to increase stability of the RNA for recombinant expression. The alterations utilize the degeneracy of the genetic code, e.g., using an alternative codon for an identical amino acid. In an embodiment, it can be desirable to alter one or more codons to encode a conservative mutation, e.g., a similar amino acid with similar chemical structure and properties and / or function as the original amino acid.

[0076] The polynucleotides can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Nucleotide sequences encoding polypeptides, antibody molecules, or portions thereof described herein, and modified versions of these polypeptides, can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the polypeptide, antibody molecule, or portion thereof. Such a polynucleotide encoding the protein can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6, herein incorporated by reference in its entirety), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the polypeptide, antibody molecule, or portion thereof, annealing, and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0077] Alternatively, a polynucleotide encoding a polypeptide, antibody molecule, or portion thereof described herein can be generated from nucleic acid from a suitable source using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the polypeptide or antibody molecule of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the polypeptide, antibody molecule, or portion thereof. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning.

[0078] If a clone containing a nucleic acid encoding a particular polypeptide is not available, but the sequence of the polypeptide is known, a nucleic acid encoding the BUSINESS.32102741.1polypeptide can be chemically synthesized or obtained from a suitable source (e.g., a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from any tissue or cells expressing the polypeptide described herein) by PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the polypeptide. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0079] DNA encoding polypeptides, antibody molecules, or portions thereof described herein can be readily isolated and sequenced using conventional procedures. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System (Lonza)), or myeloma cells that do not otherwise produce the molecules described herein.

[0080] Also provided are polynucleotides that hybridize under high stringency, intermediate, or lower stringency hybridization conditions to polynucleotides that encode a polypeptide, antibody molecule, or portion thereof described herein.

[0081] Hybridization conditions have been described in the art and are known to one of skill in the art. For example, hybridization under stringent conditions can involve hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50-65° C; hybridization under highly stringent conditions can involve hybridization to filter-bound nucleic acid in 6xSSC at about 45°C followed by one or more washes in 0.1xSSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those of skill in the art and has been described, see, e.g., Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3, which is herein incorporated by reference in its entirety.

[0082] In an aspect, provided herein are cells (e.g., host cells) expressing (e.g., recombinantly) a polypeptide or portion thereof described herein, and related polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding a polypeptide or portion thereof described herein for recombinant expression in host cells, preferably in mammalian cells (e.g., CHO cells). Also provided herein are host cells comprising such vectors for recombinantly expressing polypeptides or portions thereof described herein. In an aspect, provided herein are BUSINESS.32102741.1methods for producing a polypeptide, antibody molecule, or portion thereof described herein, comprising expressing the polypeptide, antibody molecule, or portion thereof from a host cell.

[0083] Recombinant expression of a protein described herein generally involves construction of an expression vector containing a polynucleotide that encodes the polypeptide or antibody molecule. Once a polynucleotide encoding a polypeptide or antibody molecule described herein has been obtained, the vector for the production of the polypeptide or antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a polypeptide, antibody molecule, or portion thereof by expressing a polynucleotide containing a polypeptide- or antibody molecule-encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing polypeptide coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding containing a polypeptide, antibody molecule, or portion thereof described herein, operably linked to a promoter. Such vectors can, for example, include the nucleotide sequence encoding an Fc region of the polypeptide (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No.5,122,464, which are herein incorporated by reference in their entireties).

[0084] An expression vector can be transferred to a cell (e.g., host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce a polypeptide, antibody molecule, or portion thereof described herein. Thus, provided herein are host cells containing a polynucleotide encoding containing a polypeptide or antibody molecule described herein or portions thereof.

[0085] In some embodiments, the vector is a non-viral vector. Exemplary non-viral vectors include, but are not limited to, plasmid DNA, transposons, episomal plasmids, minicircles, ministrings, and oligonucleotides (e.g., mRNA, naked DNA). In some embodiments, the vector is a DNA plasmid vector.

[0086] In some embodiments, the vector is a viral vector. Viral vectors can be replication competent or replication incompetent. Viral vectors can be integrating or non- integrating. A number of viral based systems have been developed for gene transfer into mammalian cells, and a suitable viral vector can be selected by a person of ordinary skill in the art. Exemplary viral vectors include, but are not limited to, adenovirus vectors (e.g., adenovirus 5), adeno-associated virus (AAV) vectors (e.g., AAV2, 3, 5, 6, 8, 9), retrovirus vectors BUSINESS.32102741.1(MMSV, MSCV), lentivirus vectors (e.g., HIV-1, HIV-2), gammaretrovirus vectors, herpes virus vectors (e.g., HSV1, HSV2), alphavirus vectors (e.g., SFV, SIN, VEE, M1), flavivirus (e.g., Kunjin, West Nile, Dengue virus), rhabdovirus vectors (e.g., rabies virus, VSV), measles virus vector (e.g., MV-Edm), Newcastle disease virus vectors, poxvirus vectors (e.g., VV), measles virus, and picornavirus vectors (e.g., Coxsackievirus).

[0087] In some embodiments, the vector or expression cassette comprises one or more additional elements. Additional elements include, but are not limited to, promoters, enhancers, polyadenylation (polyA) sequences, and selection genes.

[0088] In some embodiments, the vector comprises a polynucleotide sequence that encodes an amino acid sequence at least 75% identical (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to an amino acid sequence recited in Table 1. In some embodiments, the vector comprises a polynucleotide sequence that encodes an amino acid sequence recited in Table 1. Methods of Use and Uses

[0089] In another aspect, the disclosure provides a method of treating a subject using the polypeptides or antibody molecules disclosed herein. Any disease or disorder in a subject can be treated using the polypeptides or antibody molecules disclosed herein.

[0090] In another aspect, the disclosure provides a method of inhibiting the activity of disclosed herein. In another aspect, the disclosure provides a method of inhibiting the activity molecule disclosed herein.

[0091] The polypeptides or antibody molecules provided herein can be used for treating e.g., normal tissue of the same cell type. The polypeptides or antibody molecules provided herein can be treated by the polypeptides or antibody molecules provided herein include, for example, lung cancer, colon cancer, gastric cancer, breast cancer, head and neck cancer, skin cancer, liver cancer, kidney cancer, prostate cancer, pancreatic cancer, brain cancer, cancer of the skeletal muscle. BUSINESS.32102741.1

[0092] The polypeptides or antibody molecules disclosed herein can be used to inhibit tumor growth or kill tumor cells. For example, the polypeptides or antibody molecules can bind of the tumor stroma) and elicit, e.g., ADCC or other effector-mediated killing of the cancerous cells.

[0093] The polypeptides or antibody molecules can alternatively be used in order to another compound. In one aspect, a polypeptide or antibody molecule for use as a medicament is provided. In further aspects, a polypeptide or antibody molecule for use in treating a disease polypeptide or antibody molecule for use in a method of treatment is provided. In certain embodiments, the invention provides a polypeptide or antibody molecule for use in a method administering to the subject an effective amount of the polypeptide or antibody molecule. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is an anti-cancer agent, e.g., a chemotherapeutic agent, an inhibitor of tumor cell proliferation, or an activator of tumor cell apoptosis.

[0094] In a further aspect, the use of a polypeptide or antibody molecule disclosed herein in the manufacture or preparation of a medicament is provided. In one embodiment, the further embodiment, the medicament is for use in a method of treating a disease characterized such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is an anti-cancer agent, e.g., a chemotherapeutic agent, an inhibitor of tumor cell proliferation, or an activator of tumor cell apoptosis.

[0095] For the prevention or treatment of disease, the appropriate dosage of a polypeptide or antibody molecule of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody molecule, the severity and course of the disease, whether the polypeptide or antibody molecule is administered for preventive or therapeutic purposes, previous therapy, 27 BUSINESS.32102741.1the patient’s clinical history and response to the polypeptide or antibody molecule, and the discretion of the attending physician.

[0096] In one embodiment, the present invention relates to a polypeptide or antibody molecule of the present invention for use in a method of the present invention, wherein the method further comprises administering an additional therapeutic agent to the subject. In one embodiment, the present invention relates to (a) a polypeptide or antibody molecule of the present invention and (b) an additional therapeutic agent for use as a medicament. In one embodiment, the present invention relates to (a) a polypeptide or antibody molecule of the present invention and (b) an additional therapeutic agent for use in a method for the treatment of cancer. In a further embodiment, the present invention relates to a pharmaceutical composition, kit or kit-of-parts comprising (a) a polypeptide or antibody molecule of the present invention and (b) an additional therapeutic agent. In one embodiment, the additional therapeutic agent is a chemotherapeutic, a radiotherapeutic, or a checkpoint targeting agent.

[0097] A polypeptide or antibody molecule described herein may be delivered to a subject by a variety of routes. These include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, intra-arterial, and subcutaneous routes. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray. In certain embodiments, the polypeptide or antibody molecule described herein is delivered subcutaneously or intravenously. In certain embodiments, the polypeptide or antibody molecule described herein is delivered intra- arterially. In certain embodiments, the polypeptide or antibody molecule described herein is delivered intratumorally. In certain embodiments, the polypeptide or antibody molecule described herein is delivered into a tumor draining lymph node.

[0098] The amount of a polypeptide or antibody molecule which will be effective in the treatment and / or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0099] The precise dose to be employed will also depend on the route of administration, and the seriousness of the infection or disease caused by it, and should be decided according to the judgment of the practitioner and each subject’s circumstances. For example, effective doses may also vary depending upon means of administration, target site, physiological state of the patient (including age, body weight and health), whether the patient is human or an animal, other medications administered, or whether treatment is prophylactic or therapeutic. Usually, BUSINESS.32102741.1the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0100] A polypeptide or antibody molecule described herein can also be used to assay biological sample using classical immunohistological methods known to those of skill in the art, including immunoassays, such as the enzyme linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I,121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Such labels can be used to label a polypeptide or antibody molecule described herein. Alternatively, a second antibody that recognizes a polypeptide or antibody molecule described herein can be labeled and used in combination one embodiment, the present invention relates to the use of a polypeptide or antibody molecule of the present invention for in vitro In a further embodiment, the present invention relates to the use of a polypeptide or antibody biological sample in vitro, optionally wherein the polypeptide or antibody molecule is conjugated to a radionuclide or detectable label, and / or carries a label described herein, and / or wherein an immunohistological method is used.

[0101] intended to a first biological sample either directly (e.g., by determining or estimating absolute protein level) or relatively (e.g., by comparing to the disease associated protein level in a second be standard being taken, for example, from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having is known, it can be used repeatedly as a standard for comparison. Therefore, in a further embodiment, the present invention relates to an in vitro method for assaying and / or detecting levels in a biological sample, comprising qualitatively or quantitatively measuring or estimating the level of in a biological sample, by an immunohistological method. BUSINESS.32102741.1

[0102] As used herein, the term “biological sample” refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells potentially expressing human . Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans orcynomolgus monkeys) are well known in theart. Biological samples include peripheralmononuclear blood cells.

[0103] In one embodiment, a polypeptide or antibody molecule can be used in immunohistochemistry of biopsy samples. In one embodiment, the method is an in vitro method. In another embodiment, a polypeptide or antibody molecule can be used to detect surface, the levels of which can then be linked to certain disease symptoms. Polypeptide or antibody molecules described herein may carry a detectable or functional label and / or may be conjugated to a radionuclide or detectable label. When fluorescence labels are used, currently available microscopy and fluorescence-activated cell sorter analysis (FACS), or a combination of both methods known in the art, may be utilized to identify and to quantitate the specific binding members. Polypeptides or antibody molecules described herein may carry or may be conjugated to a fluorescence label. Exemplary fluorescence labels include, for example, reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes and DyLight dyes. An anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody may carry or may be conjugated to a radioactive label or radionuclide, such as the isotopes3H,14C,32P,35S,36Cl,51Cr,57Co,58Co,59Fe,67Cu,90Y,99Tc,111In,117Lu,121I,124I,125I,131I,198Au,211At,213Bi,225Ac, and186Re. When radioactive labels are used, currently available counting procedures known in the art may be utilized to identify and quantitate the specific is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques as known in the art. This can be achieved by contacting a sample or a control sample with a polypeptide or antibody molecule under conditions that allow for the formation of a complex the sample and the control. Also included herein is an assay system which may be prepared in the form of a test kit, kit, or kit-of-parts for the quantitative analysis of the extent of the presence kit, kit, or kit-of-parts may comprise a labeled component, e.g., a labeled antibody, and one or more additional immunochemical reagents. BUSINESS.32102741.1Kits

[0104] Any of the compositions described herein may be comprised in a kit. In a non- limiting example, the kit comprises one or more of a polypeptide or antibody molecule described herein, a nucleic acid molecule (e.g., an expression vector) encoding the polypeptide or antibody molecule, or a host cell expressing the polypeptide or antibody molecule.

[0105] The kit may further include reagents or instructions for using a polypeptide or antibody molecule described herein, a nucleic acid molecule (e.g., an expression vector) encoding the polypeptide or antibody molecule, or a host cell expressing the polypeptide or antibody molecule, in a subject. It may also include one or more buffers.

[0106] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third, or other additional container into which the additional components may be separately placed. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and / or other diluent. However, various combinations of components may be comprised in a vial. The kits of the present disclosure also typically include a means for containing a polypeptide or antibody molecule described herein, a nucleic acid molecule (e.g., an expression vector) encoding the polypeptide or antibody molecule, or a host cell expressing the polypeptide or antibody molecule, and any other reagent containers in close confinement for commercial sale.

[0107] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may be provided as dried powder(s). When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. EXAMPLES

[0108] The examples of the present disclosure are offered by way of illustration and explanation, and are not intended to limit the scope of the present disclosure. BUSINESS.32102741.1

[0109] This example describes the characterization of the clipping (i.e., proteolytic FAP antibodies were used, designated as AF1 and AF2.

[0110] of the human IgG1 (iso3) heavy chains with the terminal lysine removed, via a 4x(G4S)G linker combination of sequence deletion and point mutations with the aim of reducing clipping in the identified using internal mass spectrometry data of a related molecule and in silico predictions. Two truncations were designed based on removing the cleavage sites in the unstructured mutations (V56I, F58A, and S59A) were designed to reduce the recognition sequences in the the variants tested are shown in Table 1.

[0111] variants as a stable cell line. Each sequence was optimized for CHO expression, then cloned into a vector, linearized, and transfected into the cell line. Selection pressure was used to generate a pool of CHO cells stably expressing the variant. Once generated, the cell line was banked. A vial per variant was thawed and put into a 10 ml fed batch under standard culture conditions. After 10 days, the culture was harvested, cells removed by centrifugation and filtration, and the supernatant passed for purification. The supernatant was purified by drip column Protein A affinity chromatography and the concentration was determined by using the absorbance at 280 nm and the extinction coefficient.

[0112] A PerkinElmer LabChip GX II Touch HT and a Protein Express Assay Kit were used to unfold the antibodies, breaking all non-covalent interactions and any disulphide bonds, then to separate the molecules based on size using a voltage applied across a sieving matrix. BUSINESS.32102741.1

[0113] Each antibody was diluted to 0.1 mg / ml in Protein Express Sample Buffer with 40 mM DTT, then heated at 80°C for 5 minutes. Each sample was sipped by the LabChip into the microfluidics of the instrument, mixed with Protein Express Dye solution, then a few pL pulled into the separating channel by an applied voltage. The voltage separates the molecules based on size as they are pulled through the sieving matrix. Once separated, the sample is destained using the Destaining Solution and the separated molecules are detected by the fluorescence of the dye as they pass a laser. The size is determined using a ladder supplied with the Protein Express Assay Kit. Data quality was verified using parameters determined by the LabChip for successful assay run, and by visual inspection. The light chain peak at 28 kDa was identified, as was the full-length heavy chain which runs as two broad peaks at 90 – 120 kDa due to N-linked glycosylation. Peaks between 60 kDa and 90 kDa were identified as full-length

[0114] clipped differed between the variants. The area under the curve for the heavy chain species was calculated as a proportion of the total heavy chain.BUSINESS.32102741.1

[0115] The above stable cell lines were thawed, expanded, and used to inoculate a cell culture in a 7 L bioreactor under standard culture conditions. After 14 days, the culture was harvested, cells and large debris removed by centrifugation and filtration, and the supernatant passed for purification. The Protein A affinity drip columns were loaded at a product density of 25 mg / ml resin (thus the total volume of each variant used for purification is dependent on the specific titers), and concentration determined by using the absorbance at 280 nm and the extinction coefficient. Briefly, a Beckman Coulter PA800 plus and a capillary cartridge antibodies, breaking all non-covalent interactions and any disulphide bonds, then to separate the molecules based on size using a voltage applied across a sieving gel matrix.

[0116] Each bispecific antibody was diluted to 1 mg / ml in sodium dodecyl sulfate (SDS) sample buffer with 2-mercaptoethanol and a 10 kDa internal standard, then heated at 60°C for 10 minutes. Each sample was injected into the capillary that had been filled with a hydrophilic polymer gel. Applied voltage separated the molecules based on size as they were pulled through the gel sieving matrix along the length of the capillary. Once separated, the sample passed by a detector window in the capillary, and separated molecules were detected by ultraviolet (UV) absorbance. Data quality was verified using pre-defined system suitability requirements. The light chain peak at 28 kDa was identified, as was the full-length heavy chain which runs as broad peaks at 90 – 120 kDa due to N-linked glycosylation. Peaks between 60

[0117] calculated as a proportion of the total heavy chain.BUSINESS.32102741.15.64 7 L Example

[0118] SMAD reporter cells (BPS Bioscience / 60653). These cells stably expressed a firefly luciferase reporter gene under the control of SMAD-responsive elements.

[0119] HEK293 SMAD luciferase cells were expanded with Minimal Essential Medium (MEM) with Earle’s Balanced Salt Solution (EBSS) and L-Glutamine, supplemented with 10% heat-inactivated FBS, Sodium Pyruvate (1X), 400 µg / mL Geneticin, and 1% penicillin-streptomycin. At the log-growth phase, the cells were trypsinized, counted, and seeded into a white 96-well flat-bottom assay plate to have a final concentration of 4x105cells / mL, and were incubated overnight in a humidified chamber at 5% CO2and 37°C. The medium was changed to MEM with EBSS and L-Glutamine, supplemented with 0.5% heat- inactivated FBS, Sodium Pyruvate (1X), 400 µg / mL Geneticin, and 1% penicillin-streptomycin by discarding the supernatant gently. Cells were then incubated in a humidified chamber for 4.5 hrs. After incubation, the medium was gently removed and replaced by 50µL of anti-FAP- luciferase activity, 100 mL of Bio-Glo luciferase assay system (Promega / G7940) were added to each well. The luminescence signal (RLU) was measured using a Tecan Infinite M1000-Pro concentrations using GraphPad Prism software and 50% inhibitory concentration (IC50) values were calculated.

[0120] 50 values for Table 4: Calculated IC50 anti-FAP-BUSINESS.32102741.1

[0121] receptor within the described experimental system (Lopez-Casillas et al., 1993), inhibition of 50 values Table 5: Calculated IC50 anti-FAP-BUSINESS.32102741.1

[0122] which can promote tumorigenesis and metastases. In order to evaluate the ability of the anti- wound-healing assay was used as a surrogate for migration. This assay tested the ability of

[0123] Oris Pro 96-well plates harboring a non-toxic biocompatible gel (BCG) were plated with 2.5x105cells / mL (4T1 breast cancer cells or 4T1 cells overexpressing human FAP), labeled with CellTracker Red (Invitrogen), and allowed to adhere in a monolayer for 2 hours at 37°C. Subsequently, a “wound” was formed by the dissolution of the BCG to form a centrally located and temporary cell-free zone on culture surfaces. Media was removed and replaced ImageXpress Confocal microscope (Molecular Devices) to detect the invasion of the inhibition, wound closure is delayed, resulting in higher wound area.

[0124] Table 6 shows the percentage open wound area by day 4 of treatment. Blocking from 20-50% to approximately 70-90%, indicating an inhibition of invasive potential of 4T1 consistent with reduced potency in the HEK-SMAD signaling assay (Tables 4 and 5), asBUSINESS.32102741.1

[0125] epithelial-to-mesenchymal transition (EMT) in a tumor cell line, a red fluorescent protein (RFP) coding sequence was inserted at the 3’ end of the endogenous open reading frame (ORF) of vimentin gene in the lung cancer cell line A549 (ATCC CCL-185 EMT). The A549- Vimentin-RFP cell reporter line allowed the detection of vimentin protein, a marker of EMT, by measuring the RFP fluorescence.

[0126] incubated for 20 minutes at room temperature. A549-Vimentin-RFP cells were grown in F12- K media supplemented with 10% FBS at 37oC and 5% CO2and plated at a density of 20,000 bispecific variants mixture was then added to the cells and incubated for 48 hours. The cells were washed with FACS buffer (PBS, 2mM EDTA, 5% BSA, pH 7.2) and analyzed using the LSRFortessa (BD Biosciences). Flow data was analyzed using FlowJo software and were plotted with GraphPad Prism software.

[0127] variants V02 and V06 were inferior in reducing vimentin expression compared to WT. Example

[0128]

[0129] SPR experiments were performed using a Biacore T200 instrument. The association rate (ka), dissociation rate (KD), and dissociation constant (KD) were calculated from each experiment using the Biacore 200 Evaluation Software. BUSINESS.32102741.1

[0130] sensor chip (BR100530, Cytiva) immobilized with anti-human IgG1 (Fc) capture antibody HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) were captured in individual flow cells of the anti-Human Fc antibody-immobilized CM5 chip, keeping a single flow cell injection at a flow rate of 10 µL / min to reach appropriate response units (RU) (~100 RU of capture for human FAP binding). Human FAP (diluted in the running buffer to a concentration concentration of 3, 1, 0.33, 0.11, and 0 nM) were flowed over the chip surface at a flow rate of 30 µL / min with a 3-minute association phase and a 5-minute dissociation phase. The sensor chip was regenerated between cycles with a 30-second injection of 3.0 M MgCl2 at 30 µL / min followed by a stabilization period of 60 seconds. Sensorgrams were evaluated and fit to a simple Langmuir 1:1 interaction model using the Biacore Evaluation 3.1 software. Data quality was verified by visually inspecting deviations and curve fitting, and by evaluating the parameters of Rmax, Chi2, and Tc.

[0131] proteins are shown in Table 7.BUSINESS.32102741.1NBD = no binding detected.

[0132] This example describes the binding kinetics and simultaneous binding of human

[0133] SPR experiments were performed using a BiacoreTMT200 instrument. The association rate (ka), dissociation rate (kd), and dissociation constant (KD) were calculated from each experiment using a bivalent analyte model with Biacore Evaluation 3.0 software.

[0134] captured using a Series S Sensor Chip CM7 (28953828, Cytiva) immobilized with anti-human IgG1 (Fc) capture antibody (29234600, Cytiva). For human FAP, cynomolgus FAP, and mouse buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Surfactant P20) were captured in individual flow cells of the anti-Human Fc antibody immobilized CM7 chip, keeping a single flow cell as a reference. The antibodies were captured using a 30-second injection at a flow rate of 10 µL / min to reach appropriate response units (RU) (~60 RU of capture for human FAP binding). Human FAP, cynomolgus FAP, and mouse FAP (each diluted in the running buffer to a concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.098, and 0 nM), were flowed over the chip surface at a flow rate of 30 µL / min with a 3-minute association phase running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) were captured in individual flow cells of the anti-Human Fc antibody immobilized CM7 chip, keeping a single flow cell as a reference. The antibodies were captured using a 30-second injection at a flow rate of 10 µL / min to reach appropriate response units (RU) (~120 RU of running buffer to a concentration of 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0312, and 0 nM), or 0.125, 0.0625, 0.0312, and 0 nM), were flowed over the chip surface at a flow rate of 30 µL / min BUSINESS.32102741.1with a 3-minute association phase and a 20-minute dissociation phase. The sensor chip was regenerated between cycles with two 30-second injection of 3.0 M MgCl2 at 30 µL / min followed by a stabilization period of 60 seconds. Sensorgrams were evaluated and fit to a bivalent analyte model using the global data analysis option of BiacoreTMEvaluation 3.1 software. Data quality was verified by visually inspecting deviations and curve fitting, and by evaluating the parameters of Rmax, Chi2, and Tc.

[0135] The binding kinetics are shown in Table 8. Table

[0136] BiacoreTM8K+ instrument. The test antibodies were captured using a Series S Sensor Chip CM3 (BR-1005-36, Cytiva) immobilized with anti-human IgG1 (Fc) capture antibody (29234600, Cytiva).

[0137] isotype controls diluted in a running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Surfactant P20) were captured in individual flow cells of the anti-human Fc antibody immobilized CM3 chip, keeping a single flow cell as a reference. These antibodies were captured using a 60-second injection at a flow rate of 10 µL / min to reach approximately 150 alone or together in various combinations using running buffer as a control (Table 9). Each dual injection lasted for 180 sec at a flow rate of 30 µL / min, followed by a dissociation phase of 300 sec. Table 9: Combinations of injections. BUSINESS.32102741.1

[0138] The sensor chip was regenerated between cycles with 45-second injections of 10 mM glycine at pH 1.5 at 30 µL / min followed by a stabilization period of 60 sec. Data analysis was performed using BiacoreTMInsight Evaluation Software version 4.0.8.20368 for kinetic determination (GE Healthcare Life Sciences). The sensorgrams were evaluated manually using the visualization tools within the software to analyze the data and associated binding RU are reported (FIG.3). * * *

[0139] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0140] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0141] Other embodiments are within the following claims. BUSINESS.32102741.1

Claims

CLAIMS 1. (a) lacks an amino acid sequence corresponding to amino acids 1-15 of SEQ ID NO: 1; (b) lacks an amino acid sequence corresponding to amino acids 1-22 of SEQ ID NO: 1; (c) comprises Threonine and Proline at the amino acid positions corresponding to amino acids 5 and 9, respectively, of SEQ ID NO: 1; (d) comprises Isoleucine and Alanine at the amino acid positions corresponding to amino acids 33 and 35, respectively, of SEQ ID NO: 1; (e) comprises Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 33, 35, and 36, respectively, of SEQ ID NO: 1; (f) comprises Threonine, Proline, Isoleucine, and Alanine at the amino acid positions corresponding to amino acids 5, 9, 33, and 35, respectively, of SEQ ID NO: 1; (g) comprises Threonine, Proline, Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 5, 9, 33, 35, and 36, respectively, of SEQ ID NO: 1; (h) comprises Isoleucine and Alanine at the amino acid positions corresponding to amino acids 33 and 35, respectively, of SEQ ID NO: 1 and lacks an amino acid sequence corresponding to amino acids 1-15 of SEQ ID NO: 1; (i) comprises Isoleucine and Alanine at the amino acid positions corresponding to amino acids 33 and 35, respectively, of SEQ ID NO: 1 and lacks an amino acid sequence corresponding to amino acids 1-22 of SEQ ID NO: 1; (j) comprises Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 33, 35, and 36, respectively, of SEQ ID NO: 1 and lacks the amino acid sequence corresponding to amino acids 1-15 of SEQ ID NO: 1; or (k) comprises Isoleucine, Alanine, and Alanine at the amino acid positions corresponding to amino acids 33, 35, and 36, respectively, of SEQ ID NO: 1 and lacks the amino acid sequence corresponding to amino acids 1-22 of SEQ ID NO:BUSINESS.32102741.

12. is at least 75% identical (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to SEQ ID NO: 1 over the full length of the 3. The polypeptide of any one of the preceding claims, wherein the amino acid sequence forth in any one of SEQ ID NOs: 2-12.

4. The polypeptide of any one of the preceding claims, wherein the polypeptide comprises an antigen-binding region.

5. The polypeptide of any one of the preceding claims, wherein the antigen-binding region comprises an scFv, a VH, a VL, a VHH, an antibody heavy chain, or an antibody light chain.

6. The polypeptide of any one of the preceding claims, wherein the polypeptide comprises an antibody constant region.

7. The polypeptide of any one of the preceding claims, wherein the variant human region via a linker sequence.

8. The polypeptide of any one of the preceding claims, comprising, from N-terminal to C-terminal: an antibody heavy chain, or an Fc fragment thereof; and the variant human 9. The polypeptide of any one of the preceding claims, comprising, from N-terminal to C-terminal: an antibody heavy chain, or an Fc fragment thereof; a linker sequence; and the 10. The polypeptide of any one of the preceding claims, wherein the linker sequence comprises the amino acid sequence set forth in SEQ ID NO:

13. BUSINESS.32102741.

111. The polypeptide of any one of the preceding claims, wherein the linker sequence comprises the amino acid sequence set forth in SEQ ID NO:

14.

12. The polypeptide of any one of the preceding claims, comprising the amino acid sequence set forth in any one of SEQ ID NOs: 15-25.

13. An antibody molecule comprising two heavy chain polypeptides, wherein each heavy chain polypeptide comprises, from N-terminal to C-terminal: a variable region; a constant amino acid sequence set forth in any one of SEQ ID NOs: 2-12.

14. The antibody molecule of claim 13, wherein the linker sequence comprises the amino acid sequence set forth in SEQ ID NO:

13.

15. The antibody molecule of claim 13 or 14, wherein the linker sequence comprises the amino acid sequence set forth in SEQ ID NO:

14.

16. The antibody molecule of any one of claims 13-15, comprising the amino acid sequence set forth in any one of SEQ ID NOs: 15-25.

17. The antibody molecule of any one of claims 13-16, which is a full-length heterotetrameric antibody molecule comprising two heavy chain polypeptides and two light chain polypeptides.

18. The antibody molecule of any one of claims 13-16, which is a full-length heavy chain antibody molecule, comprising two heavy chain polypeptides and no light chain polypeptides.

19. The antibody molecule of any one of claims 13-18, wherein the constant region is a human heavy chain constant region.

20. The antibody molecule of claim 19, wherein the human heavy chain constant region is of the IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, or IgM isotype. BUSINESS.32102741.

121. A polynucleotide encoding the polypeptide or antibody molecule of any one of claims 1-20.

22. A vector comprising the polynucleotide of claim 21.

23. An engineered cell comprising the polynucleotide or vector of claim 21 or 22.

24. A method of producing a polypeptide, the method comprising culturing the engineered cell of claim 23 under conditions such that the polynucleotide is expressed, and the polypeptide is produced.

25. 1-20.

26. administering to the subject the polypeptide or antibody molecule of any one of claims 1-20. BUSINESS.32102741.1

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