Materials and methods for improved bioengineered pairing of antigen-binding variable regions

Abstract

Antigen-binding molecules engineered to replace the light chain constant domain (CL) and heavy chain constant domain 1 (CH1) with HLA class I histocompatibility antigen alpha chain-E alpha-3 (HLA-E) / beta2 microglobulin (B2M) or intercellular adhesion molecule 1 domain 1 (ICAM-1 D1) / ICAM-1 D1 dimerization domain are described.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 389,814, filed July 15, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. A copy of this XML was created on July 12, 2023, has the file name 253505_000341_SL, and is 391,390 bytes in size.

[0003] FIELD OF THE INVENTION Provided herein are antigen-binding molecules comprising one or more antigen-binding polypeptides that form a variable region that binds to a target antigen and have a dimerization domain described herein instead of an antibody light chain constant domain (CL) and antibody heavy chain constant domain 1 (CH1). Replacing the dimerization interface between the light chain constant domain and heavy chain constant domain 1 provides a means for selectively assembling cognate chains in a multiparatopic antigen-binding molecule. [Background technology]

[0004] The production of bispecific heterodimeric antibodies with modified heavy chain IgG constant regions to promote efficient formation of heavy chain heterodimer pairs and arm-specific pairing of the heavy and light chains has been described (WO 2018 / 237192(A1)). Summary of the Invention

[0005] In one aspect, provided herein is an antigen-binding molecule comprising one or more antigen-binding polypeptides, each comprising a variable domain that binds to a target antigen and, in place of a light chain constant domain (CL) or heavy chain constant domain 1 (CH1), a dimerization domain as described herein.

[0006] In certain embodiments, the antigen-binding molecules bind to one or more target antigens. In certain embodiments, the antigen-binding molecules described herein comprise two or more polypeptides, each comprising a variable domain, which form a paratope that binds to one or more target antigens.

[0007] In certain embodiments, the antigen-binding molecules described herein comprise 2, 3, 4, 5, 6, 7, or 8 polypeptides, optionally each of which comprises a dimerization domain and a variable domain that binds (together with its cognate variable domain) to a target antigen described herein. In certain embodiments, a dimerization domain binds to another dimerization domain to form a dimer (particularly a heterodimer, e.g., six polypeptides can form up to three dimers with each other).

[0008] In certain embodiments, the polypeptide of the antigen-binding molecule described herein comprises a light chain polypeptide comprising a light chain variable domain and a light chain dimerization domain, and a heavy chain polypeptide comprising a heavy chain variable domain and a heavy chain dimerization domain. In certain embodiments, the light chain variable domain and the heavy chain variable domain constitute a variable region that binds to a target antigen.

[0009] In certain embodiments of the antigen-binding molecules described herein, the light chain polypeptide comprises an immunoglobulin or antibody light chain, or one or more fragments thereof, and optionally, the light chain polypeptide comprises a kappa (κ), lambda (λ), sigma (σ), iota (ι), or one or more fragments thereof. In certain embodiments of the antigen-binding molecules described herein, the heavy chain polypeptide comprises an immunoglobulin or antibody heavy chain, or one or more fragments thereof, and optionally, the heavy chain polypeptide comprises a gamma (γ), delta (δ), alpha (α), mu (μ), epsilon (ε), or one or more fragments thereof.

[0010] In one aspect, an antigen binding molecule described herein comprises a first light chain polypeptide comprising, from amino-terminus to carboxy-terminus, VL1-LD1, wherein VL1 is a first light chain variable domain and LD1 is a first light chain dimerization domain; and a first heavy chain polypeptide comprising, from amino-terminus to carboxy-terminus, VH1-HD1, wherein VH1 is a first heavy chain variable domain and HD1 is a first heavy chain dimerization domain, wherein a) i) LD1 comprises a β2 microglobulin (B2M) domain and HD1 comprises an HLA-E A3 (EA3) domain, or LD1 comprises an EA3 domain and HD1 comprises a B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer, or ii) LD1 comprises a first ICAM-1 D1 domain and HD1 comprises a second ICAM-1 D1 domain and the first ICAM-1 The D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, and b) i) at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively, and ii) the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain, and the VL1 and VH1 form a first paratope.

[0011] In one aspect, the antigen-binding molecule described herein is a first light chain polypeptide comprising, in order from the amino terminus to the carboxy terminus, a first light chain variable domain, a first light chain elbow region comprising: 1) 1 to 8 contiguous amino acids selected from amino acid positions 108 to 115 of a human immunoglobulin kappa constant domain according to EU or Kabat numbering, or 2) an amino acid sequence that is at least 3 amino acids in length; and an HLA-E A3 (EA3) domain, a β2 microglobulin (B2M) domain, or a first ICAM-1 a first light chain polypeptide comprising: a first light chain dimerization domain selected from the D1 domain; and a first heavy chain polypeptide comprising, in order from amino terminus to carboxy terminus, a first heavy chain elbow region comprising: 1) 1 to 8 consecutive amino acids selected from amino acid positions 118 to 125 of human IgG1 according to EU numbering or amino acid positions 114 to 121 of human IgG1 according to Kabat numbering, or 2) an amino acid sequence that is at least 3 amino acids in length. a first heavy chain polypeptide comprising an elbow region and a first heavy chain dimerization domain, wherein i) the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, and the B2M domain and the EA3 domain bind to each other to form a dimer, or ii) the first light chain dimerization domain comprises a first ICAM-1 the first heavy chain dimerization domain comprises a second ICAM-1 D1 domain, the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, i) at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively, and ii) the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain, and the first light chain variable domain and the first heavy chain variable domain form a first paratope.

[0012] In one aspect, an antigen-binding molecule described herein comprises a dimer of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from amino terminus to carboxy terminus, (i) a first immunoglobulin fragment that does not comprise a dimerization sequence of a CH1 domain or a CL domain, and (ii) a first dimerization domain that comprises an HLA-E A3 (EA3) domain, a β2 microglobulin (B2M) domain, or a first ICAM-1 D1 domain; and the second polypeptide comprises, from amino terminus to carboxy terminus, (i) a second immunoglobulin fragment that does not comprise a dimerization sequence of a CH1 domain or a CL domain, and (ii) an EA3 domain, a B2M domain, or a second ICAM-1 D1 domain. and a second dimerization domain comprising a D1 domain, wherein i) the first dimerization domain comprises a B2M domain and the second dimerization domain comprises an EA3 domain, or the first dimerization domain comprises an EA3 domain and the second dimerization domain comprises a B2M domain, and the B2M domain and the EA3 domain bind to each other to form a dimer, or ii) the first dimerization domain comprises a first ICAM-1 D1 domain and the second dimerization domain comprises a second ICAM-1 D1 domain, and the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, and i) at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively, and ii) the first ICAM-1 D1 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively. The first immunoglobulin fragment comprises a first immunoglobulin variable domain and the second immunoglobulin fragment comprises a second immunoglobulin variable domain distinct from the D1 domain, and the first immunoglobulin variable domain and the second immunoglobulin variable domain form a first paratope.

[0013] In one aspect, provided herein is a composition comprising a plurality of polypeptide species, wherein at least one first polypeptide species comprises, from amino terminus to carboxy terminus, a first immunoglobulin fragment that does not comprise a dimerization sequence of a CH1 domain or a CL domain, and a first dimerization domain that comprises an HLA-E A3 (EA3) domain, a β2 microglobulin (B2M) domain, or a first ICAM-1 D1 domain; and at least one second polypeptide species comprises, from amino terminus to carboxy terminus, a second immunoglobulin fragment that does not comprise a dimerization sequence of a CH1 domain or a CL domain, and a first dimerization domain that comprises an HLA-E A3 (EA3) domain, a β2 microglobulin (B2M) domain, or a second ICAM-1 D1 domain. and a second polypeptide comprising a second dimerization domain comprising a D1 domain, wherein (a) the first dimerization domain comprises a B2M domain and the second dimerization domain comprises an EA3 domain, or the first dimerization domain comprises an EA3 domain and the second dimerization domain comprises a B2M domain, and the B2M domain and the EA3 domain bind to each other to form a dimer, or (b) the first dimerization domain comprises a first ICAM-1 D1 domain and the second dimerization domain comprises a second ICAM-1 D1 domain, and the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, and (a) at least one of the B2M domain or EA3 domain is different from the wild-type B2M domain or EA3 domain, respectively, and (b) the first ICAM-1 D1 domain is different from the wild-type B2M domain or EA3 domain, respectively, and The first immunoglobulin fragment comprises a first immunoglobulin variable domain and the second immunoglobulin fragment comprises a second immunoglobulin variable domain distinct from the D1 domain, and the first immunoglobulin variable domain and the second immunoglobulin variable domain form a first paratope.

[0014] In one aspect, provided herein is a composition comprising a plurality of polypeptide species, each polypeptide species comprising a means for binding to a first target antigen and a means for dimerizing, wherein in at least one polypeptide species, the means for binding comprises a first means for binding to the first target antigen and a first means for dimerizing comprising an HLA-E A3 (EA3) domain that binds to a β2 microglobulin (B2M) domain to form a dimer, a B2M domain that binds to the EA3 domain to form a dimer, or a first ICAM-1 D1 domain that binds to a second ICAM-1 D1 domain to form a dimer, and wherein in another at least one polypeptide species, the means for binding comprises a second means for binding to the first target antigen and a B2M domain that binds to the EA3 domain to form a dimer, an EA3 domain that binds to the B2M domain to form a dimer, or a second ICAM-1 D1 domain that binds to the first ICAM-1 D1 domain to form a dimer. and a second means for dimerizing comprising a D1 domain, wherein the first means for dimerizing and the second means for dimerizing bind to each other to form a dimer, at least one of the EA3 domain or the B2M domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain, and the first means for binding to the first target antigen and the second means for binding to the first target antigen form a first paratope.

[0015] In one aspect, provided herein is a method of producing a multi-paratope antibody in a single host cell, the method comprising: (a) producing a multi-paratope antibody in a single host cell; (b) producing a multi-paratope antibody in a single host cell; (c) producing a multi-paratope antibody in a single host cell; (d) producing a multi-paratope antibody in a single host cell; (e) producing a multi-paratope antibody in a single host cell; (f) producing a multi-paratope antibody in a single host cell; (g) producing a multi-paratope antibody in a single host cell; the first heavy chain dimerization domain comprises a second ICAM-1 D1 domain, the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, (i) at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively; and (ii) the first ICAM-1 D1 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively.a second antibody heavy chain comprising a first heavy chain, a second heavy chain variable domain, and a second heavy chain dimerization domain that is not an EA3 domain, a B2M domain, or an ICAM-1 domain, or is a heavy chain second constant (CH1) domain, wherein the D1 domain is different and the first light chain variable domain and the first heavy chain variable domain form a first paratope and a connection to a second heavy chain chain, wherein the second heavy chain dimerization domain dimerizes into the second light chain dimerization domain and forms a connection to the first antibody heavy chain; and a second antibody light chain comprising a second light chain variable domain and a second light chain dimerization domain that is not an EA3 domain, a B2M domain, or an ICAM-1 domain, or is a light chain constant (CL) domain, wherein the second light chain dimerization domain dimerizes into the second heavy chain dimerization domain and forms a connection to the first antibody heavy chain. delivering the one or more polynucleotide sequences to a host cell; and culturing the host cell under conditions allowing expression of the multi-paratope antibody, thereby producing the multi-paratope antibody, wherein the first light chain variable domain and the first heavy chain variable domain form a first paratope specific for a first target, the first light chain dimerization domain and the first heavy-light chain dimerization domain, the second light chain dimerization domain and the second heavy chain dimerization domain, and the first heavy chain and the second heavy chain are connected to each other, and i) at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively, and ii) the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain.

[0016] In certain embodiments, the first light chain dimerization domain comprises a β2 microglobulin (B2M) domain and the first heavy chain dimerization domain comprises an HLA-E A3 (EA3) domain. In certain embodiments, the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain. In certain embodiments, the B2M domain and the EA3 domain bind to each other to form a dimer. In certain embodiments, at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively. In certain embodiments, the B2M is different from the wild-type B2M domain (SEQ ID NO: 2). In certain embodiments, the EA3 domain is different from the wild-type EA3 domain (SEQ ID NO: 33).

[0017] In certain embodiments, the first light chain dimerization domain comprises a first ICAM-1 D1 domain, and the first heavy chain dimerization domain comprises a second ICAM-1 D1 domain. In certain embodiments, the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer. In certain embodiments, the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain.

[0018] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, the B2M domain and the EA3 domain bind to each other to form a dimer, and the B2M domain comprises or consists of the amino acid sequence of RTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 2).

[0019] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer, and the B2M domain comprises or consists of an amino acid sequence having at least 91% sequence identity to SEQ ID NO:2.

[0020] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer, and the B2M domain comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 single amino acid substitutions relative to the sequence of SEQ ID NO:2 at one or more amino acid positions 4, 8, 10, 54, 58, 60, 96, and 97 of SEQ ID NO:2.

[0021] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, wherein the B2M domain and the EA3 domain combine with each other to form a dimer, and the B2M domain comprises or consists of amino acids of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 single amino acid substitutions relative to the sequence of SEQ ID NO: 2 at one or more amino acid positions 4, 8, 10, 54, 58, 60, 96, and 97 of SEQ ID NO: 2, wherein each single amino acid substitution is independently selected from the group consisting of F, W, C, S, and T.

[0022] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises or consists of the amino acid sequence of the human B2M sequence set forth in SEQ ID NO:2, but comprises the following substitution set: F56S, W60S, and F62T, with positions numbered according to the numbering of the B2M amino acids in Table 1, and optionally further comprises 1, 2, 3, 4, or 5 single amino acid substitutions at positions K6, Y10, R12, D98, or M99, with positions numbered according to the numbering of the B2M amino acids in Table 1.

[0023] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer, and comprise or consist of the amino acid sequence of the human B2M sequence shown in SEQ ID NO: 2, but not the B2M sequence in Table 1. The set of substitutions is selected from F56S, W60S, F62T, and K6C; F56S, W60S, F62T, and any one of Y10C, Y10F, and Y10W; F56S, W60S, F62T, and R12C; F56S, W60S, F62T, and any one of D98C; D98F and D98W; F56S, W60S, F62T, and any one of M99C, M99F, and M99W, with positions numbered according to amino acid numbering.

[0024] In certain embodiments of the antigen-binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, the B2M domain and the EA3 domain bind to each other to form a dimer, and the B2M domain comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 2 to 30.

[0025] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, and the B2M domain and the EA3 domain bind to each other to form a dimer, and the EA3 domain comprises or consists of the amino acid sequence of LHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW (SEQ ID NO: 33).

[0026] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer, and the EA3 domain comprises or consists of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 33.

[0027] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer, and the EA3 domain comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, or 6 single amino acid substitutions relative to the sequence of SEQ ID NO: 33 at one or more amino acid positions 13, 23, 53, 55, 59, and 63 of SEQ ID NO: 33.

[0028] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, wherein the B2M domain and the EA3 domain combine with each other to form a dimer, and the EA3 domain comprises or consists of amino acids of an amino acid sequence having 1, 2, 3, 4, 5, or 6 single amino acid substitutions relative to the sequence of SEQ ID NO: 33 at one or more amino acid positions 13, 23, 53, 55, 59, and 63 of SEQ ID NO: 33, wherein each single amino acid substitution is independently selected from the group consisting of A, C, and L.

[0029] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, wherein the B2M domain and the EA3 domain combine with each other to form a dimer, and the EA3 domain comprises or consists of the amino acid sequence of the human EA3 sequence set forth in SEQ ID NO: 33, but contains 1, 2, 3, 4, 5, or 6 single amino acid substitutions at positions H192, R202, E232, R234, D238, or Q242, numbered according to the EA3 amino acid numbering in Table 2.

[0030] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer, and the EA3 domain is an amino acid sequence of the human EA3 sequence set forth in SEQ ID NO: 33. or R234L and Q242L, with positions numbered according to the EA3 amino acid numbering in Table 2.

[0031] In certain embodiments of the antigen-binding molecules described herein, the first light chain dimerization domain comprises a B2M domain and the first heavy chain dimerization domain comprises an EA3 domain, or the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises a B2M domain, the B2M domain and the EA3 domain bind to each other to form a dimer, and the EA3 domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 32 to 46.

[0032] In certain embodiments of the antigen-binding molecules described herein, the first light chain dimerization domain comprises a first ICAM-1 D1 domain, the first heavy chain dimerization domain comprises a second ICAM-1 D1 domain, the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, and the first ICAM-1 D1 domain or the second ICAM-1 D1 domain comprises or consists of QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVY (SEQ ID NO: 49).

[0033] In certain embodiments of the antigen binding molecules described herein, the first light chain dimerization domain comprises a first ICAM-1 D1 domain, the first heavy chain dimerization domain comprises a second ICAM-1 D1 domain, the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, and the first ICAM-1 D1 domain or the second ICAM-1 D1 domain comprises or consists of an amino acid sequence having at least 81% sequence identity to SEQ ID NO:49.

[0034] In certain embodiments of the antigen-binding molecules described herein, the first light chain dimerization domain comprises a first ICAM-1 D1 domain, the first heavy chain dimerization domain comprises a second ICAM-1 D1 domain, the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, and the first ICAM-1 D1 domain or the second ICAM-1 D1 domain comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single amino acid substitutions relative to the sequence of SEQ ID NO: 49 at one or more amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67, and 78 of SEQ ID NO: 49, and optionally further comprising a C-terminal cysteine amino acid addition.

[0035] In certain embodiments of the antigen-binding molecules described herein, the first light chain dimerization domain comprises a first ICAM-1 D1 domain, the first heavy chain dimerization domain comprises a second ICAM-1 D1 domain, the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, and the first ICAM-1 D1 domain or the second ICAM-1 D1 domain binds to each other to form a dimer. The D1 domain comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single amino acid substitutions relative to the sequence of SEQ ID NO: 49 at one or more amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67, and 78 of SEQ ID NO: 49, wherein each single amino acid substitution is independently selected from the group consisting of V, T, F, W, A, K, E, C, and R, and optionally further comprising a C-terminal cysteine amino acid addition.

[0036] In certain embodiments of the antigen-binding molecules described herein, the first light chain dimerization domain comprises a first ICAM-1 D1 domain, the first heavy chain dimerization domain comprises a second ICAM-1 D1 domain, the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, and the first ICAM-1 D1 domain or the second ICAM-1 D1 domain has the amino acid sequence of the human ICAM-1 D1 sequence shown in SEQ ID NO: 49, but is not limited to the ICAM-1 D1 domain of Table 3. or consisting of an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single amino acid substitutions at positions T2, I10, R13, L18, T20, T23, E34, P38, L42, R49, V51, E53, P63, S67, or T78, with positions numbered according to the D1 amino acid numbering, and optionally further comprising a C-terminal cysteine amino acid addition (84C), with positions numbered according to the ICAM-1 D1 amino acid numbering in Table 3.

[0037] In a specific embodiment of the antigen-binding molecule according to the present invention, the first light chain dimerization domain comprises a first ICAM-1 D1 domain, the first heavy chain dimerization domain comprises a second ICAM-1 D1 domain, the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, and the first ICAM-1 D1 domain or the second ICAM-1 D1 domain has the amino acid sequence of the human ICAM-1 D1 sequence shown in SEQ ID NO: 49, but is different from the ICAM-1 D1 sequence shown in Table 3. Positions were numbered according to the D1 amino acid numbering, E34K; T2V, I10T, T23A, E34K, P38T, P63V, S67A, and T78A; T2V, I10T, R13C, T23A, E34K, P38T, R49E, P63V, S67A, T78A, and 84C; T2V, I10T, R13C, T23A, E34K, P38T, E53R, P63V, S67A, T78A, and 84C; T2V, I10T, R13C, L1 8F, T23A, E34K, P38T, P63V, S67A, T78A, and 84C; T2V, I10T, R13C, L18A, T20A, T23A, E34K, P38T, L42A, V51A, P63V, S67A, T78A, and 84C; or T2V, I10T, R13C, L18W, T23A, E34K, P38T, P63V, S67A, T78A, and 84C.

[0038] In certain embodiments of the antigen-binding molecules described herein, the first light chain dimerization domain comprises a first ICAM-1 D1 domain, the first heavy chain dimerization domain comprises a second ICAM-1 D1 domain, the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer, and the first ICAM-1 D1 domain or the second ICAM-1 D1 domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 48 to 58.

[0039] In certain embodiments of the antigen-binding molecules described herein, a light chain polypeptide (e.g., a first light chain polypeptide) comprises an elbow region between its light chain variable domain and its light chain dimerization domain. In certain embodiments, a heavy chain polypeptide (e.g., a first heavy chain polypeptide) comprises an elbow region between its heavy chain variable domain and its heavy chain dimerization domain. In certain embodiments, the elbow region comprises an amino acid sequence at least 3 amino acids in length. In certain embodiments, the elbow region comprises or consists of an amino acid sequence 3 to 25 amino acids in length.

[0040] In certain embodiments of the antigen binding molecules described herein, the elbow region is selected from the group consisting of RTV, GGS, RTVGGS (SEQ ID NO: 59), RTVGGSRTV (SEQ ID NO: 60), AST, ASTK (SEQ ID NO: 61), ASTKG (SEQ ID NO: 62), ASTKGG (SEQ ID NO: 63), ASTKGGS (SEQ ID NO: 64), ASTKGGGS (SEQ ID NO: 65), ASTKGGGGS (SEQ ID NO: 66), ASTKGGGGSG (SEQ ID NO: 67), ASTKGGGGSGG (SEQ ID NO: 68), ASTKGGGGSGGS (SEQ ID NO: 69), ASTKGGGGSGGGS (SEQ ID NO: 70), ASTKGGGGSGGGGS (SEQ ID NO: 71), RTVA (SEQ ID NO: 72), RTVAG (SEQ ID NO: 73), RTV AGG (SEQ ID NO:74), RTVAGGS (SEQ ID NO:75), RTVAGGGS (SEQ ID NO:76), RTVAGGGGS (SEQ ID NO:77), RTVAGGGGSG (SEQ ID NO:78), RTVAGGGGSGG (SEQ ID NO:79), RTVAGGGGSGGS (SEQ ID NO:80), RTVAGGGGSGGGS (SEQ ID NO:81), RTVAGGGGSGGGGS (SEQ ID NO:82), GGGGSGGGGS (SEQ ID NO:83), GGGGSGGGGSGGGGS (SEQ ID NO:84), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:85), and GGGGSGGGGSGGGGSGGGGSGGGG (SEQ ID NO:86).

[0041] In certain embodiments of the antigen-binding molecules described herein, a polypeptide of the antigen-binding molecule comprises a spacer region fused to the C-terminus of its dimerization domain. In certain embodiments, a light chain polypeptide (e.g., a first light chain polypeptide) comprises a spacer region fused to the C-terminus of its dimerization domain. In certain embodiments, a heavy chain polypeptide (e.g., a first heavy chain polypeptide) comprises a spacer region fused to the C-terminus of its dimerization domain. In certain embodiments, the spacer region comprises an amino acid sequence at least 2 amino acids in length. In certain embodiments, the spacer region comprises or consists of an amino acid sequence 2 to 9 amino acids in length.

[0042] In certain embodiments of the antigen binding molecules described herein, the spacer region comprises or consists of an amino acid sequence selected from the group consisting of EPKSS (SEQ ID NO: 87), SG, EPKSC (SEQ ID NO: 88), GGSGECSG (SEQ ID NO: 89), GGGSGECSG (SEQ ID NO: 90), GGSGECSG (SEQ ID NO: 91), and GGGSGESSG (SEQ ID NO: 92).

[0043] In certain embodiments, the spacer region further comprises a hinge region.

[0044] In certain embodiments of the antigen-binding molecules described herein, the polypeptide of the antigen-binding molecule further comprises a C-terminal tag, and optionally, the C-terminal tag is a 6xHis tag (SEQ ID NO: 93), a streptavidin tag (e.g., a Strep-tag II tag), or a human influenza hemagglutinin tag.

[0045] In certain embodiments of the antigen-binding molecules described herein, the antigen-binding molecule is an immunoglobulin, Fab, Fab', F(ab'), antibody, biparatopic antibody, bispecific antibody, triparatopic antibody, trispecific antibody, tetraparatopic antibody, tetraspecific antibody, multiparatopic antibody, multispecific antibody, or any fragment of an antigen-binding molecule that binds to a target antigen.

[0046] In certain embodiments, the antigen-binding molecule described herein further comprises a second light chain polypeptide (VL2) comprising a second light chain variable domain (VL2) and a second heavy chain polypeptide (HC2) comprising a second heavy chain variable domain (VH2), wherein VL2 and VH2 form a second paratope. In certain embodiments, the first paratope and the second paratope bind to different antigens.

[0047] In certain embodiments, the antigen-binding molecule described herein further comprises a third light chain polypeptide (LC3) comprising a third light chain variable domain (VL3) and a third heavy chain polypeptide (HC3) comprising a third heavy chain variable domain (VH3), wherein VL3 and VH3 form a third paratope. In certain embodiments, the first paratope, the second paratope, and the third paratope bind to different antigens.

[0048] In certain embodiments, the antigen-binding molecule described herein further comprises a fourth light chain polypeptide (VL4) comprising a fourth light chain variable domain (VL4) and a fourth heavy chain polypeptide (HC4) comprising a fourth heavy chain variable domain (VH4), wherein VL4 and VH4 form a fourth paratope. In certain embodiments, the first paratope, second paratope, third paratope, and fourth paratope bind to different antigens.

[0049] In certain embodiments of the antigen binding molecules described herein, the first paratope specifically binds to a first tumor-associated antigen (TAA1), the second paratope specifically binds to a second tumor-associated antigen (TAA2), the third paratope specifically binds to a third tumor-associated antigen (TAA3), and / or the fourth paratope specifically binds to a fourth tumor-associated antigen (TAA4).

[0050] In another aspect, provided herein is an isolated polynucleotide encoding an antigen-binding molecule described herein.

[0051] In another aspect, provided herein is a vector comprising an isolated polynucleotide encoding an antigen-binding molecule described herein.

[0052] In another aspect, provided herein is a host cell containing a vector comprising an isolated polynucleotide encoding an antigen-binding molecule described herein.

[0053] In another aspect, provided herein is a pharmaceutical composition comprising an antigen-binding molecule described herein, an isolated polynucleotide described herein, or a host cell described herein, and a pharmaceutically acceptable excipient.

[0054] In another aspect, provided herein is a method for producing an antigen-binding molecule described herein, the method comprising culturing a host cell described herein under suitable conditions such that the antigen-binding molecule is expressed by the host cell, and isolating the antigen-binding molecule.

[0055] In another aspect, provided herein is a method for treating a disease or disorder in a subject, comprising administering to the subject an antigen-binding molecule or pharmaceutical composition described herein.

[0056] In another aspect, provided herein is a method for treating cancer in a subject, comprising administering to the subject an antigen-binding molecule or pharmaceutical composition described herein. [Brief explanation of the drawings]

[0057] [Figure 1A]The CH1-CL replacement strategy described herein is shown. Figure 1A shows a generalized bispecific antibody (BsAb) characterized by two unique variable domains (hatched and gray dots), whose unique VH and VL are located N-terminal to the CH or CL domains, respectively, that are native to both the light and heavy chain constant regions. Figure 1B shows the replacement of the CH1 and CL domains of one arm of a native BsAb in a "pseudo-Fab" (pFab) design, in which the CH1 and CL domains are replaced by alternative Ig domains (horizontal lines). Figure 1C shows the extension of the CH1 and CL replacement strategy to a multispecific antibody, in which the two CH1 and CL domains are replaced by different pFab regions (horizontal lines for one pFab and vertical lines for the other pFab). Standard antibody constant domains are shown as white ovals. [Figure 1B] The CH1-CL replacement strategy described herein is shown. Figure 1A shows a generalized bispecific antibody (BsAb) characterized by two unique variable domains (hatched and gray dots), whose unique VH and VL are located N-terminal to the CH or CL domains, respectively, that are native to both the light and heavy chain constant regions. Figure 1B shows the replacement of the CH1 and CL domains of one arm of a native BsAb in a "pseudo-Fab" (pFab) design, in which the CH1 and CL domains are replaced by alternative Ig domains (horizontal lines). Figure 1C shows the extension of the CH1 and CL replacement strategy to a multispecific antibody, in which the two CH1 and CL domains are replaced by different pFab regions (horizontal lines for one pFab and vertical lines for the other pFab). Standard antibody constant domains are shown as white ovals. [Figure 1C]The CH1-CL replacement strategy described herein is shown. Figure 1A shows a generalized bispecific antibody (BsAb) characterized by two unique variable domains (hatched and gray dots), whose unique VH and VL are located N-terminal to the CH or CL domains, respectively, that are native to both the light and heavy chain constant regions. Figure 1B shows the replacement of the CH1 and CL domains of one arm of a native BsAb in a "pseudo-Fab" (pFab) design, in which the CH1 and CL domains are replaced by alternative Ig domains (horizontal lines). Figure 1C shows the extension of the CH1 and CL replacement strategy to a multispecific antibody, in which the two CH1 and CL domains are replaced by different pFab regions (horizontal lines for one pFab and vertical lines for the other pFab). Standard antibody constant domains are shown as white ovals. [Figure 2A] The structure of the HLA-E A3 (EA3) domain / β2-microglobulin (B2M) domain heterodimer is compared with that of the native CH1-CL heterodimer. Figure 2A shows the Fab structure from RCSB PDB ID: 5TZ2. The heavy chain is shown in dark gray, and the light chain is shown in light gray. The distance from heavy chain VH residue S112 (Kabat) to CH1 residue S119 (EU) was 8.1 Å, and the distance from VL I106 (Kabat) to CL K111 (EU) was 13.3 Å. Figure 2B shows the EA3 / B2M domain heterodimer superimposed on the Fab structure. The distances between the above variable domain residues and the N-termini of the aligned EA3 subunit (residue L201) and B2M (residue R23) were 13.9 Å and 14.0 Å, respectively. Figure 2C shows the ICAM-1 D1 domain superimposed on the Fab structure. The distances between the above variable domain residues and the N-terminus of the aligned ICAM-1 D1 homodimer (residue Q1) were 8.0 Å and 21.9 Å, respectively. [Figure 2B]The structure of the HLA-E A3 (EA3) domain / β2-microglobulin (B2M) domain heterodimer is compared with that of the native CH1-CL heterodimer. Figure 2A shows the Fab structure from RCSB PDB ID: 5TZ2. The heavy chain is shown in dark gray, and the light chain is shown in light gray. The distance from heavy chain VH residue S112 (Kabat) to CH1 residue S119 (EU) was 8.1 Å, and the distance from VL I106 (Kabat) to CL K111 (EU) was 13.3 Å. Figure 2B shows the EA3 / B2M domain heterodimer superimposed on the Fab structure. The distances between the above variable domain residues and the N-termini of the aligned EA3 subunit (residue L201) and B2M (residue R23) were 13.9 Å and 14.0 Å, respectively. Figure 2C shows the ICAM-1 D1 domain superimposed on the Fab structure. The distances between the above variable domain residues and the N-terminus of the aligned ICAM-1 D1 homodimer (residue Q1) were 8.0 Å and 21.9 Å, respectively. [Figure 2C] The structure of the HLA-E A3 (EA3) domain / β2-microglobulin (B2M) domain heterodimer is compared with that of the native CH1-CL heterodimer. Figure 2A shows the Fab structure from RCSB PDB ID: 5TZ2. The heavy chain is shown in dark gray, and the light chain is shown in light gray. The distance from heavy chain VH residue S112 (Kabat) to CH1 residue S119 (EU) was 8.1 Å, and the distance from VL I106 (Kabat) to CL K111 (EU) was 13.3 Å. Figure 2B shows the EA3 / B2M domain heterodimer superimposed on the Fab structure. The distances between the above variable domain residues and the N-termini of the aligned EA3 subunit (residue L201) and B2M (residue R23) were 13.9 Å and 14.0 Å, respectively. Figure 2C shows the ICAM-1 D1 domain superimposed on the Fab structure. The distances between the above variable domain residues and the N-terminus of the aligned ICAM-1 D1 homodimer (residue Q1) were 8.0 Å and 21.9 Å, respectively. [Figure 3A]Figure 3 shows the sequence alignment and percent identity of the HLA-E A3 (EA3) wild-type sequence and the β2 microglobulin (B2M) wild-type sequence with the human IgG1 G1m CH1 domain, human IgG1 kappa light chain constant domain, and human IgG1 lambda light chain constant domain. Figure 3A shows the sequence alignment of the CH1 domain, kappa constant domain, and lambda constant domain with EA3 and B2M. Figure 3B shows that EA3 shows higher identity to the CH1 domain than to the CL domain, and B2M shows higher identity to the CL domain. In certain "pseudo-Fab" domains, EA3 replaces CH1 and B2M replaces CL. The figures disclose SEQ ID NOs: 98-99, 368-369, and 2, respectively, in order of appearance. [Figure 3B] Figure 3 shows the sequence alignment and percent identity of the HLA-E A3 (EA3) wild-type sequence and the β2 microglobulin (B2M) wild-type sequence with the human IgG1 G1m CH1 domain, human IgG1 kappa light chain constant domain, and human IgG1 lambda light chain constant domain. Figure 3A shows the sequence alignment of the CH1 domain, kappa constant domain, and lambda constant domain with EA3 and B2M. Figure 3B shows that EA3 shows higher identity to the CH1 domain than to the CL domain, and B2M shows higher identity to the CL domain. In certain "pseudo-Fab" domains, EA3 replaces CH1 and B2M replaces CL. The figures disclose SEQ ID NOs: 98-99, 368-369, and 2, respectively, in order of appearance. [Figure 4A] Figure 4B shows the design of the B2M(SST) variants described herein. Figure 4B shows the structure of HLA-E A3 (EA3), demonstrating the hydrophobic interactions between B2M and the α1 / α2 domains of the EA3. Figure 4B shows a sequence alignment of human B2M and B2M(SST). The figures disclose SEQ ID NOs: 2 and 4, respectively, in order of appearance. [Figure 4B]Figure 4B shows the design of the B2M(SST) variants described herein. Figure 4B shows the structure of HLA-E A3 (EA3), demonstrating the hydrophobic interactions between B2M and the α1 / α2 domains of the EA3. Figure 4B shows a sequence alignment of human B2M and B2M(SST). The figures disclose SEQ ID NOs: 2 and 4, respectively, in order of appearance. [Figure 5] Figure 1 shows an in vitro analysis of the ability of HLA-EA3 (EA3) / β2 microglobulin (B2M) pseudoFab (pFab) to bind to RSV-F glycoprotein. In the absence of engineered salt bridges or disulfide bonds, the EA3 / B2M pseudoFabs HLPPB117 and HLPPB270 show weaker binding to RSV-F glycoprotein than the B23B173 native Fab control. A wild-type EA3 / B2M dimer lacking the anti-RSV-F variable domain was used as a negative control. [Figure 6A] Figure 6 shows intact LC-MS molecular weight analysis of HLA-E A3 (EA3) / β2 microglobulin (B2M) pseudo-Fab HLPPB17 and a native Fab control (B23B173). Figure 6A shows the presence of the individual light and heavy chain arms of the EA3 / B2M pFab. Figure 6B shows the presence of intact B23B173. [Figure 6B] Figure 6 shows intact LC-MS molecular weight analysis of HLA-E A3 (EA3) / β2 microglobulin (B2M) pseudo-Fab HLPPB17 and a native Fab control (B23B173). Figure 6A shows the presence of the individual light and heavy chain arms of the EA3 / B2M pFab. Figure 6B shows the presence of intact B23B173. [Figure 7A]Figure 7 shows a comparison of expression and solubility levels of engineered pseudo-Fabs (pFabs) containing electrostatic mutations present in the HLA-E A3 (EA3) / β2 microglobulin (B2M) domain by reducing SDS-PAGE. Figure 7A shows results for pFabs HLPPW10, HLPPW13, HLPPW14, HLPPW15, HLPPW16, HLPPW17, and HLPPW18. Figure 7B shows results for pFabs HLPPW19, HLPPW20, HLPPW21, HLPPW22, HLPPW23, HLPPW24, HLPPW25, and HLPPW26. Figure 7C shows results for pFabs HLPPW27, HLPPW28, HLPPW29, HLPPW30, HLPPW32, HLPPW33, and HLPPW34. Figure 7D shows the results for pFabs HLPPW35, HLPPW36, HLPPW37, HLPPW38, HLPPW40, and HLPPW41. In Figures 7A-7D, lane S is undiluted CHO supernatant and lane P is the resuspended CHO pellet fraction. [Figure 7B] Figure 7 shows a comparison of expression and solubility levels of engineered pseudo-Fabs (pFabs) containing electrostatic mutations present in the HLA-E A3 (EA3) / β2 microglobulin (B2M) domain by reducing SDS-PAGE. Figure 7A shows results for pFabs HLPPW10, HLPPW13, HLPPW14, HLPPW15, HLPPW16, HLPPW17, and HLPPW18. Figure 7B shows results for pFabs HLPPW19, HLPPW20, HLPPW21, HLPPW22, HLPPW23, HLPPW24, HLPPW25, and HLPPW26. Figure 7C shows results for pFabs HLPPW27, HLPPW28, HLPPW29, HLPPW30, HLPPW32, HLPPW33, and HLPPW34. Figure 7D shows the results for pFabs HLPPW35, HLPPW36, HLPPW37, HLPPW38, HLPPW40, and HLPPW41. In Figures 7A-7D, lane S is undiluted CHO supernatant and lane P is the resuspended CHO pellet fraction. [Figure 7C]Figure 7 shows a comparison of expression and solubility levels of engineered pseudo-Fabs (pFabs) containing electrostatic mutations present in the HLA-E A3 (EA3) / β2 microglobulin (B2M) domain by reducing SDS-PAGE. Figure 7A shows results for pFabs HLPPW10, HLPPW13, HLPPW14, HLPPW15, HLPPW16, HLPPW17, and HLPPW18. Figure 7B shows results for pFabs HLPPW19, HLPPW20, HLPPW21, HLPPW22, HLPPW23, HLPPW24, HLPPW25, and HLPPW26. Figure 7C shows results for pFabs HLPPW27, HLPPW28, HLPPW29, HLPPW30, HLPPW32, HLPPW33, and HLPPW34. Figure 7D shows the results for pFabs HLPPW35, HLPPW36, HLPPW37, HLPPW38, HLPPW40, and HLPPW41. In Figures 7A-7D, lane S is undiluted CHO supernatant and lane P is the resuspended CHO pellet fraction. [Figure 7D] Figure 7 shows a comparison of expression and solubility levels of engineered pseudo-Fabs (pFabs) containing electrostatic mutations present in the HLA-E A3 (EA3) / β2 microglobulin (B2M) domain by reducing SDS-PAGE. Figure 7A shows results for pFabs HLPPW10, HLPPW13, HLPPW14, HLPPW15, HLPPW16, HLPPW17, and HLPPW18. Figure 7B shows results for pFabs HLPPW19, HLPPW20, HLPPW21, HLPPW22, HLPPW23, HLPPW24, HLPPW25, and HLPPW26. Figure 7C shows results for pFabs HLPPW27, HLPPW28, HLPPW29, HLPPW30, HLPPW32, HLPPW33, and HLPPW34. Figure 7D shows the results for pFabs HLPPW35, HLPPW36, HLPPW37, HLPPW38, HLPPW40, and HLPPW41. In Figures 7A-7D, lane S is undiluted CHO supernatant and lane P is the resuspended CHO pellet fraction. [Figure 8A]Figure 8 shows a comparison of the expression levels of engineered HLA-E A3 (EA3) / β2 microglobulin (B2M) pseudoFabs (pFabs) HLPPW42, HLPPW43, HLPPW54, HLPPW55, HLPPW56, HLPPW57, HLPPW48, HLPPW49, HLPPW50, HLPPW51, HLPPW52, HLPPW53, HLPPW42, HLPPB17, and HLPPB270 to the normal Fab HLPPW58, and analysis of disulfide bond integrity by SDS-PAGE. Figure 8A shows the expression levels detected by luminescence using anti-HIS capture and anti-HA / anti-Strep II detection. Figures 8B-8D show the results of reducing (R) and non-reducing (N) SDS-PAGE. In Figures 8B-8D, lane R represents the resuspended CHO pellet fraction. [Figure 8B] Figure 8 shows a comparison of the expression levels of engineered HLA-E A3 (EA3) / β2 microglobulin (B2M) pseudoFabs (pFabs) HLPPW42, HLPPW43, HLPPW54, HLPPW55, HLPPW56, HLPPW57, HLPPW48, HLPPW49, HLPPW50, HLPPW51, HLPPW52, HLPPW53, HLPPW42, HLPPB17, and HLPPB270 to the normal Fab HLPPW58, and analysis of disulfide bond integrity by SDS-PAGE. Figure 8A shows the expression levels detected by luminescence using anti-HIS capture and anti-HA / anti-Strep II detection. Figures 8B-8D show the results of reducing (R) and non-reducing (N) SDS-PAGE. In Figures 8B-8D, lane R represents the resuspended CHO pellet fraction. [Figure 8C]Figure 8 shows a comparison of the expression levels of engineered HLA-E A3 (EA3) / β2 microglobulin (B2M) pseudoFabs (pFabs) HLPPW42, HLPPW43, HLPPW54, HLPPW55, HLPPW56, HLPPW57, HLPPW48, HLPPW49, HLPPW50, HLPPW51, HLPPW52, HLPPW53, HLPPW42, HLPPB17, and HLPPB270 to the normal Fab HLPPW58, and analysis of disulfide bond integrity by SDS-PAGE. Figure 8A shows the expression levels detected by luminescence using anti-HIS capture and anti-HA / anti-Strep II detection. Figures 8B-8D show the results of reducing (R) and non-reducing (N) SDS-PAGE. In Figures 8B-8D, lane R represents the resuspended CHO pellet fraction. [Figure 8D] Figure 8 shows a comparison of the expression levels of engineered HLA-E A3 (EA3) / β2 microglobulin (B2M) pseudoFabs (pFabs) HLPPW42, HLPPW43, HLPPW54, HLPPW55, HLPPW56, HLPPW57, HLPPW48, HLPPW49, HLPPW50, HLPPW51, HLPPW52, HLPPW53, HLPPW42, HLPPB17, and HLPPB270 to the normal Fab HLPPW58, and analysis of disulfide bond integrity by SDS-PAGE. Figure 8A shows the expression levels detected by luminescence using anti-HIS capture and anti-HA / anti-Strep II detection. Figures 8B-8D show the results of reducing (R) and non-reducing (N) SDS-PAGE. In Figures 8B-8D, lane R represents the resuspended CHO pellet fraction. [Figure 9A]Figure 9A shows ELISA analysis of the ability of engineered HLA-EA3 (EA3) / β2 microglobulin (B2M) pseudo-Fabs (pFabs) derived from undiluted CHO supernatants to bind to RSV-F glycoprotein. Figure 9A shows the luminescence signals of HLPPW42, HLPPW43, HLPPW54, HLPPW55, HLPPW56, HLPPW57, HLPPW48, HLPPW49, HLPPW50, HLPPW51, HLPPW52, HLPPW53, HLPPW42, HLPPB17, and HLPPB270 compared to normal Fab HLPPW58. Figure 9B shows the calculated EC50 and 95% confidence interval values. [Figure 9B] Figure 9A shows ELISA analysis of the ability of engineered HLA-EA3 (EA3) / β2 microglobulin (B2M) pseudo-Fabs (pFabs) derived from undiluted CHO supernatants to bind to RSV-F glycoprotein. Figure 9A shows the luminescence signals of HLPPW42, HLPPW43, HLPPW54, HLPPW55, HLPPW56, HLPPW57, HLPPW48, HLPPW49, HLPPW50, HLPPW51, HLPPW52, HLPPW53, HLPPW42, HLPPB17, and HLPPB270 compared to normal Fab HLPPW58. Figure 9B shows the calculated EC50 and 95% confidence interval values. [Figure 10A] Figure 10 shows the binding of purified disulfide-engineered HLA-E A3 (EA3) / β2 microglobulin (B2M) pseudo-Fab (pFab) to RSV glycoprotein. Figure 10A shows the luminescence signals of HLPPW42, HLPPW43, HLPPW54, HLPPW55, HLPPW56, HLPPW57, HLPPW48, HLPPW49, HLPPW50, HLPPW51, HLPPW52, and HLPPW53 compared to normal Fab HLPPW58. Figure 10B shows the calculated EC50 and maximum signal values. [Figure 10B]Figure 10 shows the binding of purified disulfide-engineered HLA-E A3 (EA3) / β2 microglobulin (B2M) pseudo-Fab (pFab) to RSV glycoprotein. Figure 10A shows the luminescence signals of HLPPW42, HLPPW43, HLPPW54, HLPPW55, HLPPW56, HLPPW57, HLPPW48, HLPPW49, HLPPW50, HLPPW51, HLPPW52, and HLPPW53 compared to normal Fab HLPPW58. Figure 10B shows the calculated EC50 and maximum signal values. [Figure 11A] Size-exclusion chromatography (SEC) profiles of HLA-E A3 (EA3) / β2 microglobulin (B2M) disulfide variants are shown. Results are shown for HLPPB17 (FIG. 11A), HLPPW57 (FIG. 11B), HLPPW49 (FIG. 11C), and HLPPW53 (FIG. 11D), which appear as monodisperse species. Gel filtration standards are overlaid with dashed lines. Signals are shown relative to the maximum signal for each sample. [Figure 11B] Size-exclusion chromatography (SEC) profiles of HLA-E A3 (EA3) / β2 microglobulin (B2M) disulfide variants are shown. Results are shown for HLPPB17 (FIG. 11A), HLPPW57 (FIG. 11B), HLPPW49 (FIG. 11C), and HLPPW53 (FIG. 11D), which appear as monodisperse species. Gel filtration standards are overlaid with dashed lines. Signals are shown relative to the maximum signal for each sample. [Figure 11C] Size-exclusion chromatography (SEC) profiles of HLA-E A3 (EA3) / β2 microglobulin (B2M) disulfide variants are shown. Results are shown for HLPPB17 (FIG. 11A), HLPPW57 (FIG. 11B), HLPPW49 (FIG. 11C), and HLPPW53 (FIG. 11D), which appear as monodisperse species. Gel filtration standards are overlaid with dashed lines. Signals are shown relative to the maximum signal for each sample. [Figure 11D]Size-exclusion chromatography (SEC) profiles of HLA-E A3 (EA3) / β2 microglobulin (B2M) disulfide variants are shown. Results are shown for HLPPB17 (FIG. 11A), HLPPW57 (FIG. 11B), HLPPW49 (FIG. 11C), and HLPPW53 (FIG. 11D), which appear as monodisperse species. Gel filtration standards are overlaid with dashed lines. Signals are shown relative to the maximum signal for each sample. [Figure 12A] 12A and 12B show mass spectrometry analysis of HLA-E A3 (EA3) / β2 microglobulin (B2M) disulfide variants. Results are shown for HLPPB17 (FIG. 12A), HLPPW57 (FIG. 12B), HLPPW49 (FIG. 12C), and HLPPW53 (FIG. 12D). [Figure 12B] 12A and 12B show mass spectrometry analysis of HLA-E A3 (EA3) / β2 microglobulin (B2M) disulfide variants. Results are shown for HLPPB17 (FIG. 12A), HLPPW57 (FIG. 12B), HLPPW49 (FIG. 12C), and HLPPW53 (FIG. 12D). [Figure 12C] 12A and 12B show mass spectrometry analysis of HLA-E A3 (EA3) / β2 microglobulin (B2M) disulfide variants. Results are shown for HLPPB17 (FIG. 12A), HLPPW57 (FIG. 12B), HLPPW49 (FIG. 12C), and HLPPW53 (FIG. 12D). [Figure 12D] 12A and 12B show mass spectrometry analysis of HLA-E A3 (EA3) / β2 microglobulin (B2M) disulfide variants. Results are shown for HLPPB17 (FIG. 12A), HLPPW57 (FIG. 12B), HLPPW49 (FIG. 12C), and HLPPW53 (FIG. 12D). [Figure 13A] Figure 13 shows stability analysis by thermal melting using nanoscale differential scanning fluorimetry (NanoDSF) measurements. Figure 13A shows the melting curve and first derivative. Figure 13B shows the onset of melting (Ton), melting temperature 1 (Tm1), melting temperature 2 (Tm2), melting temperature 3 (Tm3), and aggregation onset temperature (Tagg). Two unique disulfide "pin" pairs were identified with stability comparable to that of the Fab control. [Figure 13B] Figure 13 shows stability analysis by thermal melting using nanoscale differential scanning fluorimetry (NanoDSF) measurements. Figure 13A shows the melting curve and first derivative. Figure 13B shows the onset of melting (Ton), melting temperature 1 (Tm1), melting temperature 2 (Tm2), melting temperature 3 (Tm3), and aggregation onset temperature (Tagg). Two unique disulfide "pin" pairs were identified with stability comparable to that of the Fab control. [Figure 14] Non-reducing PAGE of HLA-E A3 (EA3) / β2 microglobulin (B2M) pseudo-Fab variants HLPPW59, HLPPW60, HLPPW61, HLPPW62, HLPPW63, HLPPW64, HLPPW65, HLPPW66, HLPPW67, HLPPW68, HLPPW69, and HLPPW70 derived from HLPPW 57 is shown. [Figure 15A] Size-exclusion chromatography (SEC) profiles of selected HLA-E A3 (EA3) / β2 microglobulin (B2M) elbow variants are shown. Figures 15A-15C show the results for CHLPPW59, HLPPW60, and HLPPW63, which are shown as monodisperse species. Gel filtration standards are overlaid in light gray. Signals are shown relative to the maximum signal for each sample. [Figure 15B] Size-exclusion chromatography (SEC) profiles of selected HLA-E A3 (EA3) / β2 microglobulin (B2M) elbow variants are shown. Figures 15A-15C show the results for CHLPPW59, HLPPW60, and HLPPW63, which are shown as monodisperse species. Gel filtration standards are overlaid in light gray. Signals are shown relative to the maximum signal for each sample. [Figure 15C] Size-exclusion chromatography (SEC) profiles of selected HLA-E A3 (EA3) / β2 microglobulin (B2M) elbow variants are shown. Figures 15A-15C show the results for CHLPPW59, HLPPW60, and HLPPW63, which are shown as monodisperse species. Gel filtration standards are overlaid in light gray. Signals are shown relative to the maximum signal for each sample. [Figure 16] Figure 1 shows ELISA binding of purified disulfide-engineered HLA-E A3 (EA3) / β2 microglobulin (B2M) pseudo-Fab to RSV glycoproteins. The luminescence signals of HLPPW59, HLPPW60, HLPPW61, HLPPW62, HLPPW63, HLPPW64, HLPPW65, HLPPW66, HLPPW67, HLPPW68, HLPPW69, and HLPPW70 are shown compared to HLPPB271 and HLPPW58. [Figure 17-1] Figure 17 shows stability analysis by thermal melting using nanoscale differential scanning fluorimetry (NanoDSF) measurements to assess the effect on stability, identifying two unique disulfide "pin" pairs of interest with comparable stability to the Fab control. Figure 17A shows the melting curves of HLPPW60, HLPPW61, HLPPW62, HLPPW63, HLPPW64, HLPPW65, HLPPW66, HLPPW67, HLPPW68, HLPPW69, and HLPPW70, and Figure 17B shows their first derivatives. Figure 17C shows the melting curves of HLPPB271 and HLPPW58, and Figure 17D shows their first derivatives. [Figure 17-2] Figure 17 shows stability analysis by thermal melting using nanoscale differential scanning fluorimetry (NanoDSF) measurements to assess the effect on stability, identifying two unique disulfide "pin" pairs of interest with comparable stability to the Fab control. Figure 17A shows the melting curves of HLPPW60, HLPPW61, HLPPW62, HLPPW63, HLPPW64, HLPPW65, HLPPW66, HLPPW67, HLPPW68, HLPPW69, and HLPPW70, and Figure 17B shows their first derivatives. Figure 17C shows the melting curves of HLPPB271 and HLPPW58, and Figure 17D shows their first derivatives. [Figure 18A]Comparison of purified ICAM-1 D1 / ICAM-1 D1 dimers to identify the presence of both chains of the heterodimer is shown. Western blots were performed with purified ICAM-1 D1 / ICAM-1 D1 dimers HLPPB5, HLPPB6, HLPPB9, and HLPPB10. HLPPB17 and B23B173 were used as HLA-E A3 (EA3) / β2 microglobulin (B2M) heterodimer and native Fab controls, respectively. [Figure 18B] Comparison of purified ICAM-1 D1 / ICAM-1 D1 dimers to identify the presence of both chains of the heterodimer is shown. Western blots were performed with purified ICAM-1 D1 / ICAM-1 D1 dimers HLPPB5, HLPPB6, HLPPB9, and HLPPB10. HLPPB17 and B23B173 were used as HLA-E A3 (EA3) / β2 microglobulin (B2M) heterodimer and native Fab controls, respectively. [Figure 19A] 1 shows intact LC-MS molecular weight analysis of ICAM-1 D1 / ICAM-1 D1 pseudo-Fab variants HLPPB5, HLPPB6, HLPPB9, and HLPPB10. [Figure 19B] 1 shows intact LC-MS molecular weight analysis of ICAM-1 D1 / ICAM-1 D1 pseudo-Fab variants HLPPB5, HLPPB6, HLPPB9, and HLPPB10. [Figure 19C] 1 shows intact LC-MS molecular weight analysis of ICAM-1 D1 / ICAM-1 D1 pseudo-Fab variants HLPPB5, HLPPB6, HLPPB9, and HLPPB10. [Figure 19D] 1 shows intact LC-MS molecular weight analysis of ICAM-1 D1 / ICAM-1 D1 pseudo-Fab variants HLPPB5, HLPPB6, HLPPB9, and HLPPB10. [Figure 20] 1 shows ELISA analysis of the ability of engineered ICAM-1 D1 / ICAM-1 D1 pseudo-Fabs to bind to undiluted CHO supernatant-derived RSV-F glycoprotein. Results are shown for HLPPB6 and HLPPB10 compared to the B23B173 native Fab control. [Figure 21A]Figure 21 shows stability analysis by thermal melting using nanoscale differential scanning fluorimetry (NanoDSF) measurements for bispecific antibodies HLPPB421, HLPPB423, HLPPB425, and HLPPB426. Figure 21A shows the melting curves and first derivatives. Figure 21B shows the onset of melting (Ton), melting temperature 1 (Tm1), melting temperature 2 (Tm2), melting temperature 3 (Tm3), and aggregation onset temperature (Tagg). [Figure 21B] Figure 21 shows stability analysis by thermal melting using nanoscale differential scanning fluorimetry (NanoDSF) measurements for bispecific antibodies HLPPB421, HLPPB423, HLPPB425, and HLPPB426. Figure 21A shows the melting curves and first derivatives. Figure 21B shows the onset of melting (Ton), melting temperature 1 (Tm1), melting temperature 2 (Tm2), melting temperature 3 (Tm3), and aggregation onset temperature (Tagg). [Figure 22] Non-reducing (NR) and reducing (R) PAGE of purified bispecific antibodies HLPPB423, HLPPB425, HLPPB421, and HLPPB426, one arm consisting of HLPPW 59-derived pseudo-Fab and the other arm consisting of standard IgG Fab, are shown. All samples were normalized by total protein A280 and loaded at equal amounts. The ladder is represented by (L) and molecular weights are given in kD. [Figure 23] Figure 1 shows size exclusion chromatography (SEC) profiles of purified bispecific antibodies HLPPB423, HLPPB425, HLPPB421, and HLPPB426, where one arm consists of a pseudo-Fab derived from HLPPW59 and the other arm consists of a standard IgG Fab. Gel filtration standards are overlaid in light grey. Signals are shown relative to the maximum signal for each sample. [Figure 24A]Biolayer interferometry (BLI) binding profiles of purified bispecific antibodies are shown. Figure 24A shows the BLI binding profiles of human epidermal growth factor receptor 2 (HER2)-targeting antibodies HLPPB423, HLPPB425, HLPPB421, and HLPPB426 to HER2. Figure 24B shows the BLI binding profiles of mesenchymal-epithelial transition (MET)-targeting antibodies HLPPB423 and HLPPB425 to MET. Figure 24C shows the BLI binding profiles of cluster of differentiation 3 (CD3)-targeting antibodies HLPPB421 and HLPPB426 to CD3. [Figure 24B] Biolayer interferometry (BLI) binding profiles of purified bispecific antibodies are shown. Figure 24A shows the BLI binding profiles of human epidermal growth factor receptor 2 (HER2)-targeting antibodies HLPPB423, HLPPB425, HLPPB421, and HLPPB426 to HER2. Figure 24B shows the BLI binding profiles of mesenchymal-epithelial transition (MET)-targeting antibodies HLPPB423 and HLPPB425 to MET. Figure 24C shows the BLI binding profiles of cluster of differentiation 3 (CD3)-targeting antibodies HLPPB421 and HLPPB426 to CD3. [Figure 24C] Biolayer interferometry (BLI) binding profiles of purified bispecific antibodies are shown. Figure 24A shows the BLI binding profiles of human epidermal growth factor receptor 2 (HER2)-targeting antibodies HLPPB423, HLPPB425, HLPPB421, and HLPPB426 to HER2. Figure 24B shows the BLI binding profiles of mesenchymal-epithelial transition (MET)-targeting antibodies HLPPB423 and HLPPB425 to MET. Figure 24C shows the BLI binding profiles of cluster of differentiation 3 (CD3)-targeting antibodies HLPPB421 and HLPPB426 to CD3. DETAILED DESCRIPTION OF THE INVENTION

[0058] Provided herein are antigen-binding molecules comprising one or more polypeptides (e.g., antigen-binding polypeptides) having a variable domain that binds to a target antigen, and having a dimerization domain described herein instead of the light chain constant domain (CL) or heavy chain constant domain 1 (CH1). In certain embodiments, the antigen-binding molecule binds to one or more target antigens. In certain embodiments, the antigen-binding molecule comprises two or more polypeptides, wherein the polypeptides form a paratope that binds to one or more target antigens. In certain embodiments, each of a pair of polypeptides comprises a variable domain and a dimerization domain, wherein the variable domains form a paratope that binds to the target antigen.

[0059] 7.1 Definition The disclosed methods may be understood more readily by reference to the following detailed description taken in conjunction with the accompanying drawings, which form a part of this disclosure: It is to be understood that the disclosed methods are not limited to the specific methods described and / or illustrated herein, and further, the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting.

[0060] All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein.

[0061] Where lists are presented, unless otherwise stated, it is to be understood that each individual element of that list and every combination of that list is a separate embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0062] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0063] The transitional phrases "comprising," "consisting essentially of," and "consisting" are intended to connote their generally accepted meanings in patent language, i.e., (i) "comprising" is synonymous with "comprising," "containing," or "characterized by" and is inclusive or open-ended, not excluding additional, unrecited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not "materially affect the basic and novel characteristics" of the claimed invention. Embodiments described with the phrase "comprising" (or its equivalents) also provide as embodiments embodiments described independently with "consisting of" and "consisting essentially of."

[0064] "About" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless expressly stated otherwise in the Examples or elsewhere herein, "about" means within 1 standard deviation, or up to 5%, whichever is greater, per practice in the art.

[0065] "Antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC (antibody-dependent cell-mediated cytotoxicity)" refers to a mechanism of cell death induction that relies on the interaction of antibody-coated target cells with lytic effector cells, such as natural killer cells (NK), monocytes, macrophages, and neutrophils, via Fc gamma receptors (FcγR) expressed on the effector cells.

[0066] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells are eliminated by internalization by phagocytic cells such as macrophages or dendritic cells.

[0067] "Antigen" refers to any molecule (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portion thereof, or combination thereof) that can mediate an immune response. Exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells.

[0068] An "antigen-binding fragment" or "antigen-binding domain" refers to a portion of a protein that binds to an antigen. Antigen-binding fragments may be synthetic, enzymatically obtainable, or genetically engineered polypeptides, and include VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd, and Fv fragments, domain antibodies (dAbs) consisting of one VH domain or one VL domain, camelized VH domains, VHH domains, minimal recognition units consisting of amino acid residues mimicking the CDRs of an antibody, such as FR3-CDR3-FR4 portions, portions of immunoglobulins bound to HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3, alternative scaffolds bound to antigens, and multispecific proteins containing antigen-bound fragments. Antigen-binding fragments (such as VH and VL) can be linked together via synthetic linkers to form various types of single-chain antibody designs in which the VH / VL domains pair intramolecularly, or intermolecularly when the VH and VL domains are expressed as separate single chains, to form monovalent antigen-binding domains such as single-chain Fvs (scFvs) or antigens, or bispecific antibodies. Antigen-binding fragments can also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which can be monospecific or multispecific, to genetically engineer bispecific and multispecific proteins.

[0069] The term "antibody" has a broad meaning and includes immunoglobulin molecules, including monoclonal antibodies, including murine, human, humanized, and chimeric monoclonal antibodies; antigen-binding fragments; multispecific antibodies, such as bispecific, trispecific, and tetraspecific antibodies; dimeric, tetrameric, or multimeric antibodies; single-chain antibodies; domain antibodies; and any other modified form of an immunoglobulin molecule containing an antigen-binding site of the required specificity. A "full-length antibody" is composed of two heavy chains (HC) and two light chains (LC), inter-connected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL is composed of three CDR and four FR segments, arranged from amino- to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes, namely, IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy-chain constant domain. IgA and IgG are further subdivided into isotypes, IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0070] "Bispecific" refers to a molecule (such as an antibody) that specifically binds to two different antigens, or two different epitopes within the same antigen. Bispecific molecules may be cross-reactive to other related antigens, e.g., the same antigen (homologues), from other species such as humans or monkeys, e.g., cynomolgus monkeys (Macaca cynomolgus, cyno) or chimpanzees (Pan troglodytes), or may bind to epitopes shared between two or more different antigens.

[0071] "Complement-dependent cytotoxicity" or "CDC" refers to a cell death-inducing mechanism in which the Fc effector domain of a target-bound protein binds to and activates complement component C1q, which in turn activates the complement cascade, resulting in target cell death. Complement activation can also result in the deposition of complement components on the target cell surface, which facilitates CDC through the binding of complement receptors (e.g., CR3) to leukocytes.

[0072] The "complementarity-determining region" (CDR) is the region of an antibody that binds to an antigen. There are three CDRs in VH (HCDR1, HCDR2, and HCDR3), and there are three CDRs in VL (LCDR1, LCDR2, and LCDR3). CDRs can be defined using various descriptions, such as Kabat (Wu et al., (1970) J Exp Med 132:211-250; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al., (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton (1996) J Bmol Biol 263:800-815). The correspondence between various descriptions and the numbering of variable regions has been described (see, for example, Lefranc et al. (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-670; the International ImMunoGeneTics (IMGT) database; web resource, http: / / www_imgt_org). CDRs can be described using available programs such as abYsis by UCL Business PLC. As used herein, the terms "CDR," "HCDR1," "HCDR2," "HCDR3," "LCDR1," "LCDR2," and "LCDR3" include CDRs defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless otherwise expressly stated herein.

[0073] "Decrease," "lower," or "reduce" generally refers to the ability of a test molecule to mediate a diminished response (i.e., a downstream effect) when compared to a response mediated by a control or vehicle. Exemplary responses include binding of a protein to its antigen or receptor, enhanced binding to FcγRs or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. A reduction can be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or about a 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30-fold or greater reduction, e.g., a 500-, 600-, 700-, 800-, 900-, or 1000-fold or greater reduction.

[0074] "Enhance," "promote," or "increase" generally refer to the ability of a test molecule to mediate a greater response (i.e., a downstream effect) when compared to a control or vehicle-mediated response. Exemplary responses are binding of a protein to its antigen or receptor, enhanced binding to FcγR, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. Enhancement can be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 fold or more, e.g., a 500-, 600-, 700-, 800-, 900-, or 1000-fold or more.

[0075] An "expression vector" refers to a vector that can be utilized in a biological system or reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0076] "Heterologous" refers to a polypeptide or polynucleotide that comprises two or more polypeptides or two or more polynucleotides that are not found in the same relationship to each other in nature.

[0077] A "heterologous polynucleotide" refers to a polynucleotide that comprises two or more polynucleotides that are not found in the same relationship to each other in nature.

[0078] A "heterologous polypeptide" refers to a polypeptide that comprises two or more polypeptides that are not found in the same relationship to each other in nature.

[0079] A "human antibody" refers to an antibody optimized to minimize an immune response when administered to a human subject. The variable regions of a human antibody are derived from human immunoglobulin sequences. If a human antibody contains a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. A human antibody contains heavy and light chain variable regions "derived" from sequences of human origin when the variable regions of the human antibody are obtained from a system using human germline immunoglobulins or rearranged immunoglobulin genes. Exemplary such systems are phage-displayed human immunoglobulin gene libraries and transgenic non-human animals, such as mice or rats, carrying human immunoglobulin loci. A "human antibody" typically contains amino acid differences compared to immunoglobulins expressed in humans due to differences in the systems used to obtain human antibodies and human immunoglobulin loci, the introduction of somatic mutations or intentional substitutions into frameworks or CDRs, or both. Typically, a "human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by human germline immunoglobulin or rearranged immunoglobulin genes. Optionally, a "human antibody" can comprise a consensus framework sequence obtained from human framework sequence analysis as described, for example, in Knappik et al., (2000) J Mol Biol 296:57-86, or a synthetic HCDR3 incorporated into a library of human immunoglobulin genes displayed on phage as described, for example, in Shi et al., (2010) J Mol Biol 397:385-396 and WO 2009 / 085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of "human antibody."

[0080] A "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Humanized antibodies can contain substitutions in the framework, so that the framework may not be an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0081] "Modulate" refers to either an enhanced or decreased ability of the test molecule to mediate a greater or lesser response (i.e., a downstream effect) when compared to a control or vehicle-mediated response.

[0082] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules, i.e., a population comprising individual antibodies is identical except for possible well-known alterations such as removal of the C-terminal lysine from the antibody heavy chain, or post-translational modifications such as amino acid isomerization or deamidation, oxidation of methionine, or deamidation of asparagine or glutamine. Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, such as bispecific, and may be monovalent, bivalent, or multivalent.

[0083] "Multispecific" refers to a molecule that binds to two or more different antigens, or to two or more different epitopes within the same antigen. Multispecific molecules may be cross-reactive to other related antigens, e.g., the same antigen (homologues) from other species such as humans or monkeys, e.g., cynomolgus monkeys (Macaca fascicularis) (cynomolgus, cyno) or chimpanzees (Pan troglodytes), or may bind to an epitope shared between two or more different antigens.

[0084] "Polynucleotide" refers to a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.

[0085] As used interchangeably herein, "protein" or "polypeptide" refers to a molecule comprising one or more polypeptides, each composed of at least two amino acid residues linked by a peptide bond. A protein may be a monomer or a protein complex of two or more subunits, which may be identical or different. Small polypeptides consisting of fewer than 50 amino acids may be referred to as "peptides." A protein may be a heterologous fusion protein, a glycoprotein, or a protein modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation, or biotinylation.

[0086] "Recombinant" refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules that are prepared, expressed, created, or isolated by recombinant means.

[0087] "Specifically binds," "specific binding," "specifically binding," or "binds" refers to a protein binding to an antigen or an epitope within an antigen with higher affinity than its affinity for other antigens. Typically, a protein, such as an antigen-binding protein described herein, binds to an antigen with an affinity of about 1 x 10 -6 M or less, approximately 1×10 -7 Below, about 5×10 -8 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 or less, or about 1 x 10 -12 The equilibrium dissociation constant (K D ) binds to an antigen or an epitope within an antigen, typically Dis the K for binding to nonspecific antigens (e.g., BSA, casein) D is at least 100 times smaller than

[0088] A "subject" includes any human or non-human animal. A "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "subject" and "patient" may be used interchangeably herein.

[0089] A "therapeutically effective amount" refers to an amount effective to obtain a desired therapeutic result, at dosages and for periods of time necessary. A therapeutically effective amount may vary depending on factors such as the individual's medical condition, age, sex, and weight, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual.

[0090] "Treating," "treating," or "treatment" of a disease or disorder refers to achieving one or more of the following: reducing the severity and / or duration of the disorder, inhibiting the worsening of symptoms characteristic of the disorder being treated, limiting or preventing the recurrence of the disorder in a subject who previously had the disorder, or limiting or preventing the recurrence of symptoms in a subject who previously had symptoms of the disorder.

[0091] "Trispecific" refers to a molecule (such as an antibody) that specifically binds to three different antigens, or three different epitopes, within the same antigen. Bispecific molecules may also be cross-reactive to other related antigens, e.g., the same antigen (homologues), from other species such as humans or monkeys, e.g., cynomolgus monkeys (Macaca cynomolgus, cyno) or chimpanzees (Pan troglodytes), or may bind to epitopes shared among three or more different antigens.

[0092] "Variant," "mutant," or "altered" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, e.g., one or more substitutions, insertions, or deletions.

[0093] Throughout this specification, the numbering of amino acid residues in antibody constant regions is according to the EU index as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise expressly stated herein.

[0094] 7.2 Antigen-binding polypeptides In certain embodiments, an antigen-binding molecule comprises a polypeptide that forms a paratope that binds to one or more target antigens. In certain embodiments, the polypeptide of the antigen-binding molecule is an antigen-binding polypeptide. In certain embodiments, the polypeptide of the antigen-binding molecule comprises a variable domain that binds to the target antigen. In certain embodiments, the polypeptide of the antigen-binding molecule comprises at least one dimerization domain described herein. In certain embodiments, at least one dimerization domain binds to another dimerization domain of an antigen-binding polypeptide described herein to form a dimer. In a preferred embodiment, the polypeptide of the antigen-binding molecule comprises a variable domain and at least one dimerization domain.

[0095] In certain embodiments, the antigen-binding molecule comprises 2, 3, 4, 5, 6, 7, or 8 polypeptides. In certain embodiments, each of the 2, 3, 4, 5, 6, 7, or 8 polypeptides comprises a variable domain that binds to a target antigen. In certain embodiments, each of the 2, 3, 4, 5, 6, 7, or 8 polypeptides comprises at least one dimerization domain. In certain embodiments, each of the 2, 3, 4, 5, 6, 7, or 8 polypeptides comprises a variable domain that binds to a target antigen and at least one dimerization domain. In certain embodiments, a dimerization domain of an antigen-binding polypeptide binds to another dimerization domain of another antigen-binding polypeptide to form a dimer (particularly a heterodimer; e.g., 6 polypeptides can form up to 3 dimers with each other). In a preferred embodiment, the antigen-binding molecule comprises two polypeptides, each comprising a variable domain and at least one dimerization domain, wherein the variable domains form a variable region that binds to a target antigen, and the dimerization domains bind to each other to form a dimer, preferably a heterodimer. In a preferred embodiment, the antigen-binding molecule comprises four polypeptides, each comprising a variable domain and at least one dimerization domain, wherein the variable domains form two variable regions that each bind to a target antigen, and the dimerization domains bind to each other to form two dimers. In a preferred embodiment, the antigen-binding molecule comprises six polypeptides, each comprising a variable domain and at least one dimerization domain, wherein the variable domains form three variable regions that each bind to a target antigen, and the dimerization domains bind to each other to form three dimers.

[0096] In certain embodiments, the polypeptide of the antigen-binding molecule comprises a light chain polypeptide comprising a variable domain and a heavy chain polypeptide comprising a variable domain. In certain embodiments, the variable domains of the light and heavy chains form a variable region comprising a paratope that binds to a target antigen. In certain embodiments, the light chain polypeptide is a first light chain polypeptide (LC1), a second light chain polypeptide (LC2), a third light chain polypeptide (LC3), a fourth light chain polypeptide (LC4), or an additional light chain polypeptide (e.g., LC5+) of the antigen-binding molecule. In certain embodiments, the heavy chain polypeptide is a first heavy chain polypeptide (HC1), a second heavy chain polypeptide (HC2), a third heavy chain polypeptide (HC3), a fourth heavy chain polypeptide (HC4), or an additional heavy chain polypeptide (e.g., HC5+) of the antigen-binding molecule.

[0097] In certain embodiments, the light chain polypeptide comprises an immunoglobulin or antibody light chain, or one or more fragments thereof. In certain embodiments, the light chain polypeptide comprises an immunoglobulin or antibody kappa (κ), lambda (λ), sigma (σ), or iota (ι) chain, or one or more fragments thereof. In certain embodiments, the light chain polypeptide is an intact immunoglobulin light chain comprising a light chain variable domain (VL) and a light chain constant domain (CL). In preferred embodiments, the light chain constant domain is completely or partially replaced with a dimerization domain described herein. In certain embodiments, the light chain polypeptide may, but need not, comprise an immunoglobulin light chain constant region or a fragment thereof. In certain embodiments, the light chain polypeptide is not an intact immunoglobulin light chain. In certain embodiments, the light chain polypeptide does not comprise the constant domain (CL) of an immunoglobulin light chain or a fragment thereof. In certain embodiments, the light chain polypeptide does not comprise the dimerization sequence of the light chain constant domain. In certain embodiments, the dimerization sequence of the light chain constant domain binds to the heavy chain constant domain to form a dimer. In certain embodiments, the dimerization sequence of the light chain constant domain mediates dimerization between the light chain and the heavy chain. In certain embodiments, the light chain polypeptide comprises a fragment of the light chain constant domain (CL). In certain embodiments, the light chain polypeptide comprises 1 to 8 contiguous amino acids selected from amino acid positions 108-115 of the human immunoglobulin kappa constant domain according to EU numbering. In certain embodiments, the light chain polypeptide comprises 1 to 8 contiguous amino acids selected from amino acid positions 108-115 of the human immunoglobulin kappa constant domain according to Kabat numbering.

[0098] In certain embodiments, the heavy chain polypeptide comprises an immunoglobulin or antibody heavy chain, or one or more fragments thereof. In certain embodiments, the heavy chain polypeptide comprises an immunoglobulin or antibody gamma (γ), delta (δ), alpha (α), mu (μ), or epsilon (ε) chain, or one or more fragments thereof. In certain embodiments, the heavy chain polypeptide is an intact immunoglobulin heavy chain comprising a heavy chain variable domain (VH), heavy chain constant domain 1 (CH1), a hinge region, heavy chain constant domain 2 (CH2), and heavy chain constant domain 3 (CH3). In preferred embodiments, heavy chain constant domain 1 (CH1) is completely or partially replaced with a dimerization domain described herein. In certain embodiments, the heavy chain polypeptide may, but need not, contain immunoglobulin heavy chain constant domain 1 (CH1) or a fragment thereof. In certain embodiments, the heavy chain polypeptide may, but need not contain immunoglobulin heavy chain constant domain 2 (CH2) or a fragment thereof. In certain embodiments, a heavy chain polypeptide may, but need not, contain immunoglobulin heavy chain constant domain 3 (CH3) or a fragment thereof. In certain embodiments, a heavy chain polypeptide may, but need not contain immunoglobulin heavy chain constant domain 2 (CH2) or a fragment thereof and immunoglobulin heavy chain constant domain 3 (CH3). In certain embodiments, a heavy chain polypeptide is not an intact immunoglobulin heavy chain. In certain embodiments, a heavy chain polypeptide does not contain immunoglobulin heavy chain constant domain 1 (CH1) or a fragment thereof. In certain embodiments, a heavy chain polypeptide does not contain a dimerization sequence of heavy chain constant domain 1 (CH1). In certain embodiments, the dimerization sequence of heavy chain constant domain 1 (CH1) binds to a light chain constant domain (CL) to form a dimer. In certain embodiments, the dimerization sequence of the heavy chain constant domain mediates dimerization between the heavy chain and the light chain. In certain embodiments, a heavy chain polypeptide comprises a fragment of a heavy chain constant domain (CH1). In certain embodiments, the heavy chain polypeptide comprises 1 to 8 contiguous amino acids selected from amino acid positions 118 to 125 of human IgG1 according to EU numbering.In certain embodiments, the light chain polypeptide comprises 1 to 8 contiguous amino acids selected from amino acid positions 114 to 121 of human IgG1 according to the Kabat numbering system.

[0099] In certain embodiments, the light chain polypeptide is a first light chain polypeptide comprising, from amino- to carboxy-terminus, VL1-LD1, where VL1 is the first light chain variable domain and LD1 is the first light chain dimerization domain. In certain embodiments, the heavy chain polypeptide is a first heavy chain polypeptide comprising, from amino- to carboxy-terminus, VH1-HD1, where VH1 is the first heavy chain variable domain and HD1 is the first heavy chain dimerization domain. In certain embodiments, VL1 and VH1 form a first paratope that binds to a first target antigen. In certain embodiments, VL1 and VH1 form a first variable region that comprises a first paratope that binds to a first target antigen. In certain embodiments, LD1 comprises a β2-microglobulin (B2M) domain and HD1 comprises an HLA-E A3 (EA3) domain. In certain embodiments, LD1 comprises an HLA-E A3 (EA3) domain, and HD1 comprises a β2 microglobulin (B2M) domain. In certain embodiments, the β2 microglobulin (B2M) domain and the HLA-E A3 (EA3) domain bind to each other to form a dimer. In certain embodiments, at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively. In certain embodiments, B2M is different from the wild-type B2M domain (SEQ ID NO: 2). In certain embodiments, the EA3 domain is different from the wild-type EA3 domain (SEQ ID NO: 33). In certain embodiments, at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively. In certain embodiments, LD1 comprises a first ICAM-1 D1 domain, and HD1 comprises a second ICAM-1 D1 domain. In certain embodiments, the first ICAM-1 D1 domain and the second ICAM-1 D1 domain associate with each other to form a dimer, and in certain embodiments, the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain.

[0100] In certain embodiments, VH1 and / or VL1 are derived from antibodies of any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2. In certain embodiments, VH1 and / or VL1 are derived from an IgG antibody, such as an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody (e.g., an IgG4 nullbody and a variant of an IgG4 antibody).

[0101] In certain embodiments, VH1 is derived from an immunoglobulin or antibody gamma (γ), delta (δ), alpha (α), mu (μ), or epsilon (ε) heavy chain. In certain embodiments, VL1 is derived from an immunoglobulin or antibody kappa (κ), lambda (λ), sigma (σ), or iota (ι) light chain.

[0102] In certain embodiments, VH1 and / or VL1 are derived from a human antibody, a humanized antibody, or an antibody derived from a non-human animal, such as a mouse antibody, a rat antibody, a camel antibody, a llama antibody, or a chimeric antibody thereof.

[0103] In certain embodiments, the first light chain polypeptide comprises an elbow region between VL1 and LD1. In certain embodiments, the first light chain polypeptide comprises, from amino-terminal to carboxy-terminal order, VL1-LE1-LD1, where VL1 is the first light chain variable domain, LE1 is the first light chain elbow region, and LD1 is the first light chain dimerization domain.

[0104] In certain embodiments, the first light chain polypeptide comprises a first light chain spacer region fused to the C-terminus of LD1. In certain embodiments, the first light chain polypeptide comprises, from amino- to carboxy-terminus, VL1-LD1-LS1, where VL1 is the first light chain variable domain, LD1 is the first light chain dimerization domain, and LS1 is the first light chain spacer region.

[0105] In certain embodiments, the first light chain polypeptide comprises, from amino-terminus to carboxy-terminus, VL1-LE1-LD1-LS1, where VL1 is the first light chain variable domain, LE1 is the first light chain elbow region, LD1 is the first light chain dimerization domain, and LS1 is the first light chain spacer region.

[0106] In certain embodiments, the first heavy chain polypeptide comprises an elbow region between VH1 and HD1. In certain embodiments, the first heavy chain polypeptide comprises, from amino- to carboxy-terminus, V1-HE1-HD1, where VH1 is the first heavy chain variable domain, HE1 is the first heavy chain elbow region, and HD1 is the first heavy chain dimerization domain.

[0107] In certain embodiments, the first heavy chain polypeptide comprises a first heavy chain spacer region fused to the C-terminus of HD1. In certain embodiments, the first heavy chain polypeptide comprises, from amino- to carboxy-terminus, VH1-HD1-HS1, where VH1 is the first heavy chain variable domain, HD1 is the first heavy chain dimerization domain, and HS1 is the first heavy chain spacer region.

[0108] In certain embodiments, the first heavy chain polypeptide comprises, from amino-terminus to carboxy-terminus, VH1-HE1-HD1-HS1, where VH1 is the first heavy chain variable domain, HE1 is the first heavy chain elbow region, HD1 is the first heavy chain dimerization domain, and HS1 is the first heavy chain spacer region.

[0109] The light chain polypeptides described herein may be the first light chain polypeptide, second light chain polypeptide, third light chain polypeptide, or fourth light chain polypeptide of an antigen-binding molecule, each of which is intended to be embodied as described herein for the first light chain polypeptide, but renumbered accordingly (e.g., VLn, LEn, LDn, and LSn, where n is 1 for the first light chain polypeptide, n is 2 for the second light chain polypeptide, n is 3 for the third light chain polypeptide, or n is 4 for the fourth light chain polypeptide). The heavy chain polypeptides described herein may be the first heavy chain polypeptide, second heavy chain polypeptide, third heavy chain polypeptide, or fourth heavy chain polypeptide of an antigen-binding molecule, each of which is intended to be embodied as described herein for the first heavy chain polypeptide, but renumbered accordingly (e.g., VHn, HEn, HDn, and HSn, where n is 1 for the first heavy chain polypeptide, n is 2 for the second heavy chain polypeptide, n is 3 for the third heavy chain polypeptide, or n is 4 for the fourth heavy chain polypeptide).

[0110] In certain embodiments, the VHn and / or VLn (n is 1 for the first light chain polypeptide, n is 2 for the second light chain polypeptide, n is 3 for the third light chain polypeptide, or n is 4 for the fourth light chain polypeptide) are derived from antibodies of any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2. In certain embodiments, the VHn and / or VLn are derived from an IgG antibody, such as an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody (e.g., IgG4 nullbodies and variants of IgG4 antibodies).

[0111] In certain embodiments, the VHn (n is 1 for the first light chain polypeptide, n is 2 for the second light chain polypeptide, n is 3 for the third light chain polypeptide, or n is 4 for the fourth light chain polypeptide) is derived from an immunoglobulin or antibody gamma (γ), delta (δ), alpha (α), mu (μ), or epsilon (ε) heavy chain. In certain embodiments, the VLn (n is 1 for the first light chain polypeptide, n is 2 for the second light chain polypeptide, n is 3 for the third light chain polypeptide, or n is 4 for the fourth light chain polypeptide) is derived from an immunoglobulin or antibody kappa (κ), lambda (λ), sigma (σ), or iota (ι) light chain.

[0112] In certain embodiments, the VHn and / or VLn (n is 1 for the first light chain polypeptide, n is 2 for the second light chain polypeptide, n is 3 for the third light chain polypeptide, or n is 4 for the fourth light chain polypeptide) are derived from a human antibody, a humanized antibody, or an antibody derived from a non-human animal, such as a mouse antibody, a rat antibody, a camel antibody, a llama antibody, or a chimeric antibody thereof.

[0113] In certain embodiments, the first heavy chain polypeptide further comprises an immunoglobulin constant domain. In certain embodiments, the first heavy chain polypeptide further comprises antibody heavy chain constant domain 2 (CH2) or a fragment thereof, optionally fused to the C-terminus of the first heavy chain dimerization domain or the first heavy chain spacer region. In certain embodiments, the first heavy chain polypeptide further comprises antibody heavy chain constant domain 3 (CH3) or a fragment thereof, optionally fused to the C-terminus of the first heavy chain dimerization domain, the C-terminus of the first heavy chain spacer region, or, if present, the C-terminus of the CH2 domain. In certain embodiments, the first heavy chain polypeptide further comprises an antibody fourth heavy chain constant (CH4) domain or fragment thereof, optionally fused to the C-terminus of the first heavy chain dimerization domain, the C-terminus of the first heavy chain spacer region, the C-terminus of the CH2 domain, if present, or the C-terminus of the CH3 domain, if present.

[0114] In certain embodiments, CH2 and / or CH3 are derived from antibodies of any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2. In certain embodiments, CH2 and / or CH3 are derived from an IgG antibody, such as an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody (e.g., an IgG4 nullbody and a variant of an IgG4 antibody).

[0115] In certain embodiments, CH2 and / or CH3 are derived from an immunoglobulin or antibody gamma (γ), delta (δ), alpha (α), mu (μ), or epsilon (ε) heavy chain.

[0116] In certain embodiments, CH2 and / or CH3 are derived from a human antibody, a humanized antibody, or an antibody derived from a non-human animal, such as a mouse antibody, a rat antibody, a camel antibody, a llama antibody, or a chimeric antibody thereof.

[0117] 7.3 Dimerization Domains In certain aspects, a dimerization domain of a polypeptide of an antigen-binding molecule forms a dimer with another dimerization domain of another polypeptide of the antigen-binding molecule. In certain embodiments, a polypeptide of an antigen-binding molecule described herein comprises a means for dimerizing with another polypeptide of the antigen-binding molecule. In certain embodiments, the means for dimerization is a dimerization domain described herein.

[0118] In certain embodiments, a dimerization domain binds to another dimerization domain to form a dimer. In certain embodiments, a light chain dimerization domain completely or partially replaces a light chain constant domain (CL). In certain embodiments, a light chain dimerization domain completely or partially replaces the dimerization sequence of a light chain constant domain (CL). In certain embodiments, a heavy chain dimerization domain completely or partially replaces heavy chain constant domain 1 (CH1). In certain embodiments, a heavy chain dimerization domain completely or partially replaces the dimerization sequence of heavy chain constant domain 1 (CH1).

[0119] 7.3.1 β2-microglobulin (B2M) domain In certain embodiments, the dimerization domain described herein comprises a β2 microglobulin (B2M) domain. In certain embodiments, the B2M domain is a human B2M domain. In certain embodiments, the B2M domain is a wild-type human B2M domain or a fragment thereof. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of B2M or a fragment thereof (e.g., mature B2M lacking the signal sequence) set forth in UniProt Accession No. P61769. The amino acid sequence of B2M set forth in UniProt Accession No. P61769 is MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 1). In certain embodiments, the B2M domain comprises or consists of the amino acid sequence RTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 2).

[0120] In certain embodiments, the B2M domain is a variant B2M domain comprising an amino acid sequence that differs from the wild-type B2M domain of SEQ ID NO: 2. In certain embodiments, the variant B2M domain comprises at least one amino acid substitution, deletion, or insertion compared to the wild-type B2M domain at at least one position numbered according to the B2M amino acid numbering in Table 1 below. In certain embodiments, the variant B2M domain comprises at least one amino acid substitution, deletion, or insertion compared to the wild-type B2M domain at at least one position numbered according to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the variant B2M domain differs from the wild-type B2M domain of SEQ ID NO: 1 or SEQ ID NO: 2 by 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, deletions, or insertions.

[0121] [Table 1]

[0122] In certain embodiments, the B2M domain comprises or consists of an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In a preferred embodiment, the B2M domain comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO:2.

[0123] In certain embodiments, the B2M domain comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 single amino acid substitutions relative to the sequence of SEQ ID NO: 2 at one or more amino acid positions 4, 8, 10, 54, 58, 60, 96, and 97 of SEQ ID NO: 2. In certain embodiments, the B2M domain comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 single amino acid substitutions relative to the sequence of SEQ ID NO: 2 at one or more amino acid positions 4, 8, 10, 54, 58, 60, 96, and 97 of SEQ ID NO: 2, wherein each of the single amino acid substitutions is independently selected from the group consisting of F, W, C, S, and T.

[0124] In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of the human B2M sequence set forth in SEQ ID NO: 2, but comprises the substitution set of F56S, W60S, and F62T, with positions numbered according to the B2M amino acid numbering of Table 1, and optionally further comprises 1, 2, 3, 4, or 5 single amino acid substitutions at positions K6, Y10, R12, D98, or M99, with positions numbered according to the B2M amino acid numbering of Table 1. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of the human B2M sequence set forth in SEQ ID NO:2, but comprises a set of substitutions selected from: i) F56S, W60S, F62T, and K6C; ii) F56S, W60S, F62T, and any one of Y10C, Y10F, or Y10W; iii) F56S, W60S, F62T, and R12C; iv) F56S, W60S, F62T, and any one of D98C, D98F, or D98W; or v) F56S, W60S, F62T, and any one of M99C, M99F, or M99W, with positions numbered according to the B2M amino acid numbering in Table 1.

[0125] In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 2-30. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO:22.In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 23. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 25. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 26. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 29. In certain embodiments, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 30.

[0126] 7.3.2 HLA-E A3 (EA3) domain In certain embodiments, the dimerization domain described herein comprises an HLA class I histocompatibility antigen alpha chain-E alpha3 domain (referred to herein as HLA class I histocompatibility antigen A3 or EA3). In certain embodiments, the EA3 domain is a human EA3 domain. In certain embodiments, the EA3 domain is a wild-type EA3 domain or a fragment thereof. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of EA3 set forth in UniProt Accession No. P13747, or a fragment thereof. The amino acid sequence of the entire HLA class I histocompatibility antigen alpha chain-E (HLA-E) set forth in UniProt Accession No. P13747, including the EA3 domain, is MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNE The amino acid sequence of EA3 shown in UniProt accession number P13747 (e.g., positions 204 to 295 of SEQ ID NO: 31) is EPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL (SEQ ID NO: 32). In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence LHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW (SEQ ID NO: 33).

[0127] In certain embodiments, the EA3 domain is a variant EA3 domain comprising an amino acid sequence that differs from the wild-type EA3 domain of SEQ ID NO: 33. In certain embodiments, the variant EA3 domain comprises at least one amino acid substitution, deletion, or insertion compared to the wild-type EA3 domain at at least one position numbered according to the EA3 amino acid numbering in Table 2 below. In certain embodiments, the variant EA3 domain comprises at least one amino acid substitution, deletion, or insertion compared to the wild-type EA3 domain at at least one position numbered according to the amino acid sequence of SEQ ID NO: 33. In certain embodiments, the variant EA3 domain differs from the wild-type EA3 domain of SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33 by 1, 2, 3, 4, 5, or 6 amino acid substitutions, deletions, or insertions.

[0128] [Table 2]

[0129] In certain embodiments, the EA3 domain comprises or consists of an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33. In a preferred embodiment, the EA3 domain comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 33.

[0130] In certain embodiments, the EA3 domain comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, or 6 single amino acid substitutions relative to the sequence of SEQ ID NO: 33 at one or more amino acid positions 13, 23, 53, 55, 59, and 63 of SEQ ID NO: 33. In certain embodiments, the EA3 domain comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, or 6 single amino acid substitutions relative to the sequence of SEQ ID NO: 33 at one or more amino acid positions 13, 23, 53, 55, 59, and 63 of SEQ ID NO: 33, wherein each of the single amino acid substitutions is independently selected from the group consisting of A, C, and L.

[0131] In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of the human EA3 sequence set forth in SEQ ID NO: 33, but contains 1, 2, 3, 4, 5, or 6 single amino acid substitutions at positions H192, R202, E232, R234, D238, or Q242, with positions numbered according to the EA3 amino acid numbering in Table 2. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of the human EA3 sequence set forth in SEQ ID NO: 33, but comprises a substitution set selected from: i) H192C, ii) R202A or R202C, iii) E232C, iv) R234A, R234L, or R234C, v) D238C, vi) Q242A or Q242L, vii) R234A and Q242A, viii) R234A and Q242L, ix) R234A and Q242A, x) R234L and Q242L, with positions numbered according to the EA3 amino acid numbering in Table 2.

[0132] In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 32-33 and 35-46. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 33. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 35. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 37. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 40. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 41. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 43. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 44. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 45. In certain embodiments, the EA3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 46.

[0133] 7.3.3 ICAM-1 D1 domain In certain embodiments, the dimerization domain described herein comprises or consists of an intercellular adhesion molecule 1 domain 1 (ICAM-1 D1) domain. In certain embodiments, the ICAM-1 D1 domain is a human ICAM-1 D1 domain. In certain embodiments, the ICAM-1 D1 domain is a wild-type human ICAM-1 D1 domain or a fragment thereof. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO:49.

[0134] In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of ICAM-1 D1 set forth in UniProt Accession No. P05362, or a fragment thereof. The amino acid sequence of ICAM-1 set forth in UniProt Accession No. P05362, including the intercellular adhesion molecule 1 (ICAM-1) D1 domain, is (SEQ ID NO: 47). The amino acid sequence of ICAM-1 D1 set forth in UniProt Accession No. P05362 (e.g., positions 41-103 of SEQ ID NO: 47) is GGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTA (SEQ ID NO: 48). In a specific embodiment, the ICAM-1 D1 domain comprises or consists of the amino acid sequence QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVY (SEQ ID NO: 49).

[0135] In certain embodiments, the ICAM-1 D1 domain is a variant ICAM-1 D1 domain comprising an amino acid sequence that differs from the wild-type ICAM-1 D1 domain of SEQ ID NO: 49. In certain embodiments, the variant ICAM-1 D1 domain comprises at least one amino acid substitution, deletion, or insertion compared to the wild-type EA3 domain at at least one position numbered according to the ICAM-1 D1 amino acid numbering in Table 3 below. In certain embodiments, the variant ICAM-1 D1 domain comprises at least one amino acid substitution, deletion, or insertion compared to the wild-type ICAM-1 D1 domain at at least one position numbered according to the amino acid sequence of SEQ ID NO: 49. In certain embodiments, the variant ICAM-1 D1 domain differs from the wild-type ICAM-1 D1 domain of SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, deletions, or insertions.

[0136] [Table 3]

[0137] In certain embodiments, the ICAM-1 D1 domain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 49. In a preferred embodiment, the ICAM-1 D1 domain comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO:49.

[0138] In certain embodiments, the ICAM-1 D1 domain comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single amino acid substitutions relative to the sequence of SEQ ID NO: 49 at one or more amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67, and 78 of SEQ ID NO: 49, and optionally further comprising a C-terminal cysteine amino acid addition. In certain embodiments, the ICAM-1 D1 domain comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single amino acid substitutions relative to the sequence of SEQ ID NO: 49 at one or more amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67, and 78 of SEQ ID NO: 49, wherein each single amino acid substitution is independently selected from the group consisting of V, T, F, W, A, K, E, C, and R, and optionally further comprises a C-terminal cysteine amino acid addition.

[0139] In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of the human ICAM-1 D1 sequence set forth in SEQ ID NO:49, but comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single amino acid substitutions at positions T2, I10, R13, L18, T20, T23, E34, P38, L42, R49, V51, E53, P63, S67, or T78, with positions numbered according to the ICAM-1 D1 amino acid numbering in Table 3, and optionally further comprises a C-terminal cysteine amino acid addition (84C), with positions numbered according to the ICAM-1 D1 amino acid numbering in Table 3. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of the human ICAM-1 D1 sequence set forth in SEQ ID NO:49, but comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single amino acid substitutions at positions T2, I10, R13, L18, T20, T23, E34, P38, L42, R49, V51, E53, P63, S67, or T78, with positions numbered according to the ICAM-1 D1 amino acid numbering in Table 3. Positions were numbered according to the D1 amino acid numbering: i) E34K, ii) T2V, I10T, T23A, E34K, P38T, P63V, S67A, and T78A, iii) T2V, I10T, R13C, T23A, E34K, P38T, R49E, P63V, S67A, T78A, and 84C, iv) T2V, I10T, R13C, T23A, E34K, P38T, E53R, P63V, S67A, T78A, 84C, R234A, R234L, and R234L. 4C, v) T2V, I10T, R13C, L18F, T23A, E34K, P38T, P63V, S67A, T78A, and 84C, vi) T2V, I10T, R13C, L18A, T20A, T23A, E34K, P38T, L42A, V51A, P63V, S67A, T78A, and 84C, or vii) T2V, I10T, R13C, L18W, T23A, E34K, P38T, P63V, S67A, T78A, and 84C.

[0140] In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 48-49 and 51-58. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 48. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 49. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 51. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 52. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 53. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 54. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 55. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 56. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 57. In certain embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 58.

[0141] 7.4 Elbow Area In certain aspects, an elbow region of a polypeptide of an antigen-binding molecule connects its variable domain to its dimerization domain. In certain embodiments, the light chain polypeptide or heavy chain polypeptide of an antigen-binding molecule comprises an elbow region. In certain embodiments, the elbow region is located between the variable domain and the dimerization domain of the light chain, or between the variable domain and the dimerization domain of the heavy chain polypeptide. In certain embodiments, a light chain polypeptide (e.g., a first light chain polypeptide, a second light chain polypeptide, a third light chain polypeptide, a fourth light chain polypeptide, or an additional light chain polypeptide) comprises a light chain elbow region (e.g., a first light chain elbow region, a second light chain elbow region, a third light chain elbow region, a fourth light chain elbow region, or an additional light chain elbow region, respectively). In certain embodiments, a heavy chain polypeptide (e.g., a first heavy chain polypeptide, a second heavy chain polypeptide, a third heavy chain polypeptide, a fourth heavy chain polypeptide, or an additional heavy chain polypeptide) comprises a heavy chain elbow region (e.g., a first heavy chain elbow region, a second heavy chain elbow region, a third heavy chain elbow region, a fourth heavy chain elbow region, or an additional heavy chain elbow region, respectively).

[0142] In certain embodiments, the light chain elbow region comprises or consists of an amino acid sequence between 3 and 25 amino acids in length. In some embodiments, the elbow region is at least 3 amino acids in length. In certain embodiments, the light chain elbow region comprises or consists of an amino acid sequence selected from the group consisting of RTV, GGS, RTVGGS (SEQ ID NO: 59), RTVGGSRTV (SEQ ID NO: 60), AST, ASTK (SEQ ID NO: 61), ASTKG (SEQ ID NO: 62), ASTKGG (SEQ ID NO: 63), ASTKGGS (SEQ ID NO: 64), ASTKGGGS (SEQ ID NO: 65), ASTKGGGGS (SEQ ID NO: 66), ASTKGGGGSG (SEQ ID NO: 67), ASTKGGGGSGG (SEQ ID NO: 68), ASTKGGGGSGGS (SEQ ID NO: 69), ASTKGGGGSGGGS (SEQ ID NO: 70), ASTKGGGGSGGGGS (SEQ ID NO: 71), RTVA (SEQ ID NO: 72), RTVAG (SEQ ID NO: 73), RTVAGG (SEQ ID NO: 74), RTVAGG (SEQ ID NO: 75), RTVAGG (SEQ ID NO: 76), RTVAGG (SEQ ID NO: 77), RTVAGG (SEQ ID NO: 78), RTVAGG (SEQ ID NO: 79), RTVAGG (SEQ ID NO: 80), RTVAGG (SEQ ID NO: 81), RTVAGG (SEQ ID NO: 82), RTVAGG (SEQ ID NO: 83), RTVAGG (SEQ ID NO: 84), RTVAGG (SEQ ID NO: 85), RTVAGG (SEQ ID NO: 86), RTVAGG (SEQ ID NO: 87), RTVAGG (SEQ ID NO SEQ ID NO: 74), RTVAGGS (SEQ ID NO: 75), RTVAGGGS (SEQ ID NO: 76), RTVAGGGGS (SEQ ID NO: 77), RTVAGGGGSG (SEQ ID NO: 78), RTVAGGGGSGG (SEQ ID NO: 79), RTVAGGGGSGGS (SEQ ID NO: 80), RTVAGGGGSGGGS (SEQ ID NO: 81), RTVAGGGGSGGGGS (SEQ ID NO: 82), GGGGSGGGGS (SEQ ID NO: 83), GGGGSGGGGSGGGGS (SEQ ID NO: 84), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 85), and GGGGSGGGGSGGGGSGGGGSGGGG (SEQ ID NO: 86).

[0143] In certain embodiments, the light chain dimerization domain is a B2M domain and the light chain elbow region comprises or consists of an amino acid sequence selected from the group consisting of RTV, GGS, RTVGGS (SEQ ID NO:59), RTVGGSRTV (SEQ ID NO:60), RTVA (SEQ ID NO:72), RTVAG (SEQ ID NO:73), RTVAGG (SEQ ID NO:74), RTVAGGS (SEQ ID NO:75), RTVAGGGS (SEQ ID NO:76), RTVAGGGGS (SEQ ID NO:77), RTVAGGGGSG (SEQ ID NO:78), RTVAGGGGSGG (SEQ ID NO:79), RTVAGGGGSGGS (SEQ ID NO:80), RTVAGGGGSGGGS (SEQ ID NO:81), and RTVAGGGGSGGGGS (SEQ ID NO:82).

[0144] In certain embodiments, the heavy chain dimerization domain is an EA3 domain and the heavy chain elbow region comprises or consists of an amino acid sequence selected from the group consisting of GGS, AST, ASTK (SEQ ID NO: 61), ASTKG (SEQ ID NO: 62), ASTKGG (SEQ ID NO: 63), ASTKGGS (SEQ ID NO: 64), ASTKGGGS (SEQ ID NO: 65), ASTKGGGGS (SEQ ID NO: 66), ASTKGGGGSG (SEQ ID NO: 67), ASTKGGGGSGG (SEQ ID NO: 68), ASTKGGGGSGGS (SEQ ID NO: 69) ASTKGGGGSGGGS (SEQ ID NO: 70), or ASTKGGGGSGGGGS (SEQ ID NO: 71).

[0145] In certain embodiments, the light chain dimerization domain is a first ICAM-1 D1 domain and the light chain elbow region comprises or consists of an amino acid sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 83), GGGGSGGGGSGGGGGS (SEQ ID NO: 84), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 85), and GGGGSGGGGSGGGGSGGGGSGGGG (SEQ ID NO: 86). In certain embodiments, the heavy chain dimerization domain is a second ICAM-1 D1 domain and the heavy chain elbow region comprises or consists of an amino acid sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 83), GGGGSGGGGSGGGGGS (SEQ ID NO: 84), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 85), and GGGGSGGGGSGGGGSGGGGSGGGG (SEQ ID NO: 86).

[0146] 7.5 Spacers and Tags In certain aspects, the spacer region of the polypeptide of the antigen-binding molecule is fused to the C-terminus of its dimerization domain. In certain embodiments, the light chain spacer region is fused to the C-terminus of the light chain dimerization domain of the antigen-binding molecule described herein. In certain embodiments, the heavy chain spacer region is fused to the C-terminus of the heavy chain dimerization domain of the antigen-binding molecule described herein.

[0147] In certain embodiments, the light chain spacer region comprises a hinge region. In certain embodiments, the light chain spacer region is connected to a hinge region. In certain embodiments, the heavy chain spacer region comprises a hinge region. In certain embodiments, the heavy chain spacer region is connected to a hinge region. In certain embodiments, the hinge region is an antibody hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region).

[0148] In certain embodiments, the light chain spacer region or heavy chain spacer region comprises or consists of an amino acid sequence 2 to 9 amino acids in length, or comprises or consists of an amino acid sequence selected from the group consisting of EPKSS (SEQ ID NO: 87), G, SG, EPKSC (SEQ ID NO: 88), GGSGECSG (SEQ ID NO: 89), GGGSGECSG (SEQ ID NO: 90), GGSGECSG (SEQ ID NO: 91), and GGGSGESSG (SEQ ID NO: 92).

[0149] In certain embodiments, the spacer region of the polypeptide of the antigen-binding molecule is connected to another moiety (e.g., another protein). In certain embodiments, the spacer region of the polypeptide of the antigen-binding molecule is connected to a C-terminal tag, and optionally, the C-terminal tag is an affinity tag or a purification tag. In certain embodiments, the C-terminal tag is a 6xHis tag (SEQ ID NO: 93), a streptavidin tag (e.g., a Strep-tag II tag), or a human influenza hemagglutinin tag. In certain embodiments, the 6xHis tag is a 6xHis poly-histidine (i.e., His-His-His-His-His-His (SEQ ID NO: 93)). In certain embodiments, the C-terminal tag comprises or consists of the amino acid sequence of HHHHHH (SEQ ID NO: 93). In certain embodiments, the C-terminal tag comprises or consists of the amino acid sequence of WSHPQFEK (SEQ ID NO: 94). In certain embodiments, the C-terminal tag comprises or consists of the amino acid sequence of YPYDVPDYA (SEQ ID NO: 95).

[0150] 7.6 Target antigen In certain embodiments, the paratope of an antigen-binding molecule described herein binds to an epitope of a target antigen. In certain embodiments, the paratope is formed by the variable region of an antigen-binding molecule described herein, preferably consisting of a light chain variable domain associated with its heavy chain variable domain. In certain embodiments, the paratope is formed by two variable domains (e.g., a light chain variable domain and a heavy chain variable domain) of an antigen-binding molecule described herein.

[0151] In certain embodiments, the target antigen is an antigen associated with a disease or disorder in a subject. In certain embodiments, the target antigen is an antigen associated with a cancer in a subject. In certain embodiments, the target antigen is an antigen specific to a cancer in a subject. In certain embodiments, the target antigen is an antigen of respiratory syncytial virus (RSV).

[0152] In certain embodiments, the target antigen is a tumor-associated antigen (TAA), which is an antigen that is overexpressed in tumor cells (preferably cancer) compared to non-tumor cells in a subject. In certain embodiments, the first paratope of the antigen-binding molecule described herein binds to the first tumor-associated antigen (TAA1), if present, the second paratope of the antigen-binding molecule binds to the second tumor-associated antigen (TAA2), if present, the third paratope of the antigen-binding molecule binds to the third tumor-associated antigen (TAA3), and if present, the fourth paratope of the antigen-binding molecule binds to the fourth tumor-associated antigen (TAA4).

[0153] In certain embodiments, the target antigen is a tumor-specific antigen (TSA), which is unique to tumor cells or is expressed only on tumor cells in a subject. In certain embodiments, the first paratope of the antigen-binding molecule described herein binds to the first tumor-specific antigen (TSA1), if present, the second paratope of the antigen-binding molecule binds to the second tumor-specific antigen (TSA2), if present, the third paratope of the antigen-binding molecule binds to the third tumor-specific antigen (TSA3), and if present, the fourth paratope of the antigen-binding molecule binds to the fourth tumor-specific antigen (TAA4).

[0154] 7.7 Variable Regions, Variable Domains, and Paratopes In one aspect, the antigen-binding molecules of the present invention bind to a target antigen (i.e., the paratope of the antigen-binding molecule binds to an epitope of the target antigen). In certain embodiments, the antigen-binding molecules described herein comprise one or more variable regions that form one or more paratopes that bind to one or more target antigens. In certain embodiments, the variable region comprises two variable domains (e.g., two variable domains derived from two antigen-binding polypeptides described herein that bind to the target antigen). In certain embodiments, the variable region comprises the light chain variable domain and heavy chain variable domain of an antibody that binds to the target antigen. In certain embodiments, the variable region comprises the light chain variable domain and heavy chain variable domain of one arm of an antibody that binds to the target antigen.

[0155] In certain embodiments, the polypeptide of the antigen-binding molecule described herein comprises a means for binding to a target antigen. In certain embodiments, the means for binding is a variable domain (e.g., a light chain variable domain or a heavy chain variable domain). In certain embodiments, the means for linking the two polypeptide chains of the antigen-binding molecule described herein forms a paratope.

[0156] In certain embodiments, the variable regions of the antigen-binding molecules described herein form a paratope that binds to a target antigen (e.g., an epitope of the target antigen). In certain embodiments, the variable domains of the two polypeptide chains of the antigen-binding molecules described herein form a paratope that binds to a target antigen (e.g., an epitope of the target antigen). In certain embodiments, the light chain complementarity-determining region 1 (LCDR1), the light chain complementarity-determining region 2 (LCDR2), the light chain complementarity-determining region 3 (LCDR3), the heavy chain complementarity-determining region 1 (HCDR1), the heavy chain complementarity-determining region 2 (HCDR2), and the heavy chain complementarity-determining region 3 (HCDR3) form a paratope that binds to a target antigen (e.g., an epitope of the target antigen).

[0157] In certain embodiments, the paratopes described herein bind to a target antigen. In certain embodiments, the paratopes described herein bind to an epitope of the target antigen. In certain embodiments, different paratopes (e.g., a first paratope, a second paratope, a third paratope, or a fourth paratope) of the same antigen-binding molecule each bind to a different target antigen.

[0158] In certain embodiments, an antigen-binding molecule comprises two variable regions that bind to two epitopes. In certain embodiments, an antigen-binding molecule comprises two variable regions that bind to two target antigens. In certain embodiments, an antigen-binding molecule comprises two variable regions that bind to two different epitopes, optionally, the two different epitopes are derived from different target antigens.

[0159] In certain embodiments, the antigen-binding molecule comprises three variable regions that bind to three epitopes. In certain embodiments, the antigen-binding molecule comprises three variable regions that bind to three target antigens. In certain embodiments, the antigen-binding molecule comprises three variable regions that bind to three different epitopes, optionally each of the three different epitopes is derived from a different target antigen.

[0160] In certain embodiments, the antigen-binding molecule comprises four variable regions that bind to four epitopes. In certain embodiments, the antigen-binding molecule comprises four variable regions that bind to four target antigens. In certain embodiments, the antigen-binding molecule comprises four variable regions that bind to four different epitopes, optionally each of the four different epitopes is derived from a different target antigen.

[0161] In certain embodiments, the variable domain of the polypeptide described herein comprises an antibody light chain variable domain or a fragment thereof. In certain embodiments, the variable domain or a fragment thereof comprises the amino acid sequence of an antibody light chain framework region 1 (LFR1), light chain complementarity determining region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity determining region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity determining region 3 (LCDR3), light chain framework region 4 (LFR4), or any combination thereof, which binds to a target antigen or forms a paratope that binds to a target antigen.

[0162] In certain embodiments, the variable domain of the polypeptide described herein comprises an antibody heavy chain variable domain or a fragment thereof. In certain embodiments, the variable domain or fragment thereof comprises the amino acid sequence of antibody heavy chain framework region 1 (HFR1), heavy chain complementarity determining region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determining region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determining region 3 (HCDR3), heavy chain framework region 4 (HFR4), or any combination thereof, which binds to a target antigen or forms a paratope that binds to a target antigen.

[0163] In certain embodiments, the antigen-binding molecules described herein bind to respiratory syncytial virus (RSV). In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a light chain variable domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 97. In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 96. In certain embodiments, the antigen-binding molecules described herein comprise a light chain variable domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 97 and a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 96.

[0164] In certain embodiments, the antigen-binding molecules described herein bind to human epidermal growth factor receptor 2 (HER2). In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a light chain variable domain (VL) comprising or consisting of the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 362). In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 363). In certain embodiments, the antigen-binding molecule described herein comprises a light chain variable domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 362 and a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 363.

[0165] In certain embodiments, the antigen binding molecules described herein bind to the mesenchymal-epithelial transition (MET) region. In certain embodiments, the polypeptides of the antigen binding molecules described herein comprise a light chain variable domain (VL) comprising or consisting of the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIK (SEQ ID NO: 364). In certain embodiments, the polypeptides of the antigen binding molecules described herein comprise a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS (SEQ ID NO: 365). In certain embodiments, the antigen-binding molecule described herein comprises a light chain variable domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 364 and a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 365.

[0166] In certain embodiments, the antigen-binding molecules described herein bind to cluster of differentiation 3 (CD3). In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a light chain variable domain (VL) comprising or consisting of the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID NO: 366). In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO: 367). In certain embodiments, the antigen-binding molecule described herein comprises a light chain variable domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 366 and a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 367.

[0167] In certain embodiments, the antigen-binding molecules described herein bind to both HER2 and MET. In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a light chain variable domain (VL1) comprising or consisting of the amino acid sequence of SEQ ID NO: 362. In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a first heavy chain variable domain (VH1) comprising or consisting of the amino acid sequence of SEQ ID NO: 363. In certain embodiments, the antigen-binding molecules described herein comprise a first light chain variable domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 362 and a first heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 363. In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a second light chain variable domain (VL2) comprising or consisting of the amino acid sequence of SEQ ID NO: 364. In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a second heavy chain variable domain (VH2) comprising or consisting of the amino acid sequence of SEQ ID NO: 365. In certain embodiments, the antigen binding molecule described herein comprises a second light chain variable domain (VL2) comprising or consisting of the amino acid sequence of SEQ ID NO: 364 and a second heavy chain variable domain (VH2) comprising or consisting of the amino acid sequence of SEQ ID NO: 365.

[0168] In certain embodiments, the antigen-binding molecules described herein bind to both HER2 and CD3. In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a light chain variable domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 362. In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 363. In certain embodiments, the antigen-binding molecules described herein comprise a light chain variable domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 362 and a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 363. In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a second light chain variable domain (VL2) comprising or consisting of the amino acid sequence of SEQ ID NO: 366. In certain embodiments, the polypeptides of the antigen-binding molecules described herein comprise a second heavy chain variable domain (VH2) comprising or consisting of the amino acid sequence of SEQ ID NO: 367. In certain embodiments, the antigen-binding molecule described herein comprises a second light chain variable domain (VL2) comprising or consisting of the amino acid sequence of SEQ ID NO: 366 and a second heavy chain variable domain (VH2) comprising or consisting of the amino acid sequence of SEQ ID NO: 367.

[0169] 7.8 Antigen-binding molecules In one embodiment, an antigen-binding molecule of the present invention comprises a dimer of two polypeptides, each comprising a variable domain and a dimerization domain described herein, where the variable domains form a paratope and the dimerization domains bind to each other to form a dimer. Complete or partial replacement of the dimerization interface between the light chain constant domain (CL) and heavy chain constant domain 1 (CH1) provides a means for selectively assembling cognate antibody chains. For example, a dual-paratope antibody has one arm (e.g., a light chain polypeptide and a heavy chain polypeptide) modified with either the B2M / EA3 dimerization domain or the ICAM-1 D1 / ICAM-1 D1 dimerization domain described herein, while the other arm is unmodified. In another example, a triple paratope antibody has one arm (e.g., a light chain polypeptide and a heavy chain polypeptide) modified with a B2M / EA3 dimerization domain described herein and one arm (e.g., a light chain polypeptide and a heavy chain polypeptide) modified with an ICAM-1 D1 / ICAM-1 D1 dimerization domain described herein, and the other arm is unmodified.

[0170] In certain embodiments, the antigen-binding domain molecule is an immunoglobulin or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding molecule is an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding molecule is an immunoglobulin fragment or antibody fragment comprising a variable region and at least one constant domain and binding to a target antigen. In certain embodiments, the immunoglobulin or antibody fragment is a Fab, Fab', F(ab')2, or a bispecific Fab. In certain embodiments, the antibody is a biparatopic antibody, a bispecific antibody, a triparatopic antibody, a trispecific antibody, a tetraparatopic antibody, a tetraspecific antibody, a multiparatopic antibody, or a multispecific antibody. In certain embodiments, the antigen-binding molecule is an intact immunoglobulin or antibody. In certain embodiments, the antigen-binding molecule is not an intact immunoglobulin or antibody. In certain embodiments, the antigen-binding molecule is any fragment of the antigen-binding molecules described herein that binds to a target antigen.

[0171] 7.8.1 Fab-based antigen-binding molecules In certain embodiments, the antigen-binding molecule is a Fab. In certain embodiments, the Fab comprises an antibody fragment having a variable region that binds to a target antigen, and comprises fragments of a light chain and a heavy chain cross-linked by a disulfide bond. In certain embodiments, the antigen-binding molecule is a pseudo-Fab (pFab). In certain embodiments, the pFab comprises a variable region that binds to a target antigen and two dimerization domains as described herein. In certain embodiments, the antigen-binding molecule is a Fab'. In certain embodiments, the Fab' comprises an antibody fragment having a single variable region that binds to a target antigen, and comprises a Fab and an additional portion of a heavy chain via a hinge region. In certain embodiments, the antigen-binding molecule is a pseudo-Fab' (pFab'). In certain embodiments, the pFab' comprises a variable region that binds to a target antigen, two dimerization domains as described herein, and a heavy chain hinge region. In certain embodiments, the antigen-binding molecule is a F(ab')2. In certain embodiments, an F(ab')2 comprises two Fab' molecules linked by an interchain disulfide bond in the hinge region of the heavy chain. In certain embodiments, the Fab' molecules of an F(ab')2 can be directed against the same epitope or different epitopes. In certain embodiments, the antigen-binding molecule is a pseudo-F(ab')2 (pF(ab')2). In certain embodiments, a pF(ab')2 comprises two variable regions, each binding to a target antigen, two antigen-binding domains, two or more dimerization domains described herein, and a heavy chain hinge region. In certain embodiments, the antigen-binding molecule is a bispecific Fab. In certain embodiments, a bispecific Fab comprises a Fab molecule having two variable regions, each binding to a target antigen, each of which can be directed against a different epitope.

[0172] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17, and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. Fab-like antibody fragments can be expressed and secreted in E. coli or yeast cells, allowing for the large-scale production of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. By another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives containing salvage receptor-binding epitope residues are described, for example, in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. An antibody fragment may also be a "linear antibody," e.g., as described in the references cited above. Such linear antibodies may be monospecific or multispecific, e.g., bispecific.

[0173] Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules comprising an antigen-binding site that binds to a target antigen. The immunoglobulin molecules provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules that comprise an antigen-binding site that binds to a target antigen. In certain embodiments, the antibodies provided herein are IgG antibodies, such as IgG1 antibodies, IgG2 antibodies, or IgG4 antibodies (e.g., IgG4 nullbodies and IgG4 antibody variants). In a preferred embodiment, the IgG antibody is an IgG1 antibody.

[0174] In certain embodiments, the antigen-binding molecule is a Fab, Fab', F(ab')2, or bispecific Fab comprising variable regions derived from an antibody specific for one or more target antigens. In certain embodiments, the antigen-binding molecule comprises variable regions derived from an antibody specific for one or more target antigens and a dimerization domain as described herein.

[0175] 7.8.2 Antibody-Based Antigen-Binding Molecules The antigen-binding molecules of the present disclosure can be derived from antibodies by complete or partial replacement of the dimerization interface between the light chain constant domain and heavy chain constant domain 1 (CH1) of the antibody.

[0176] 7.8.2.1 Monoclonal antibodies In certain embodiments, the antigen-binding molecules provided herein comprise monoclonal antibodies or fragments thereof. Monoclonal antibodies may be made using the hybridoma method first described by Kohler, et al., Nature, 1975, 256:495-97, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

[0177] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized with a target antigen as described above to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to an epitope of the target antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (1986)).

[0178] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that, in certain embodiments, contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as the fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), then the selective culture medium for the hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0179] Exemplary fusion partner myeloma cells are those that fuse efficiently, support stable high-level antibody production by the selected antibody-producing cells, and are sensitive to selective media that select against the unfused parent cells. Exemplary myeloma cell lines are mouse myeloma lines such as SP-2 cells and derivatives, e.g., X63-Ag8-653 cells, available from the American Type Culture Collection (Manassas, VA), and mouse myeloma lines derived from the MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center (San Diego, CA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, 1984, Immunol. 133:3001-05, and Brodeur et al., 1987, Monoclonal Antibody Production Techniques and Applications 51-63).

[0180] The culture medium in which the hybridoma cells grow is assayed for the production of monoclonal antibodies against the target antigen. The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or an in vitro binding assay such as RIA or ELISA. The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis as described in Munson et al., Anal. Biochem., 1980, 107:220-39.

[0181] Once hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, DMEM medium or RPMI-1640 medium. Additionally, the hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by i.p. injection of the cells into mice.

[0182] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (using, for example, protein A-Sepharose or protein G-Sepharose), ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, or the like.

[0183] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells can be used as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which can then be introduced into host cells that do not normally produce antibody protein, such as Escherichia coli (E. coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra, et al., 1993, Curr. Opinion in Immunol. 5:256-62 and Pluckthun, 1992, Immunol. Rev. 130:151-88.

[0184] In a further embodiment, monoclonal antibodies can be isolated from antibody phage libraries generated using, for example, the techniques described in Antibody Phage Display: Methods and Protocols (O'Brien and Aitken, eds., 2002), in which functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. Examples of phage display methods that can be used to generate the antibodies described herein are described in Brinkman, et al. 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic, et al., Gene, 1997, 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280, PCT Application No. GB91 / 01134, WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401, and WO 97 / 13844, and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403, 484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

[0185] In principle, synthetic antibody clones are selected by screening phage libraries containing phage displaying diverse fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are screened against a desired target antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. Binding clones can then be eluted from the antigen and further enriched by additional cycles of antigen adsorption / elution.

[0186] Variable domains can be functionally displayed on phage as single-chain Fv (scFv) fragments in which VH and VL are covalently linked by a short flexible peptide, or as Fab fragments in which each is fused to a constant domain and interacts non-covalently, as described, for example, in Winter et al., 1994, Ann. Rev. Immunol. 12:433-55.

[0187] Repertoires of VH and VL genes can be individually cloned by PCR and randomly recombined into phage libraries, as described by Winter et al. (supra), which can then be screened for antigen-binding clones. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned to provide a single source of human antibodies to a broad range of non-self antigens, as described by Griffiths et al., 1993, EMBO J 12:725-34, and also to provide a single source of human antibodies to self-antigens without immunization. Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments derived from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve in vitro rearrangement, as described, for example, by Hoogenboom and Winter, J. Mol. Biol., 1992, 227:381-88.

[0188] Screening of libraries can be accomplished by a variety of techniques known in the art. For example, target antigens can be used to coat the wells of adsorption plates, expressed on host cells immobilized on adsorption plates, used in cell sorting, conjugated to biotin for capture by Strep-tag II-coated beads, or any other method for panning display libraries. Selection of antibodies with slow dissociation kinetics (e.g., good binding affinity) can be facilitated by the use of extended washes and monovalent phage display, as described in Bass, et al., Proteins, 1990, 8:309-14 and WO 92 / 09690, and by the use of low antigen coating density, as described in Marks et al., Biotechnol., 1992, 10:779-83.

[0189] Antibodies can be obtained by designing the construction of full-length antibody clones using VH and / or VL sequences (e.g., Fv sequences) derived from the phage clone of interest and suitable constant region (e.g., Fc) sequences, or various CDR sequences derived from the VH and VL sequences, as described in Kabat et al. (supra), followed by a suitable antigen screening procedure to select a phage clone of interest.

[0190] The antibodies described herein may also include, for example, chimeric antibodies. Chimeric antibodies are molecules in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may contain the variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science, 1985, 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

[0191] Antibodies or antigen-binding fragments produced using techniques such as those described herein can be isolated using well-known standard techniques. For example, antibodies or antigen-binding fragments can be suitably separated from, e.g., culture medium, ascites fluid, serum, cell lysates, synthesis reaction materials, etc., by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. In certain embodiments, isolated or purified antibodies are substantially free of cellular material or other proteins from the cell or tissue source from which the antibody is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals.

[0192] 7.8.2.2 Humanized Antibodies In certain embodiments, the antigen-binding molecules provided herein comprise humanized antibodies (e.g., deimmunized antibodies or composite human antibodies) or fragments thereof. In certain embodiments, the humanized antibodies comprise human framework region sequences and / or human constant region sequences. In certain embodiments, the humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4 (e.g., IgG4 variants and IgG4 nullbodies). In certain embodiments, the humanized antibodies may comprise a κ light chain constant sequence or a λ light chain constant sequence.

[0193] Humanized antibodies can be produced by a variety of techniques, including CDR grafting (EP 239,400, WO 91 / 09967, U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106, 519,596, Padlan, 1991, Molecular Immunology 28(4 / 5):489-498, Studnicka et al., 1994, Protein Engineering 7(6):805-814, and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and, for example, U.S. Pat. Nos. 6,407,213, 5,766,886, WO 93 / 17105, Tan et al., J. Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng. 13 (5):353-60 (2000), Morea et al., Methods 20 (3):267 79 (2000), Baca et al., J. Biol. Chem. 272 (16):10678-84 (1997), Roguska et al., Protein Eng. 9 (10):895 904 (1996), Couto et al., Cancer Res. 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent Application Publication No. 2005 / 0042664(A1) (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0194] Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically taken from an "import" variable domain. Humanization can be performed, for example, by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the methods of Jones et al., 1986, Nature 321:522-25; Riechmann, et al., Nature, 1988, 332:323-27; and Verhoeyen, et al., Science, 1988, 239:1534-36).

[0195] In certain embodiments, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of a parent non-human antibody (e.g., a rodent) are grafted onto a human antibody framework. For example, Padlan et al. determined that only about one-third of the CDR residues actually contact the antigen, and called these "specificity-determining residues" or SDRs (Padlan et al., 1995, FASEB J. 9:13339). In the SDR grafting technique, only the SDR residues are grafted onto a human antibody framework (see, e.g., Kashmiri et al., 2005, Methods 36:2534).

[0196] The selection of human variable domains, both light and heavy, used to create a humanized antibody can be important to reduce antigenicity. For example, according to the so-called "best-fit" method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent human sequence can be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol. 151:2296-308, and Chothia et al., 1987, J. Mol. Biol. 196:90117). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:428589, and Presta et al., 1993, J. Immunol. 151:2623-32). In certain cases, the framework is derived from the consensus sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) and V subgroup III (VHIII). Alternatively, human germline genes are used as the source of the framework regions.

[0197] In an alternative paradigm based on CDR comparison, called superhumanization, the homology of FRs is irrelevant. This method involves comparing non-human sequences with a functional human germline gene repertoire. Genes encoding canonical structures identical to or closely related to the mouse sequence are then selected. Next, among genes that share canonical structures with non-human antibodies, the gene with the highest homology within the CDR is selected as the FR donor. Finally, non-human CDRs are grafted onto these FRs (see, for example, Tan et al., 2002, J. Immunol. 169:1119-25).

[0198] Furthermore, it is generally desirable for antibodies to be humanized with retention of affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol. 234:779-815), and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). Inspection of these displays permits analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, for example, analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the hypervariable region residues are directly and most substantially involved in antigen binding.

[0199] Another method for antibody humanization is based on a metric for antibody humanity called Human String Content (HSC). This method compares the mouse sequence with the human germline gene repertoire and scores the differences as HSC. The target sequence is then humanized by maximizing its HSC, rather than using an overall identity measure to generate diverse humanized variants (Lazar et al., 2007, Mol. Immunol. 44:1986-98).

[0200] In addition to the methods described above, empirical methods can be used to generate and select humanized antibodies. These methods involve generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosomal, and yeast display libraries, as well as by bacterial colony screening (see, e.g., Hoogenboom, 2005, Nat. Biotechnol. 23:1105-16; Dufner et al., 2006, Trends Biotechnol. 24:523-29; Feldhaus et al., 2003, Nat. Biotechnol. 21:163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sel. 17:847-60).

[0201] In the FR library approach, multiple residue variants are introduced at specific positions in the FR, and the library is then screened to select the FR that best supports the grafted CDR. The substituted residues can include some or all of the "vernier" residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol. 224:48799), or those from the more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. 272:10678-84).

[0202] In FR shuffling, entire FRs are combined with non-human CDRs rather than creating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., Methods, 2005, 36:43-60). The library can be screened for binding in a two-step process, first humanizing the VL, followed by humanizing the VH. Alternatively, a one-step FR shuffling process can be used. Such a process has been shown to be more efficient than two-step screening, as the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol. 44:3049-60).

[0203] The "humaneering" method is based on the experimental identification of essential minimum specificity determinants (MSDs) and the sequential substitution of non-human fragments into a human FR library and evaluation of binding. Humaneering begins with the CDR3 regions of the non-human VH and VL chains, and gradually substitutes other regions of the non-human antibody, including CDR1 and CDR2 of both VH and VL, with human FRs. This approach typically identifies multiple subclasses of antibodies that retain the epitope but have distinct CDRs in the human V segments. Humaneering allows the isolation of antibodies that are 91-96% homologous to human germline antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0204] The "human engineering" method involves modifying non-human antibodies or antibody fragments, such as murine or chimeric antibodies or antibody fragments, by making specific changes to the antibody's amino acid sequence to generate an altered antibody that reduces immunogenicity in humans while retaining the desired binding characteristics of the original non-human antibody. Generally, this technique involves classifying amino acid residues in a non-human (e.g., murine) antibody as "low risk," "moderate risk," or "high risk" residues. Classification is performed using a global risk / benefit calculation that assesses the predicted benefit of making a particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the folding of the resulting antibody. Specific human amino acid residues to be substituted at a given position (e.g., low risk or moderate risk) in a non-human (e.g., murine) antibody sequence can be selected by aligning amino acid sequences from the variable regions of the non-human antibody with the corresponding regions of specific or consensus human antibody sequences. Amino acid residues at low or moderate risk positions in the non-human sequence can be substituted with the corresponding residue in the human antibody sequence depending on the alignment. Techniques for producing human engineered proteins are described in detail in Studnicka et al., 1994, Protein Engineering 7:80514; U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and WO 93 / 11794.

[0205] Composite human antibodies can be produced, for example, using Composite Human Antibody™ technology (Antitope Ltd., Cambridge, United Kingdom). To produce a composite human antibody, fragments of multiple human antibody variable region sequences are engineered to minimize the immunogenicity of the resulting antibody by avoiding T-cell epitopes. Such antibodies may contain human constant region sequences, such as a human light chain constant region and / or a human heavy chain constant region.

[0206] In certain embodiments, the antigen-binding molecule comprises a deimmunized antibody from which T cell epitopes have been removed. Methods for producing deimmunized antibodies have been described (see, for example, Jones et al., Methods Mol Biol. 2009;525:405-23, xiv and De Groot et al., Cell. Immunol. 244:148-153 (2006)). Deimmunized antibodies comprise a T cell epitope-depleted variable region and a human constant region. Briefly, T cell epitopes are identified by cloning the antibody VH and VL, and then examining overlapping peptides derived from the antibody VH and VL in a T cell proliferation assay. T cell epitopes are identified by in silico methods to identify peptides that bind to human MHC class II. Mutations are introduced into the VH and VL to abolish binding to human MHC class II. The VH and VL are then used to generate deimmunized antibodies.

[0207] 7.8.2.3 Human antibodies In certain embodiments, the antigen-binding molecules provided herein comprise fully human anti-human antibodies or fragments thereof. Fully human antibodies can be determined by any method known in the art. The human antibodies provided herein can be constructed by combining Fv clone variable domain sequences selected from a human-derived phage display library with known human constant domain sequences. Alternatively, the human monoclonal antibodies of the present disclosure can be produced by hybridoma technology. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are described, for example, in Kozbor, 1984, J. Immunol. 133:3001-05; Brodeuret et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987); and Gillies et al., 1991, J. Immunol. 147:86-95.

[0208] It is also possible to produce transgenic animals (e.g., mice) that, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. Transgenic mice expressing human antibody repertoires have been used to produce high-affinity, human-sequence monoclonal antibodies against a wide variety of potential drug targets (see, e.g., Jakobovits, A., 1995, Curr. Opin. Biotechnol. 6(5):561-66; Bruggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; U.S. Patent Nos. 6,075,181 and 6,150,584; and Lonberg et al., 2005, Nature Biotechnol. 23:1117-25).

[0209] Alternatively, human antibodies can be prepared through immortalization of human B lymphocytes that produce antibodies against a target antigen (e.g., such B lymphocytes may be harvested from an individual or immunized in vitro) (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy (1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and J. Immunol. 5,750,373).

[0210] Gene shuffling can also be used to derive human antibodies from nonhuman (e.g., rodent) antibodies, where the human antibody has similar affinity and specificity to the starting nonhuman antibody. According to this method, also referred to as "epitope imprinting" or "guided selection," either the heavy or light chain variable region of a nonhuman antibody fragment obtained by the phage display technology described herein is replaced with a repertoire of human V domain genes, generating a population of nonhuman chain / human chain chimeric scFv or Fab chimeras. Antigen-driven selection results in the isolation of nonhuman chain / human chain chimeric scFv or Fab, with the human chain restoring the antigen-binding site destroyed upon removal of the corresponding nonhuman chain in the primary phage display clone (e.g., the epitope guides (imprints) the selection of the human chain partner). Repeating the process to replace the remaining non-human chains results in a human antibody (see, e.g., WO 93 / 06213 and Osbourn et al., 2005, Methods 36:61-68). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides fully human antibodies with no FR or CDR residues of non-human origin. Examples of guided selection for humanizing murine antibodies against cell surface antigens include folate-binding protein present on ovarian cancer cells (see, e.g., Figini et al., 1998, Cancer Res. 58:991-96) and CD147, which is highly expressed on hepatocellular carcinoma (see, e.g., Bao et al., 2005, Cancer Biol. Ther. 4:1374-80).

[0211] A potential drawback of the guided selection approach is that shuffling one antibody chain while keeping the other constant can result in epitope drift. To maintain the epitope recognized by the nonhuman antibody, CDR retention can be applied (see, for example, Klimka et al., 2000, Br. J. Cancer. 83:252-60, and Chothia et al., 2000, J. Mol. Biol. 296:833-49). In this method, the nonhuman VH CDR3 is generally retained, as this CDR may be at the center of the antigen-binding site and may be the most important region of the antibody for antigen recognition. However, in certain cases, the VH CDR3 and VL CDR3, as well as the VH CDR2, VL CDR2, and VL CDR1 of the nonhuman antibody may be retained.

[0212] 7.8.2.4 Multispecific antibodies Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificities for at least two different antigens. In certain embodiments, the multispecific antibodies provided herein are bispecific antibodies. In certain embodiments, the bispecific antibodies are murine, chimeric, human, or humanized antibodies. In certain embodiments, one of the binding specificities is for one target and / or target antigen and the other is for another target and / or target antigen. In certain embodiments, the bispecific antibodies can bind to two different epitopes of the same target and / or target antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0213] Methods for producing multispecific antibodies are known in the art, for example, by co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details on the generation of multispecific antibodies (e.g., bispecific antibodies), see, e.g., Bispecific Antibodies (Kontermann ed., 2011).

[0214] 7.8.2.5 Fc operation It may be desirable to alter the antibodies provided herein by Fc engineering. In certain embodiments, modifications to the Fc region of the antibody result in a reduction or elimination of the effector function of the antibody. In certain embodiments, the effector function is ADCC, ADCP, and / or CDC. In certain embodiments, the effector function is ADCC. In other embodiments, the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector function is ADCC and ADCP. In one embodiment, the effector function is ADCC and CDC. In one embodiment, the effector function is ADCP and CDC. In one embodiment, the effector function is ADCC, ADCP, and CDC. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody.

[0215] In certain embodiments, modifications to the Fc region of the antibody result in enhanced effector function of the antibody. In certain embodiments, the effector function is ADCC, ADCP, and / or CDC. In some embodiments, the effector function is ADCC. In other embodiments, the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector function is ADCC and ADCP. In one embodiment, the effector function is ADCC and CDC. In one embodiment, the effector function is ADCP and CDC. In one embodiment, the effector function is ADCC, ADCP, and CDC. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody.

[0216] In certain embodiments, antibodies can be engineered using knob-in-hole (KiH) technology. The "knob-in-hole" technology can include one or more mutations selected from Y349C, T366S, L368A, and Y407V in the CH3 domain of the Fc region of the hole arm, and mutations S354C and / or T366W in the CH3 domain of the Fc region of the knob arm. The mutations can promote the formation of heteromultimers. Knob-into-hole technology is described in U.S. Patent Nos. 5,731,168 and 8,216,805, which are incorporated herein by reference in their entireties.

[0217] In certain embodiments, one or both Fc regions of an antibody can be engineered to contain an RF mutation. "RF mutation" generally refers to a mutation of amino acid HY to RF in the CH3 domain of the Fc region, such as the mutations H435R and Y436F in the CH3 domain, as described by Jendeberg, L. et al. (1997, J. Immunological Meth., 201:25-34). RF mutations have been described as advantageous for purification purposes, as they abolish binding to Protein A. In some embodiments, one Fc region of an antibody contains an RF mutation and the other Fc region does not.

[0218] In certain embodiments, the antigen-binding molecules described herein comprise a knob arm comprising a T366W mutation in the CH3 domain of the Fc region. In one embodiment, the knob arm comprises or consists of the following amino acid sequence:

[0219] TIFF2025525530000005.tif16160

[0220] In some embodiments, the antigen-binding molecules described herein comprise a Hole arm comprising the mutations T366S, L368A, and Y407V in the CH3 domain of the Fc region. The Hole arm may further comprise an RF mutation. In one embodiment, the Hole arm comprises or consists of the following amino acid sequence:

[0221] TIFF2025525530000006.tif16160

[0222] To increase the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly an antibody fragment), as described, for example, in U.S. Patent No. 5,739,277. The term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0223] 7.8.2.6 Antibody Variants In certain embodiments, modifications of the amino acid sequence of the antibodies or antigen-binding fragments provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody (including, but not limited to, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility). Thus, in addition to the antibodies described herein, it is contemplated that antibody variants may also be prepared. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or polypeptide. Those skilled in the art will understand that amino acid changes may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring properties.

[0224] In certain embodiments, the antibodies provided herein are chemically modified, for example, by covalently attaching any type of molecule to the antibody. Antibody derivatives can include antibodies chemically modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody can contain one or more non-classical amino acids.

[0225] Mutations can be substitutions, deletions, or insertions of one or more codons encoding the antibody or polypeptide, resulting in a change in the amino acid sequence compared to the native sequence antibody or polypeptide. Amino acid substitutions can be the result of substituting one amino acid for another amino acid with similar structural and / or chemical properties, such as substituting a leucine with a serine, e.g., a conservative amino acid substitution. Standard techniques known to those of skill in the art can be used to introduce mutations into the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Insertions or deletions can optionally range from about 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions comprise fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the original molecule. In certain embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Permissible variations can be determined by systematically making amino acid insertions, deletions, or substitutions in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

[0226] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.

[0227] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues with similarly charged side chains have been defined. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resulting mutants screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein determined.

[0228] Substantial alterations in the biological properties of antibodies can be achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structural backbone of the polypeptide in the area of substitution, e.g., sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chains. Alternatively, conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) can be made that maintain or do not significantly change the properties. Amino acids can be grouped according to the similarity of their side chain properties (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H)).

[0229] Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0230] Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class. Such substituted residues also may be introduced into the conservative substitution sites or into the remaining (non-conserved) sites. Thus, in one embodiment, an antibody or antigen-binding fragment thereof that binds to a target epitope comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of an antibody described herein, e.g., an antibody described in Section 7, infra. In another embodiment, the antibody or antigen-binding fragment thereof that binds to the target antigen comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of an antibody described herein, e.g., an antibody described in Section 7, below.

[0231] Modifications can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, PCR mutagenesis, etc. Site-directed mutagenesis (see, e.g., Carter, 1986, Biochem J. 237:1-7, and Zoller et al., 1982, Nucl. Acids Res. 10:6487-500), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques can be performed on cloned DNA to produce antigen-binding molecule variant DNA.

[0232] Any cysteine residue not involved in maintaining the proper conformation of the antibodies provided herein can be substituted with another amino acid, such as, for example, alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the antibody to improve its stability (e.g., where the antibody is an antibody fragment such as an Fv fragment).

[0233] In certain embodiments, the antibody molecules of the present disclosure are "deimmunized" antibodies. A "deimmunized" antibody is an antibody derived from a humanized or chimeric antibody and has one or more modifications in its amino acid sequence that result in reduced immunogenicity of the antibody compared to the respective original, non-deimmunized antibody. One procedure for generating such antibody variants involves identifying and removing T cell epitopes from the antibody molecule. In a first step, the immunogenicity of an antibody molecule can be determined by several methods, for example, by in vitro determination of T cell epitopes or in silico prediction of such epitopes, as known in the art. Once residues important for T cell epitope function have been identified, mutations can be performed to remove immunogenicity and retain antibody activity. For a review, see, e.g., Jones et al., 2009, Methods in Molecular Biology 525:405-23.

[0234] 7.8.2.7 In vitro affinity maturation In some embodiments, antibody variants with improved properties, such as affinity, stability, or expression level, compared to the parent antibody can be prepared by in vitro affinity maturation. Similar to natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. Libraries of antibodies are displayed on the surface of organisms (e.g., phage, bacteria, yeast, or mammalian cells) or in association with the encoding mRNA or DNA (e.g., covalently or noncovalently). Affinity selection of the displayed antibodies allows for the isolation of organisms or complexes carrying the genetic information encoding the antibody. Using display methods such as phage display, two to three rounds of mutation and selection typically yield antibody fragments with affinities in the low nanomolar range. Affinity-matured antibodies can have nanomolar or picomolar affinities for their target antigens.

[0235] Phage display is a widespread method for antibody display and selection. Antibodies are displayed on the surface of Fd or M13 bacteriophage as fusions to bacteriophage coat proteins. Selection involves exposure to antigen and allowing the phage-displayed antibodies to bind to the target, a process called "panning." Antigen-bound phage are recovered and used to infect bacteria to generate phage for further rounds of selection. For reviews, see, e.g., Hoogenboom, 2002, Methods. Mol. Biol. 178:1-37, and Bradbury and Marks, 2004, J. Immunol. Methods 290:29-49.

[0236] In yeast display systems (see, e.g., Boder et al., 1997, Nat. Biotech. 15:553-57, and Chao et al., 2006, Nat. Protocols 1:755-68), antibodies can be fused to the adhesive subunit of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall via a disulfide bond with Aga1p. Display of the protein via Aga2p keeps the protein away from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen the library to select antibodies with improved affinity or stability. Binding to the soluble antigen of interest is determined by labeling the yeast with biotinylated antigen and a secondary reagent, such as Strep-tag II conjugated to a fluorophore. Variations in antibody surface expression can be measured by immunofluorescently labeling either hemagglutinin or c-Myc epitope tags adjacent to the scFv. Expression has been shown to correlate with the stability of the displayed protein, allowing antibodies to be selected for improved stability and affinity (see, e.g., Shusta et al., 1999, J. Mol. Biol. 292:949-56). An additional advantage of yeast display is that the displayed protein folds in the endoplasmic reticulum of the eukaryotic yeast cell, utilizing endoplasmic reticulum chaperones and quality control machinery. Once maturation is complete, antibody affinity can be conveniently "titrated" while displayed on the yeast surface, eliminating the need for expression and purification of each clone. A theoretical limitation of yeast surface display is the potentially smaller size of functional libraries compared to other display methods. However, recent approaches have utilized yeast cell mating systems to generate libraries of up to 10 ... 14 The size and estimated combinatorial diversity have been generated (see, for example, US Patent Application Publication No. 2003 / 0186374, and Blaise et al., 2004, Gene 342:211-18).

[0237] In ribosome display, antibody-ribosome-mRNA (ARM) complexes are generated and selected in a cell-free system. A DNA library encoding a specific antibody library is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence remains attached to peptidyl-tRNA during translation and occupies the ribosomal tunnel, allowing the target protein to protrude from the ribosome and fold. The resulting mRNA, ribosome, and protein complex binds to a surface-bound ligand, allowing for simultaneous isolation of the antibody and its encoding mRNA by affinity capture. The ribosome-bound mRNA can then be reverse-transcribed back to cDNA, which can then be subjected to mutagenesis and used for the next round of selection (e.g., Fukuda et al., 2006, Nucleic Acids Res. 34:e127). For mRNA display, puromycin is used as an adapter molecule to form a covalent link between the antibody and the mRNA (Wilson et al., 2001, Proc. Natl. Acad. Sci. USA 98:3750-55).

[0238] Because these methods are performed entirely in vitro, they offer two main advantages over other selection techniques. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be easily introduced after each round of selection, for example, by non-proofreading polymerases, because there is no need to convert the library after the diversity step. In certain embodiments, mammalian display systems can be used.

[0239] Diversity can also be introduced into the CDRs of antibody libraries in a targeted manner or through random introduction. The former approach includes sequentially targeting all CDRs of an antibody through high- or low-level mutagenesis, or by targeting isolated hotspots of somatic hypermutation (see, e.g., Ho et al., 2005, J. Biol. Chem. 280:607-17), or residues suspected of affecting affinity for experimental or structural reasons. Diversity can also be introduced by replacing naturally diverse regions through DNA shuffling or similar techniques (see, e.g., Lu et al., 2003, J. Biol. Chem. 278:43496-507; U.S. Patent Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops extending into framework region residues (see, e.g., Bond et al., 2005, J. Mol. Biol. 348:699-709), utilize loop deletions and insertions in the CDRs, or use hybridization-based diversification (see, e.g., U.S. Patent Publication No. 2004 / 0005709). Additional methods for generating diversity in CDRs are disclosed, for example, in U.S. Patent No. 7,985,840. Additional methods that can be used for generating antibody libraries and / or antibody affinity maturation are disclosed, for example, in U.S. Pat. Nos. 8,685,897 and 8,603,930, and U.S. Patent Application Publication Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0240] Screening of libraries can be accomplished by a variety of techniques known in the art, for example, antibodies can be immobilized on solid supports, columns, pins, or cellulose / poly(vinylidene fluoride) membranes / other filters, expressed in host cells immobilized on adsorption plates or used in cell sorting, or conjugated to biotin for capture on Strep-tag II coated beads, or used in any other method for panning display libraries.

[0241] For reviews of in vitro affinity maturation methods, see, e.g., Hoogenboom, 2005, Nature Biotechnology 23:1105-16; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia 4:39-51, and references therein.

[0242] 7.8.2.8 Antibody modification Covalent modification of antibodies that bind to target antigens is included within the scope of the present disclosure. Covalent modifications include reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or the N- or C-terminal residues of the antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of N-terminal amines, and amidation of C-terminal carboxyl groups.

[0243] Other types of covalent modifications of the antibodies provided herein that are within the scope of this disclosure include altering the native glycosylation pattern of the antibody or polypeptide (see, e.g., Beck et al., 2008, Curr. Pharm. Biotechnol. 9:482-501, and Walsh, 2010, Drug Discov. Today 15:773-80), and linking the antibody to one of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, e.g., by methods described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0244] The antibodies or fragments thereof of the present disclosure can also be modified to form chimeric molecules comprising the antibody or fragment thereof fused to another, heterologous polypeptide or amino acid sequence, such as an epitope tag (see, e.g., Terpe, 2003, Appl. Microbiol. Biotechnol. 60:523-33), or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)).

[0245] Also provided herein are fusion proteins comprising, for example, an antigen-binding molecule provided herein that binds to a target antigen and a heterologous polypeptide.

[0246] Also provided herein are panels of antigen-binding molecules that bind to one or more target antigens. In certain embodiments, the panel of antigen-binding domains has different on-rates, dissociation rates, affinities, and / or specificities for the target antigen. In certain embodiments, the panel comprises or consists of about 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 or more antibodies. The panel of antigen-binding domains can be used in assays such as ELISA, for example, in 96- or 384-well plates.

[0247] 7.8.2.9 Immunoconjugates The present disclosure also provides conjugates comprising any one of the antibodies or antigen-binding fragments thereof of the present disclosure covalently attached by a synthetic linker to one or more non-antibody agents.

[0248] In certain embodiments, the antibodies provided herein are conjugated or recombinantly fused to, for example, a therapeutic agent (e.g., a cytotoxic agent) or a diagnostic or detectable molecule. The conjugated or recombinantly fused antibody may be useful, for example, for treating or preventing a disease or disorder. The conjugated or recombinantly fused antibody may be useful, for example, for monitoring or prognosing the onset, development, progression, and / or severity of a disease or disorder.

[0249] Such diagnosis and detection can be accomplished, for example, by conjugating the antibody to a detectable substance, which can include various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, Strep-tag; II / biotin or avidin / biotin, fluorescent substances such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin, luminescent substances such as, but not limited to, luminol, bioluminescent substances such as, but not limited to, luciferase, luciferin, or aequorin, chemiluminescent substances such as, but not limited to, acridinium-based compounds or HALOTAG, iodine (I, I, I, and I), carbon (C), sulfur (S), tritium (H), indium (In, In, In, and In), technetium (Tc), thallium (Ti), gallium (Ga and Ga), palladium (Ga), palladium (Ga), iodine (I, I, I, and I), carbon (C), sulfur (S), tritium (H), indium (In, In, In, and In), technetium (Tc), thallium (Ti), gallium (Ga), palladium (Ga), palladium (Ga), iodine ... Positron emission tomography (PET) imaging techniques include radioactive materials such as Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, or 117Sn, various positron-emitting metals using positron emission tomography; and non-radioactive paramagnetic metal ions.

[0250] Also provided herein are antibodies that are recombinantly fused or chemically conjugated (covalently or non-covalently) to a heterologous protein or polypeptide (or fragment thereof, e.g., a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to produce fusion proteins, and uses thereof. Specifically provided herein are fusion proteins comprising an antigen-binding fragment (e.g., CDR1, CDR2, and / or CDR3) of an antibody provided herein and a heterologous protein, polypeptide, or peptide. In one embodiment, the heterologous protein, polypeptide, or peptide to which the antibody is fused is useful for targeting the antibody to a specific cell type.

[0251] Additionally, the antibodies provided herein can be fused to a marker or "tag" sequence, such as a peptide, to facilitate purification. In certain embodiments, the amino acid sequence of the marker or tag is a hexahistidine peptide (SEQ ID NO: 93), such as the tag provided in the pQE vector (see, e.g., QIAGEN, Inc.), among others, many of which are commercially available. For example, as described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexahistidine (SEQ ID NO: 93) provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767-78), and the "FLAG" tag.

[0252] Methods for fusing or conjugating moieties (including polypeptides) to antibodies are known (e.g., Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, in Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al. eds., 1985); Hellstrom et al., Antibodies for Drug Delivery, in Controlled Drug Delivery 623-53 (Robinson et al. eds., 2d ed. 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, in Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al. eds., 1985); Analysis, Results, and Future Prospect of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy, in Monoclonal Antibodies for Cancer Detection and Therapy 475-506 (Pinchera et al. eds., 1985)). 303-16 (Baldwin et al. eds., 1985); Thorpe et al. al., 1982, Immunol.Rev.62:119-58, U.S. Patent No. 5,336,603, U.S. Patent No. 5,622,929, U.S. Patent No. 5,359,046, U.S. Patent No. No. 5,349,053, No. 5,447,851, No. 5,723,125, No. 5,783,181, No. 5,908,626, No. 5,844,095, and 5,112,946, European Patent Nos. 307,434, 367,166, 394,827, International Publication Nos. 91 / 06570, 96 / 04388, 96 / 22024, 97 / 34631, and 99 / 04813, Ashkenazi et al., 1991, Proc. Natl. Acad. Sci.USA 88:10535-39; Traunecker et al., 1988, Nature 331:84-86; Zheng et al., 1995, J. Immunol. 154:5590-600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-41).

[0253] Fusion proteins can be generated, for example, by techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activities of the antibodies provided herein, including, for example, antibodies with higher affinities and lower dissociation rates (see, e.g., U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13). Antibodies, or the encoded antibodies, may be altered by subjecting them to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. Polynucleotides encoding the antibodies provided herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

[0254] The antibodies (e.g., VHH domains) provided herein can be conjugated to a second antibody to form an antibody heteroconjugate, for example, as described in US Pat. No. 4,676,980.

[0255] Antibodies as provided herein can also be bound to solid supports, which are particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0256] The linker can be a "cleavable linker" that facilitates release of the conjugated drug within the cell, although non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates of the present disclosure include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids such as valine and / or citrulline, e.g., citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers (see, e.g., Chari et al., 1992, Cancer Res. 52:127-31; and U.S. Pat. No. 5,208,020), thioether linkers, or hydrophilic linkers designed to circumvent multidrug transporter-mediated resistance (see, e.g., Kovtun et al., 2010, Cancer Res. 70:2528-37).

[0257] Conjugates of antibodies and drugs can be made using a variety of bifunctional protein coupling agents, such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate). The present disclosure further contemplates that conjugates of antibodies and drugs can be prepared using any suitable method disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2d ed. 2008)).

[0258] Traditional antibody and drug conjugation strategies are based on random conjugation chemistry involving the ε-amino group of Lys residues or the thiol group of Cys residues, resulting in heterogeneous conjugates. Recently developed technologies enable site-specific conjugation to antibodies, resulting in uniform loading and avoiding conjugate subpopulations with altered antigen binding or pharmacokinetics. These include engineering "thiomab" antibodies that contain cysteine substitutions at positions on the heavy and light chains that provide reactive thiol groups and do not inhibit immunoglobulin folding and assembly or alter antigen binding (see, e.g., Junutula et al., 2008, J. Immunol. Meth. 332:41-52; and Junutula et al., 2008, Nature Biotechnol. 26:925-32). In another method, selenocysteine is co-translationally inserted into the antibody sequence by recoding the stop codon UGA from termination to a selenocysteine insertion, allowing site-specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of other natural amino acids (see, e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105:12451-56; and Hofer et al., 2009, Biochemistry 48(50):12047-57).

[0259] 7.9 Polynucleotides In certain embodiments, the present disclosure encompasses polynucleotides (interchangeably referred to herein as nucleic acids) encoding the antigen-binding molecules described herein or fragments thereof. The term "polynucleotide encoding a polypeptide" encompasses polynucleotides comprising only the coding sequence of a polypeptide, as well as polynucleotides comprising additional coding and / or non-coding sequences. Polynucleotides of the present disclosure may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, which may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand.

[0260] In certain embodiments, a polynucleotide comprises a coding sequence for a polypeptide, e.g., fused in frame with a polynucleotide that aids in the expression and secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence to control transport of the polypeptide). The polypeptide can have a leader sequence that is cleaved by the host cell to form a "mature" form of the polypeptide.

[0261] In certain embodiments, the polynucleotide comprises the coding sequence for the polypeptide fused in-frame to a marker or tag sequence. For example, in certain embodiments, in the case of a bacterial host, the marker sequence is a hexahistidine tag (SEQ ID NO: 93) provided by the vector, which allows for efficient purification of the polypeptide fused to the marker. In certain embodiments, the marker is used in conjunction with other affinity tags.

[0262] The present disclosure further relates to variants of the polynucleotides described herein, where the variants encode, for example, fragments, analogs, and / or derivatives of the polynucleotides. In certain embodiments, the present disclosure provides polynucleotides, including polynucleotides having a nucleotide sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in certain embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding a polypeptide comprising an antibody or antigen-binding fragment thereof described herein.

[0263] As used herein, the phrase "a polynucleotide having a nucleotide sequence at least, e.g., 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides of the reference sequence may be deleted or replaced with alternative nucleotides, or up to 5% of the total number of nucleotides of the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, and may be dispersed individually among the nucleotides of the reference sequence or in one or more contiguous groups within the reference sequence.

[0264] Polynucleotide variants can contain alterations in coding regions, non-coding regions, or both. In certain embodiments, polynucleotide variants contain alterations that result in silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In certain embodiments, polynucleotide variants contain silent substitutions that do not result in changes to the amino acid sequence of a polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be produced for a variety of reasons, such as to optimize codon expression for a particular host (i.e., to change the codons of human mRNA to those preferred by a bacterial host, such as E. coli). In certain embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0265] In some embodiments, polynucleotide variants are produced to modulate or alter expression (or expression levels) of an encoded polypeptide. In certain embodiments, polynucleotide variants are produced to increase expression of an encoded polypeptide. In certain embodiments, polynucleotide variants are produced to decrease expression of an encoded polypeptide. In certain embodiments, polynucleotide variants increase expression of an encoded polypeptide compared to the parent polynucleotide sequence. In certain embodiments, polynucleotide variants decrease expression of an encoded polypeptide compared to the parent polynucleotide sequence.

[0266] In certain embodiments, the present disclosure provides polynucleotides comprising a nucleotide sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in certain embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide listed in the sequence listing provided herein (e.g., SEQ ID NOs: 3 and 263-343).

[0267] In certain embodiments, the present disclosure provides polynucleotides comprising a nucleotide sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in certain embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide selected from those provided herein (e.g., SEQ ID NOs: 3 and 263-343).

[0268] In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure.

[0269] Also provided are vectors and cells comprising the polynucleotides described herein. In certain embodiments, an expression vector comprises the polynucleotide molecule. In certain embodiments, a host cell comprises an expression vector comprising the polynucleotide molecule. In certain embodiments, a host cell comprises one or more expression vectors comprising the polynucleotide molecule. In certain embodiments, a host cell comprises a polynucleotide molecule described herein. In certain embodiments, a host cell comprises one or more polynucleotide molecules described herein.

[0270] 7.10 Method or process for producing antigen-binding molecules In yet another aspect, the present specification provides a method or process for producing the antigen-binding molecules described herein. Although the following describes an antigen-binding molecule that is an antibody (e.g., a dual paratope antibody comprising a dimerization domain described herein), it is understood that the following is also applicable to other antigen-binding molecules of the present invention, unless understood to be inapplicable from the context.

[0271] Recombinant expression of the antigen-binding molecules provided herein can be achieved by constructing an expression vector containing one or more polynucleotides encoding the antigen-binding molecule. Once a polynucleotide encoding the antigen-binding molecule provided herein, the heavy or light chain of the antigen-binding molecule, or a fragment thereof (e.g., including, but not necessarily, the heavy and / or light chain variable domain) is obtained, a vector for producing the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Accordingly, methods for preparing a protein by expressing a polynucleotide comprising an antigen-binding molecule-encoding nucleotide sequence are described herein. Expression vectors containing antigen-binding molecule-encoding sequences and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding the antigen-binding molecule provided herein, the heavy or light chain of an anti-antigen-binding molecule, the heavy or light chain variable domain of an antigen-binding molecule or a fragment thereof, or the heavy or light chain CDRs, operably linked to a promoter. Such vectors may contain nucleotide sequences encoding the constant region of an antigen-binding molecule (see, e.g., WO 86 / 05807 and WO 89 / 01036 and U.S. Pat. No. 5,122,464), and antibody variable domains may be cloned into such vectors for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains. In certain embodiments, the antigen-binding molecule comprises one or more antigen-binding polypeptides having an antibody variable domain and having a dimerization domain described herein instead of the light chain constant domain (CL) or heavy chain constant domain 1 (CH1).

[0272] The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antigen-binding molecule provided herein. Accordingly, also provided herein is a host cell comprising a polynucleotide encoding an antigen-binding molecule provided herein or a fragment thereof, or a heavy or light chain thereof, or a fragment thereof, operably linked to a heterologous promoter. The host cell can be any type of cell, for example, a primary cell, a cell in culture, or a cell derived from a cell line. In certain embodiments, the host cell is a cell transfected with a nucleic acid molecule (e.g., a vector) provided herein. In certain embodiments, the host cell is the progeny or potential progeny of a cell transfected with a nucleic acid molecule (e.g., a vector) provided herein. In certain embodiments for expressing a double-chain antigen-binding molecule (e.g., a single paratope antigen-binding molecule), vectors encoding both the heavy and light chains can be co-expressed in the host cell for expression of the entire immunoglobulin molecule. In certain embodiments for expressing a quadruple-chain antigen-binding molecule (e.g., a double-paratope antigen-binding molecule), vectors encoding each of the heavy and light chains can be co-expressed in a host cell for expression of the entire immunoglobulin molecule. In certain embodiments for expressing a hexa-chain antigen-binding molecule (e.g., a triple-paratope antigen-binding molecule), vectors encoding each of the heavy and light chains can be co-expressed in a host cell for expression of the entire immunoglobulin molecule, as described in detail below. In certain embodiments for expressing a hexa-chain antigen-binding molecule, vectors encoding each of the heavy and light chains can be co-expressed in a host cell for expression of the entire immunoglobulin molecule, as described in detail below. In certain embodiments, the multi-paratope antigen-binding molecules described herein are produced in a single host cell.

[0273] Various host-expression vector systems can be utilized to express the antigen-binding molecules provided herein (see, for example, U.S. Pat. No. 5,807,715). Such host-expression systems represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also represent cells that can express the antigen-binding molecules provided herein in situ when transformed or transfected with an appropriate nucleotide coding sequence. These host-expression systems include microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with a recombinant bacteriophage DNA expression vector, plasmid DNA expression vector, or cosmid DNA expression vector containing the antigen-binding molecule coding sequence, and yeast (e.g., Saccharomyces pyogenes) transformed with a recombinant yeast expression vector containing the antigen-binding molecule coding sequence. Examples of suitable vectors include, but are not limited to, insect cells infected with a recombinant virus expression vector (e.g., baculovirus) containing an antigen-binding molecule coding sequence, plant cell systems infected with a recombinant virus expression vector (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) containing an antigen-binding molecule coding sequence or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing an antigen-binding molecule coding sequence, and mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter). Particularly for expression of whole recombinant antigen-binding molecules, bacterial cells such as Escherichia coli or eukaryotic cells can be used to express recombinant antigen-binding molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) can be used as an effective expression system for antigen-binding molecules when used in combination with a vector such as the major intermediate-early gene promoter element derived from human cytomegalovirus (Foecking et al., 1986, Gene 45:101, and Cockett et al., 1990, Bio / Technology 8:2).In certain embodiments, the antigen-binding molecules provided herein are produced in CHO cells. In certain embodiments, the expression of a nucleotide sequence encoding an antigen-binding molecule provided herein that immunospecifically binds to a target antigen is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter. In certain embodiments, the expression of a nucleotide sequence encoding an antigen-binding molecule provided herein that immunospecifically binds to a target antigen is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0274] In bacterial systems, many expression vectors can be advantageously selected depending on the intended use of the antigen-binding molecule to be expressed. For example, when producing large quantities of such antigen-binding molecules, a vector that directs the expression of a high-level fusion protein product that is easily purified to produce a pharmaceutical composition of the antigen-binding molecule may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther, et al., EMBO, 1983, 12:1791) and the pIN vector (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509), into which the antigen-binding molecule-encoding sequence can be ligated in frame with and separately from the lac Z coding region to produce a fusion protein. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0275] In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. This virus grows in Spodoptera frugiperda cells. The antigen-binding molecule coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0276] Many viral-based expression systems can be used in mammalian host cells. When adenovirus is used as an expression vector, the antigen-binding molecule coding sequence of interest can be ligated into an adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) can produce a viable recombinant virus capable of expressing the antibody molecule in infected hosts (see, for example, Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiation signals may also be required for efficient translation of the inserted antigen-binding molecule coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0277] Additionally, a host cell line can be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in the specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. By selecting the appropriate cell line or host system, the expressed foreign protein can be correctly modified and processed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In certain embodiments, the fully human antigen-binding molecules provided herein are produced in mammalian cells, such as CHO cells.

[0278] Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express antigen-binding molecules can be engineered. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) and a selectable marker. After introduction of the foreign DNA, engineered cells can be grown in an enriched medium for 1 to 2 days and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antigen-binding molecules. Such engineered cell lines can be particularly useful for screening and evaluating compositions that interact directly or indirectly with the antigen-binding molecule.

[0279] A number of selection systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17), genes that can be used in tk-, hgprt-, or aprt- cells, respectively. Antimetabolite resistance can also be used as a selection criterion for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al. 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al. 1981, Proc. Natl. Acad. Sci. USA 78:1527), gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072), and neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932, and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217, 1993, TIB TECH 11(5):155-215), and hygro, which confers resistance to hygromycin (Santerre, et al., Gene, 1984, 30:147).Methods generally known in the art in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clones, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli, et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated herein by reference in their entireties.

[0280] The expression level of antigen-binding molecules can be increased by vector amplification (for a review, see Bebbington and Hentschel, "The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning," Vol. 3 (Academic Press, New York, 1987)). If the marker in a vector system expressing an antigen-binding molecule is amplifiable, the copy number of the marker gene will increase when the level of an inhibitor present in host cell culture is increased. Because the amplified region is associated with the antigen-binding molecule gene, production of the antigen-binding molecule will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

[0281] A host cell can be co-transfected with two or more expression vectors provided herein. The two or more vectors can contain, for example, identical selectable markers that enable equal expression of heavy and light chain polypeptides of an antigen-binding molecule. Alternatively, a single vector can be used that encodes and expresses different component polypeptides of the antigen-binding molecule, for example, both the heavy and light chain polypeptides of the antigen-binding molecule. The coding sequence can comprise cDNA or genomic DNA.

[0282] Once the antigen-binding molecules provided herein are produced by recombinant expression, they can be purified or isolated by any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly Protein A chromatography followed by affinity chromatography for a specific antigen, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Furthermore, the antigen-binding molecules provided herein can be fused to heterologous amino acid sequences described herein or known in the art to facilitate purification.

[0283] 7.11 Pharmaceutical Compositions In one aspect, the present disclosure further provides a pharmaceutical composition comprising at least one antigen-binding molecule of the present disclosure. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an antigen-binding molecule provided herein and a pharmaceutically acceptable excipient. In certain embodiments, the antigen-binding molecule is isolated. In certain embodiments, the antigen-binding molecule is purified. Any of the antigen-binding molecules provided herein are contemplated to be in a pharmaceutical composition.

[0284] Pharmaceutical compositions containing the antigen-binding molecule or a fragment thereof are prepared by mixing the protein having the desired purity with any physiologically acceptable excipient (see, for example, Remington, Remington's Pharmaceutical Sciences (18th ed. 1980)) in the form of an aqueous solution or in lyophilized or other dried form for storage.

[0285] The antigen-binding molecules of the present disclosure can be formulated in any form suitable for delivery to target cells / tissues, for example, as microcapsules or macroemulsions (Remington, supra; Park, et al., Molecules, 2005, 10:146-61; Malik et al., 2007, Curr. Drug. Deliv. 4:141-51), sustained-release formulations ((Putney and Burke, 1998, Nature Biotechnol. 16:153-57), or in liposomes (Maclean et al., 1997, Int. J. Oncol. 11:325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8:39-45).

[0286] The antigen-binding molecules provided herein can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, prepared by, for example, coacervation or interfacial polymerization, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.), or in macroemulsions. Such techniques are disclosed, for example, in Remington (supra).

[0287] Various compositions and delivery systems are known and can be used with the antigen-binding molecules described herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antigen-binding molecule, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-32), construction of nucleic acids as part of retroviral or other vectors, etc. In another embodiment, the composition can be provided as a controlled-release or sustained-release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see, e.g., Langer (supra); Sefton, 1987, Crit. Ref. Biomed. Eng. 14:201-40; Buchwald et al., 1980, Surgery 88:507-16; and Saudek et al., 1989, N. Engl. J. Med. 321:569-74). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., antibodies or antigen-binding fragments thereof described herein), or compositions provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise, eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126; Levy et al., Science 228:190-92 (1985); During et al., 1989, Ann. Neurol. 25:351-56; Howard et al. al., 1989, J. Neurosurg. 71:105-12; U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326; WO 99 / 15154 and WO 99 / 20253).Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymers used in the sustained release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable.

[0288] In yet another embodiment, a controlled-release or sustained-release system can be placed in close proximity to a specific target tissue, such as the nasal cavity or lungs, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled-release systems are discussed, for example, by Langer, 1990, Science 249:1527-33. Any technique known to those of skill in the art can be used to prepare sustained-release formulations comprising one or more antibodies or antigen-binding fragments thereof described herein (e.g., U.S. Pat. No. 4,526,938; WO 91 / 05548 and WO 96 / 20698; Ning, et al., Radiotherapy & Oncology, 39:179-89 (1996); Song et al., 1995, PDA J. of Pharma. Sci. & Tech. 50:372-97; Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60).

[0289] 7.12 How to use In another aspect, provided herein are methods and uses for using the antigen-binding molecules provided herein. Such methods and uses include, for example, therapeutic methods and uses comprising administering the antigen-binding molecule or a composition containing the antigen-binding molecule to a subject having a disease or disorder. In certain embodiments, the subject is in need of treatment. In certain embodiments, the composition is administered in an amount effective to provide treatment for the disease or disorder in the subject. Uses include use of the composition in such methods and treatments, and in the preparation of a medicament for carrying out such treatments. In certain embodiments, the method is carried out by administering the composition to a subject having or suspected of having a disease or condition. In certain embodiments, the method thereby treats the disease or disorder in the subject.

[0290] In certain embodiments, the treatments provided herein result in a complete or partial improvement or reduction of a disease or disorder, or its associated symptoms, adverse effects or outcomes, or phenotype. Desirable effects of treatment include, but are not limited to, suppression of disease occurrence or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, suppression of metastasis, reduction of the rate of disease progression, improvement or amelioration of the disease state, and remission or improved prognosis. These terms include, but do not imply, complete cure of the disease or complete elimination of any symptoms or the effects of all symptoms or outcomes.

[0291] As used herein, in certain embodiments, the treatments provided herein delay the onset of a disease or disorder, e.g., postpone, prevent, slow, retard, stabilize, inhibit, and / or postpone the onset of a disease (such as cancer). This delay can be of varying duration, depending on the disease history and / or the individual receiving treatment. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease or disorder. For example, late-stage cancer, such as the development of metastases, can be delayed. In other embodiments, the methods or uses provided herein prevent a disease or disorder.

[0292] In yet another aspect, provided herein is a method for enriching, isolating, separating, purifying, sorting, selecting, capturing, detecting, or depleting cells expressing one or more target antigens, the method comprising: providing a sample containing cells expressing one or more target antigens; contacting the sample with the target antigens; and enriching, isolating, separating, purifying, sorting, selecting, capturing, detecting, or depleting cells that express the target antigens and that are bound to an antigen-binding molecule, wherein the antigen-binding molecule comprises a first paratope capable of binding to a first target antigen, optionally a second paratope capable of binding to a second target antigen, optionally a third paratope capable of binding to a third target antigen, and optionally a fourth paratope capable of binding to a fourth target antigen. In certain embodiments, the sample is a blood sample. In other embodiments, the sample is a tissue sample.

[0293] In another aspect, provided herein is a method for inhibiting or depleting cancer cells or T cells, the method comprising contacting cancer cells or T cells with an effective amount of an antigen-binding molecule that binds to at least one tumor-associated or tumor-specific antigen.

[0294] In another aspect, provided herein is a method for inhibiting or depleting cancer cells or T cells in a subject having cancer, the method comprising administering to the subject an effective amount of an antigen-binding molecule that binds to at least one tumor-associated or tumor-specific antigen.

[0295] In another aspect, provided herein is a method for treating a disease or disorder in a subject, comprising administering to the subject an effective amount of an antigen-binding molecule that binds to at least one target antigen.

[0296] In another aspect, provided herein is a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an antigen-binding molecule that binds to at least one tumor-associated or tumor-specific antigen.

[0297] In another aspect, provided herein is a method for treating a disease or disorder, wherein a subject is administered one or more therapeutic agents in combination with an effective amount of an antigen-binding molecule that binds to at least one target antigen.

[0298] In another aspect, provided herein is the use of an antibody provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0299] In another aspect, provided herein is the use of a pharmaceutical composition provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0300] In certain embodiments, provided herein are compositions comprising the antigen-binding molecules provided herein for use in the prevention and / or treatment of a disease or condition. In certain embodiments, the subject is a subject in need of such treatment. In certain embodiments, the subject has the disease or condition. In other embodiments, the subject is at risk of having the disease or condition. In certain embodiments, administration results in the prevention, management, treatment, or amelioration of the disease or condition.

[0301] In another embodiment, provided herein is a method for preventing and / or treating symptoms of a disease or condition in a subject, the method comprising administering an effective amount of an antigen binding molecule provided herein. In one embodiment, provided herein is a method for preventing symptoms of a disease or condition in a subject, the method comprising administering an effective amount of an antigen binding molecule provided herein. In certain embodiments, the subject is in need of treatment thereof. In certain embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of having a disease or condition. In certain embodiments, the administration results in prevention or treatment of symptoms of the disease or condition.

[0302] In certain embodiments, the disease is cancer, and the antigen-binding molecule is multiparatopic or multispecific. In certain embodiments, the disease is cancer, and the antigen-binding molecule is biparatopic and monospecific (e.g., both paratopes bind to a single target antigen) for one target antigen that is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). In certain embodiments, the disease is cancer, and the antigen-binding molecule is biparatopic and bispecific (e.g., each paratope binds to a different target antigen) for two target antigens selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), or a combination thereof. In certain embodiments, the disease is cancer, and the antigen-binding molecule is triparatopic and monospecific (e.g., each paratope binds to a single target antigen) for one target antigen that is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). In certain embodiments, the disease is cancer, and the antigen-binding molecule is tritopic and bispecific for two target antigens selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), or a combination thereof (e.g., two paratopes bind to a single target antigen and one paratope binds to a different target antigen). In certain embodiments, the disease is cancer, and the antigen-binding molecule is tritopic and trispecific for three target antigens selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), or a combination thereof (e.g., each paratope binds to a different target antigen). In certain embodiments, the disease is cancer, and the antigen-binding molecule is quadruple-paratopic and bispecific for two target antigens selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), or a combination thereof (e.g., two paratopes bind to a target antigen and two paratopes bind to different target antigens). In certain embodiments, the disease is cancer, and the antigen-binding molecule is quadruple paratopic and quadruple specific (e.g., each paratope binds to a different target antigen) for four target antigens selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), or a combination thereof. In certain embodiments, the disease is cancer, and the antigen-binding molecule is as shown in Table 4 below.In certain embodiments, the disease is cancer and the antigen-binding molecule is as shown in Table 4 below, except that each of the target antigens TAA1, TAA2, TAA3, or TAA4 is independently and optionally replaced with a tumor-specific antigen (e.g., TSA1, TSA2, TSA3, or TSA4). In certain embodiments, the paratope, or at least the first paratope, of the antigen-binding molecule described herein binds to i) a target antigen associated with cancer, or ii) a target antigen specific to cancer.

[0303] [Table 4]

[0304] In certain embodiments, the tumor-associated antigen (TAA) or tumor-specific antigen (TSA) is present on the surface of a cancer cell.

[0305] In certain embodiments, the cancer cell is a cell of adrenal gland cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational trophoblastic, head and neck cancer, Hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma spinal cancer, stomach cancer, testicular cancer, pharyngeal cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer.

[0306] In certain embodiments, the tumor-associated antigen (TAA) or tumor-specific antigen (TSA) is an angiopoietin, BCMA, CD19, CD20, CD22, CD25 (IL2-R), CD30, CD33, CD37, CD38, CD52, CD56, CD123 (IL-3R), cMET, DLL / Notch, EGFR, EpCAM, FGF, FGF-R, GD2, HER2, mesothelin, nectin-4, PAP, PDGFRα, PSA, PSA3, PSMA, RANKL, SLAMF7, STEAPI, TARP, TROP2, VEGF, or VEGF-R antigen. In some embodiments, the tumor-associated antigen (TAA) or tumor-specific antigen (TSA) is CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EpCAM, EphA3, Her2 / neu, telomerase, mesothelin, SAP-1, survivin, BAGE family antigens, CAGE family antigens, GAGE family antigens, MAGE family antigens, SAGE family antigens, XAGE family antigens, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A, MART-1, Gp100, pmel17, tyrosinase, TRP-1, TRP-2, P polypeptide, MC1R, prostate-specific antigen, β-catenin, or BRCA1 antigen.

[0307] In certain embodiments, the tumor-associated antigen (TAA) or tumor-specific antigen (TSA) is HER2 or MET.

[0308] Provided herein are methods for preventing and / or treating a disease or condition by administering an effective amount of an antigen-binding molecule provided herein or a pharmaceutical composition comprising an antigen-binding molecule provided herein to a subject. In one aspect, the antigen-binding molecule is substantially purified (i.e., substantially free from substances that limit its effect or cause undesirable side effects). In certain embodiments, the subject is any animal, preferably a mammal (e.g., any mammal), most preferably a human. In certain embodiments, the mammal is a non-primate or a primate. In certain embodiments, the mammal is selected from the group consisting of cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans. In preferred embodiments, the subject is a human. In certain embodiments, the subject is a human with a disease or condition. In certain embodiments, the subject is a human with cancer.

[0309] Various delivery systems are known and can be used to administer prophylactic or therapeutic agents (e.g., antigen-binding molecules provided herein), including, but not limited to, liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing antibodies or antigen-binding fragments thereof, receptor-mediated endocytosis (e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), and construction of nucleic acids as part of retroviral or other vectors. Methods of administering prophylactic or therapeutic agents (e.g., antigen-binding molecules provided herein) or pharmaceutical compositions include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In certain embodiments, prophylactic or therapeutic agents (e.g., antigen-binding molecules provided herein) or pharmaceutical compositions are administered intranasally, intramuscularly, intravenously, or subcutaneously. The prophylactic or therapeutic agents or compositions can be administered by any convenient route, e.g., by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, nasal, rectal, and intestinal mucosa), and can be administered in conjunction with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can be employed, e.g., by use of an inhaler or nebulizer and formulation with an aerosolizing agent. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078, and WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[0310] In certain embodiments, it may be desirable to administer the prophylactic or therapeutic agents or pharmaceutical compositions provided herein locally to the area in need of treatment. This can be achieved, for example, but not by way of limitation, by local infusion, by topical administration (e.g., by nasal spray), by injection, or using an implant, which can be a porous, non-porous, or gelatinous material, including membranes such as silastic membranes or fibers. In certain embodiments, when administering the antigen-binding molecules provided herein, care must be taken to use materials to which the antigen-binding molecules do not adsorb.

[0311] In another embodiment, the prophylactic or therapeutic agents or compositions provided herein can be delivered in a vesicle, in particular a liposome (see Langer, Science, 1990, 249:1527-1533; Treat, et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally, ibid.).

[0312] In another embodiment, the prophylactic or therapeutic agents, or compositions provided herein can be delivered in a controlled or sustained release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., Surgery, 1980, 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., antibodies provided herein), or compositions provided herein (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23-61; also, Levy, et al., Science, 1985, 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al. al., 1989, J. Neurosurg. 7:1-105), see also U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, 5,128,326, WO 99 / 15154, and WO 99 / 20253).Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In certain embodiments, the polymers used in sustained-release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled-release or sustained-release system can be placed in close proximity to the therapeutic target, i.e., the nasal cavity or lungs, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Release (supra), vol. 2, pp. 115-138 (1984)). Controlled-release systems are discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to those skilled in the art can be used to produce sustained-release formulations containing one or more antigen-binding molecules provided herein.See, for example, U.S. Pat. No. 4,526,938, WO 91 / 05548, WO 96 / 20698, Ning et al., 1996, "Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy & Oncology 39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Proc. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al., 1997, "Microencapsulation of Recombinant See, "Humanized Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in its entirety.

[0313] In certain embodiments in which the compositions provided herein are polynucleotides encoding prophylactic or therapeutic agents (e.g., antigen-binding molecules provided herein), the polynucleotides can be constructed as part of an appropriate polynucleotide expression vector and administered in vivo to promote expression of the encoded prophylactic or therapeutic agent, for example, by using a retroviral vector, administering the vector so that the polynucleotide becomes an intracellular polynucleotide (see U.S. Pat. No. 4,980,286), or by direct injection, or by microparticle bombardment (e.g., a gene gun; Biolistic, DuPont), or by using coating with lipids or cell surface receptors or transfection agents, or by administering the nucleic acid in linkage to a homeobox-like peptide known to enter the nucleus (see, e.g., Joliot, et al., 1991, Proc. Natl. Acad. Sci. USA, 88:1864-1868). Alternatively, the polynucleotides can be introduced intracellularly and integrated into host cell DNA for expression by homologous recombination.

[0314] In certain embodiments, the compositions provided herein comprise one, two or more antigen-binding molecules provided herein. In another embodiment, the compositions provided herein comprise one, two or more antigen-binding molecules provided herein and a prophylactic or therapeutic agent other than the antigen-binding molecules provided herein. In one embodiment, the agent is known to be useful for, is being used for, or is currently being used for the prevention, management, treatment, and / or amelioration of a disease or condition. In addition to the prophylactic or therapeutic agent, the compositions provided herein may also comprise an excipient.

[0315] The compositions provided herein include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., compositions suitable for administration to a subject or patient) that can be used to prepare unit dosage forms. In some embodiments, the compositions provided herein are pharmaceutical compositions. Such compositions comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., an antigen-binding molecule provided herein or other prophylactic or therapeutic agent) and a pharmaceutically acceptable excipient. Pharmaceutical compositions can be formulated to be suitable for the route of administration to a subject.

[0316] In certain embodiments, the term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle. Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a typical excipient when pharmaceutical compositions are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, glycol, water, ethanol, and the like. If desired, the composition can further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions contain a prophylactically or therapeutically effective amount of the antigen-binding molecule provided herein, such as in purified form, in combination with appropriate amounts of excipients to provide a form for proper administration to a patient. The formulation should suit the mode of administration.

[0317] In embodiments, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile aqueous isotonic buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. However, such compositions may be administered via routes other than the intravenous route.

[0318] The components of the compositions provided herein are supplied individually or mixed together in unit dosage form, for example, as a dry lyophilized powder or a moisture-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of active ingredient.When the composition is administered by injection, the composition can be dispensed using an infusion bottle containing pharmaceutical-grade sterile water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0319] The antigen-binding molecules provided herein may be packaged in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of the antigen-binding molecule. In one embodiment, the antigen-binding molecules are supplied as a dry, sterilized, lyophilized powder or water-free concentrate in a hermetically sealed container, which can be reconstituted with, for example, water or saline, to the appropriate concentration for administration to a subject. The lyophilized antigen-binding molecules can be stored in their original container at 2-8°C, and the antibodies or antigen-binding fragments thereof can be administered within 12 hours (e.g., within 6 hours, within 5 hours, within 3 hours, or within 1 hour) after reconstitution. In an alternative embodiment, the antigen-binding molecules provided herein are supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the antigen-binding molecule.

[0320] The compositions provided herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0321] The amount of a prophylactic or therapeutic agent (e.g., an antigen-binding molecule provided herein) or a composition provided herein that is effective in the prevention and / or treatment of a disease or condition can be determined by standard clinical techniques. In addition, in vitro assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances.

[0322] Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0323] In certain embodiments, the route of administration of a dose of an antigen-binding molecule provided herein to a patient is intranasal, intramuscular, intravenous, subcutaneous, or a combination thereof, although other routes described herein are also acceptable. Each dose may or may not be administered via the same route of administration. In certain embodiments, an antigen-binding molecule provided herein may be administered via multiple routes of administration simultaneously with or after other doses of the same or different antigen-binding molecule provided herein.

[0324] In certain embodiments, the antigen-binding molecules provided herein are administered to a subject prophylactically or therapeutically. The antigen-binding molecules provided herein can be administered to a subject prophylactically or therapeutically to prevent, reduce, or ameliorate a disease or its symptoms.

[0325] 7.13 Gene Therapy In certain embodiments, polynucleotides comprising sequences encoding antigen-binding molecules or functional derivatives thereof are administered to a subject for use in the methods provided herein to prevent, manage, treat, and / or ameliorate a disease, disorder, or condition, e.g., by gene therapy. Such treatments include those performed by administering expressed or expressible polynucleotides to a subject. In one embodiment, the polynucleotides produce their encoded antigen-binding molecules, and the antigen-binding molecules mediate a prophylactic or therapeutic effect. Any method for recombinant gene expression (or gene therapy) available in the art can be used.

[0326] For general reviews of gene therapy methods, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIBTECH 11(5):155-215. Methods generally known in the field of recombinant DNA technology that can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993) and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0327] In certain embodiments, a composition comprises a polynucleotide encoding an antigen-binding molecule provided herein, wherein the polynucleotide is part of an expression vector that expresses the antigen-binding molecule or its heavy or light chain polypeptide in a suitable host. Specifically, such a polynucleotide has a promoter (e.g., a heterologous promoter) operably linked to the antigen-binding molecule-encoding region, which promoter is inducible or constitutive, and optionally, tissue-specific. In another specific embodiment, a polynucleotide molecule is used in which the antigen-binding molecule-encoding sequence and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing intrachromosomal expression of the antibody-encoding polynucleotide (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

[0328] Delivery of a polynucleotide (e.g., nucleic acid) to a subject can be either direct (where the subject is directly exposed to the polynucleotide or polynucleotide-carrying vector) or indirect (where cells are first transformed with the polynucleotide in vitro and then transplanted into the subject). These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

[0329] In certain embodiments, polynucleotide sequences are directly administered in vivo, where they are expressed to produce the encoded product. This can be achieved by any of a number of methods known in the art, for example, by constructing them as part of a suitable nucleic acid expression vector and administering the vector so that the sequences become intracellular, for example, by infection using a defective or attenuated retroviral vector or other viral vector (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by using microparticle bombardment (e.g., a gene gun) (Biolistic, DuPont), or by coating with lipids or cell surface receptors or gene transfer agents, encapsulating in liposomes, microparticles, or microcapsules, or by administering them in conjunction with a peptide known to enter the nucleus, or by administering them in conjunction with a ligand that undergoes receptor-mediated endocytosis (e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), which can be used to target cell types that specifically express the receptor. In another embodiment, the ligand can comprise a fusogenic viral peptide for disrupting endosomes, forming a nucleic acid-ligand complex that allows the nucleic acid to avoid lysosomal degradation. In yet another embodiment, nucleic acids can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (see, e.g., WO 92 / 06180, WO 92 / 22635, WO 92 / 20316, WO 93 / 14188, WO 93 / 20221). Alternatively, nucleic acids can be introduced into cells by homologous recombination and integrated into host cell DNA for expression (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935, and Zijlstra et al., 1989, Nature 342:435-438).

[0330] In certain embodiments, viral vectors containing polynucleotide sequences encoding antigen-binding molecules are used. For example, retroviral vectors can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for accurate packaging of the viral genome and integration into host cell DNA. Polynucleotide sequences encoding antigen-binding molecules used in gene therapy can be cloned into one or more vectors, which facilitate delivery of the gene to a subject. Further details about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of retroviral vectors to deliver the MDR1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references describing the use of retroviral vectors in gene therapy are as follows: Clowes et al., 1994, J.Clin.Invest.93:644-651, Klein et al., (1994)Blood 83:1467-1473), Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141, and Grossman and Wilson, 1993, Curr.Opin.in Genetics and Devel.3:110-114.

[0331] Adenovirus is another viral vector that can be used in the recombinant production of antigen-binding molecules. Adenovirus is a particularly attractive vehicle for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia, where they cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, present a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10, demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld, et al., 1992, Cell 68:143-155; Zheng et al., 1993, J. Immunol. Invest. 91:225-234; WO 94 / 12649; and Wang et al., 1995, Gene Therapy 2:775-783. In some embodiments, adenovirus vectors are used.

[0332] Adeno-associated viruses (AAV) can also be used (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300, and U.S. Pat. No. 5,436,146). In certain embodiments, AAV vectors are used to express the antigen-binding molecules provided herein. In certain embodiments, the AAV comprises a polynucleotide encoding one or more polypeptides of the antigen-binding molecule. In certain embodiments, the AAV comprises a polynucleotide encoding one or more light chain polypeptides of the antigen-binding molecule. In certain embodiments, the AAV comprises a polynucleotide encoding one or more heavy chain polypeptides of the antigen-binding molecule.

[0333] Another approach to gene therapy involves transferring a gene into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated gene transfer, or viral infection. Usually, the transfer method includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate cells that have taken up and are expressing the transferred gene. These cells are then delivered to a subject.

[0334] In certain embodiments, a polynucleotide (e.g., a nucleic acid) is introduced into a cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be accomplished by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing a polynucleotide sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques for the introduction of foreign genes into cells are known in the art (e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Clin. Pharma. Ther. 29:69-92 (1985)), and can be used in accordance with the methods provided herein, provided that the necessary developmental and physiological functions of the recipient cell are not disrupted. The technique should provide for the stable transfer of a nucleic acid (a nucleic acid) to the cell, so that the polynucleotide is expressible by the cell (e.g., heritable and expressible by its cell progeny).

[0335] The resulting recombinant cells can be delivered to a subject by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) can be administered intravenously. The amount of cells envisioned for use depends on the desired effect, the patient's condition, etc., and can be determined by one skilled in the art.

[0336] Cells into which polynucleotides can be introduced for purposes of gene therapy include any desired available cell type, including, but not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes, and various stem or progenitor cells, particularly hematopoietic stem or progenitor cells, such as those obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

[0337] In certain embodiments, the cells used in gene therapy are autologous to the subject.

[0338] In embodiments in which recombinant cells are used in gene therapy, polynucleotide sequences encoding antigen-binding molecules are introduced into the cells so that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In certain embodiments, stem or progenitor cells are used. Any stem and / or progenitor cells that can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the methods provided herein (see, e.g., WO 94 / 08598; Stemple and Anderson, 1992, Cell 7 1:973-985; Rheinwald, 1980, Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).

[0339] In certain embodiments, a polynucleotide introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the polynucleotide can be controlled by controlling the presence or absence of the appropriate transcription inducer.

[0340] 7.14 Diagnostic Assays and Methods Labeled antigen-binding molecules and derivatives and analogs thereof that immunospecifically bind to a target antigen can be used for diagnostic purposes to detect, diagnose, or monitor a disease or disorder.

[0341] The antigen-binding molecules provided herein can be used to assay antigen levels in biological samples using classical immunohistological methods described herein or known to those skilled in the art (see, e.g., Jalkanen et al., 1985, J. Cell. Biol. 101:976-985, and Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096). Other antigen-binding molecule-based methods useful for detecting protein gene expression include enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include radioisotopes such as glucose oxidase, iodine (I, I), carbon (C), sulfur (S), tritium (H), indium (In), technetium (Tc), luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodamine, and enzyme labels such as biotin. One aspect provided herein is the detection and diagnosis of disease or disorders in humans.

[0342] It is understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is typically in the range of about 5 to 20 millicuries of 99Tc. The labeled antigen-binding molecule then accumulates at the location of cells containing the specific protein. In vivo tumor imaging is described in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 of Tumor Imaging: The Radiochemical Detection of Cancer, SW Burchiel and BA Rhodes, eds., Masson Publishing Inc. (1982)).

[0343] Depending on several variables, including the type of label used and the mode of administration, the time interval after administration to allow the labeled antibody to concentrate at the site of interest and for unbound labeled antibody to clear to background levels is 6 to 48 hours, or 6 to 24 hours, or 6 to 12 hours, hi other embodiments, the time interval after administration is 5 to 20 days, or 5 to 10 days.

[0344] In one embodiment, monitoring of the disease or disorder is performed by repeating the method for diagnosing the disease or disorder, e.g., one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

[0345] The presence of labeled antigen-binding molecules can be detected in a subject using methods known in the art for in vivo scanning.These methods depend on the type of label used.Those skilled in the art can determine the appropriate method for detecting a particular label.Methods and devices that can be used in the diagnostic methods provided herein include, but are not limited to, whole-body scanning such as computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound examination.

[0346] In a specific embodiment, the antigen-binding molecule is labeled with a radioisotope and is detected in the patient using a radiation-responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the antigen-binding molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence-responsive scanning instrument. In another embodiment, the antigen-binding molecule is labeled with a positron-emitting metal and is detected in the patient using positron emission tomography. In yet another embodiment, the antigen-binding molecule is labeled with a paramagnetic label and is detected in the patient using magnetic resonance imaging (MRI).

[0347] 7.15 Kit Also provided herein are kits containing the antigen-binding molecules provided herein or compositions thereof (e.g., pharmaceutical compositions) packaged in suitable packaging material. The kits optionally include a label or package insert containing a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components therein.

[0348] The term "packaging material" refers to a physical structure that contains the components of the kit. The packaging material can maintain the sterility of the components and can be made from materials commonly used for such purposes (e.g., paper, cardboard, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0349] The kits provided herein can include a label or insert. A label or insert includes "printed matter," such as paper or cardboard, separate from or attached to a component, kit, or packaging material (e.g., a box), or, for example, attached to an ampoule, tube, or vial containing a kit component. The label or insert can further include a computer-readable medium, such as a disk (e.g., hard disk, card, memory disk), CD or DVD-ROM / RAM, DVD, MP3, optical disk such as magnetic tape, or electronic storage medium such as RAM and ROM, or hybrids thereof, such as magnetic / optical storage medium, FLASH media, or memory-type cards. The label or insert can include information identifying the manufacturer, lot number, manufacturer's address, and date.

[0350] The kits provided herein can further include other components. Each component of the kit can be enclosed in an individual container, and all of the various containers can be in a single package. The kits can also be designed for refrigeration. The kits can further be designed to include cells comprising an antibody provided herein or a polynucleotide encoding an antibody provided herein. The cells in the kit can be maintained under appropriate storage conditions until ready for use.

[0351] Also provided herein are panels of antigen-binding molecules that immunospecifically bind to a target antigen. In certain embodiments, provided herein are panels of antigen-binding molecules having different association rate constants, different dissociation rate constants, different affinities, and / or different specificities for the target antigen. In certain embodiments, provided herein are panels of about 10, preferably about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 or more. Panels of antigen-binding molecules can be used in assays such as ELISA, for example, in 96-well or 384-well plates.

[0352] As used herein, numerical values are often presented in range format throughout this document. The use of the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention unless the context clearly dictates otherwise. Thus, the use of a range explicitly includes all possible subranges, all individual numerical values within that range, and all numerical values or ranges, including integers within such ranges and fractions of values or integers within the range, unless the context clearly dictates otherwise. This configuration applies in all contexts throughout this patent document, regardless of the breadth of the range. Thus, for example, a reference to a range of 90 to 100% includes 91 to 99%, 92 to 98%, 93 to 95%, 91 to 98%, 91 to 97%, 91 to 96%, 91 to 95%, 91 to 94%, 91 to 93%, etc. References to the range 90-100% include 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc.

[0353] For brevity, certain abbreviations are used herein. One example is the single letter abbreviations that represent amino acid residues. The amino acids and their corresponding three letter and one letter abbreviations are shown in Table 5 below.

[0354] [Table 5]

[0355] As used herein, the term "percent identity" in the context of two or more polypeptide or nucleotide sequences (e.g., antigen-binding molecules or fragments thereof described herein, and the polynucleotides encoding them) refers to two or more sequences or subsequences that have a specified percentage of identical amino acid residues or nucleotides. Percent identity can be determined by aligning two related sequences for maximum correspondence using sequence comparison algorithms known in the art, or by visual inspection.

[0356] Optimal alignment of sequences for comparison can be determined by sequence comparison algorithms, which can be the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or visual inspection (see generally, Current Protocols in Molecular Biology, FMAusubel et al., eds., Current Protocols, Greene Publishing Associates, Inc. and John This can be done through a collaboration with Wiley & Sons, Inc. (1995 Supplement) (see Ausubel).

[0357] Suitable comparison algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match when aligned with words of the same length in database sequences or satisfy some positive threshold score T, where T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0358] For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score drops by an amount X from its maximum achieved value, when the accumulation of one or more alignments of negatively scoring residues causes the cumulative score to fall below zero, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0359] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or two amino acid sequences will occur by chance. For example, a polynucleotide (e.g., nucleic acid) is considered similar to a reference sequence if the smallest sum probability in a comparison of the test polynucleotide with the reference polynucleotide is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0360] The present invention is generally disclosed herein using affirmative language to describe numerous embodiments. The present invention also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays or analyses, is excluded in whole or in part. Thus, the present specification generally discloses aspects not explicitly included in the present invention, even if the specification does not expressly state otherwise.

[0361] Many embodiments of the present invention have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate, but not limit, the scope of the invention as claimed.

[0362] 8. Implementation The present invention provides the following non-limiting embodiments: As contemplated herein, any of embodiments A1-A39, B1-B33, C1-C39, D1-D31, and E1-E16 may be combined and / or applied to the claims described herein.

[0363] In one set of embodiments (Embodiment Set A), the following are provided: A1. An antigen-binding molecule, a first light chain polypeptide comprising, in amino- to carboxy-terminal order, VL1-LD1, where VL1 is a first light chain variable domain and LD1 is a first light chain dimerization domain; a first heavy chain polypeptide comprising, in order from amino terminus to carboxy terminus, VH1-HD1, where VH1 is a first heavy chain variable domain and HD1 is a first heavy chain dimerization domain; where: a) i) LD1 comprises a β2 microglobulin (B2M) domain and HD1 comprises an HLA-E A3 (EA3) domain, or LD1 comprises an EA3 domain and HD1 comprises a B2M domain, and the B2M domain and the EA3 domain bind to each other to form a dimer, or ii) LD1 comprises a first ICAM-1 D1 domain, HD1 comprises a second ICAM-1 D1 domain, and the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer; b) i) at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively; ii) the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain; c) An antigen-binding molecule, wherein VL1 and VH1 form a first paratope. A2. LD1 comprises a B2M domain and HD1 comprises an EA3 domain, or LD1 comprises an EA3 domain and HD1 comprises a B2M domain, and the B2M domain and the EA3 domain bind to each other to form a dimer; B2M domains are a) the amino acid sequence of RTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 2); b) an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 2; c) an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 single amino acid substitutions relative to the sequence of SEQ ID NO: 2 at one or more amino acid positions 4, 8, 10, 54, 58, 60, 96, and 97 of SEQ ID NO: 2; c) an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 single amino acid substitutions relative to the sequence of SEQ ID NO: 2 at one or more amino acid positions 4, 8, 10, 54, 58, 60, 96, and 97 of SEQ ID NO: 2, wherein each single amino acid substitution is independently selected from the group consisting of F, W, C, S, and T; e) the amino acid sequence of the human B2M sequence shown in SEQ ID NO: 2, but comprising the set of substitutions F56S, W60S, and F62T, with the positions numbered according to the numbering of the B2M amino acids in Table 1, and optionally further comprising 1, 2, 3, 4, or 5 single amino acid substitutions at positions K6, Y10, R12, D98, or M99, with the positions numbered according to the numbering of the B2M amino acids in Table 1; f) The amino acid sequence of the human B2M sequence shown in SEQ ID NO: 2, but with positions numbered according to the B2M amino acid numbering in Table 1; i) F56S, W60S, F62T, and K6C; ii) F56S, W60S, F62T, and one of Y10C, Y10F, and Y10W; iii) F56S, W60S, F62T, and R12C; iv) F56S, W60S, F62T, and one of D98C, D98F, and D98W; v) an amino acid sequence comprising a set of substitutions selected from F56S, W60S, F62T, and any one of M99C, M99F, and M99W; or The antigen-binding molecule of embodiment A1, comprising any one of the amino acid sequences of SEQ ID NOs: 4 to 30. A3. LD1 comprises a B2M domain and HD1 comprises an EA3 domain, or LD1 comprises an EA3 domain and HD1 comprises a B2M domain, and the B2M domain and the EA3 domain bind to each other to form a dimer; The EA3 domain is a) the amino acid sequence of LHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW (SEQ ID NO: 33); b) an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 33; c) an amino acid sequence having 1, 2, 3, 4, 5, or 6 single amino acid substitutions relative to the sequence of SEQ ID NO: 33 at one or more amino acid positions 13, 23, 53, 55, 59, and 63 of SEQ ID NO: 33; d) an amino acid sequence having 1, 2, 3, 4, 5, or 6 single amino acid substitutions relative to the sequence of SEQ ID NO: 33 at one or more amino acid positions 13, 23, 53, 55, 59, and 63 of SEQ ID NO: 33, wherein each single amino acid substitution is independently selected from the group consisting of A, C, and L; or e) The amino acid sequence of the human EA3 sequence shown in SEQ ID NO: 33, but containing 1, 2, 3, 4, 5, or 6 single amino acid substitutions at positions H192, R202, E232, R234, D238, or Q242, with positions numbered according to the EA3 amino acid numbering in Table 2. f) The amino acid sequence of the human EA3 sequence shown in SEQ ID NO: 33, with positions numbered according to the EA3 amino acid numbering in Table 2; i) H192C, ii) R202A or R202C, iii) E232C, iv) R234A, R234L, or R234C; v) D238C, vi) Q242A or Q242L, vii) R234A and Q242A, viii) R234A and Q242L, ix) R234A and Q242A, or x) an amino acid sequence comprising a substitution or set of substitutions selected from R234L and Q242L, or g) The antigen-binding molecule of embodiment A1 or A2, comprising any one of the amino acid sequences of SEQ ID NOs: 35 to 46. A4. LD1 comprises a first ICAM-1 D1 domain, HD1 comprises a second ICAM-1 D1 domain, and the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer; Each of the first ICAM-1 D1 domain and the second ICAM-1 D1 domain comprises: a) the amino acid sequence of QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVY (SEQ ID NO: 49); b) an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 49; c) an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single amino acid substitutions relative to the sequence of SEQ ID NO: 49 at one or more amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67, and 78 of SEQ ID NO: 49, and optionally further comprising a C-terminal cysteine amino acid addition; d) an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single ami...

Claims

1. An antigen-binding molecule, a first light chain polypeptide comprising, in amino- to carboxy-terminal order, VL1-LD1, where VL1 is a first light chain variable domain and LD1 is a first light chain dimerization domain; a first heavy chain polypeptide comprising, in amino-terminal to carboxy-terminal order, VH1-HD1, where VH1 is a first heavy chain variable domain and HD1 is a first heavy chain dimerization domain; a) i) LD1 comprises a β2 microglobulin (B2M) domain and HD1 comprises an HLA-E A3 (EA3) domain, or LD1 comprises an EA3 domain and HD1 comprises a B2M domain, and the B2M domain and the EA3 domain bind to each other to form a dimer; or ii) LD1 comprises a first ICAM-1 D1 domain, and HD1 comprises a second ICAM-1 D1 domain, and the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer; b) i) at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 33), respectively; ii) the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain; c) An antigen-binding molecule, wherein VL1 and VH1 form a first paratope.

2. LD1 comprises the B2M domain and HD1 comprises the EA3 domain, or LD1 comprises the EA3 domain and HD1 comprises the B2M domain, and the B2M domain and the EA3 domain bind to each other to form a dimer; The B2M domain a) the amino acid sequence of RTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 2); b) an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2; c) an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 single amino acid substitutions relative to the sequence of SEQ ID NO: 2 at one or more amino acid positions 4, 8, 10, 54, 58, 60, 96, and 97 of SEQ ID NO: 2; d) an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 single amino acid substitutions relative to the sequence of SEQ ID NO: 2 at one or more amino acid positions 4, 8, 10, 54, 58, 60, 96, and 97 of SEQ ID NO: 2, wherein each of said single amino acid substitutions is independently selected from the group consisting of F, W, C, S, and T; e) an amino acid sequence of the human B2M sequence shown in SEQ ID NO: 2, but comprising the set of substitutions F56S, W60S, and F62T, with the positions numbered according to the numbering of said B2M amino acids in the table below, and optionally further comprising 1, 2, 3, 4, or 5 single amino acid substitutions at positions K6, Y10, R12, D98, or M99, with the positions numbered according to the numbering of said B2M amino acids in the table below; 【Table 1】 f) an amino acid sequence of the human B2M sequence shown in SEQ ID NO:2, but comprising a set of substitutions selected from the following, with positions numbered according to the numbering of said B2M amino acids in the table below: i) F56S, W60S, F62T, and K6C; ii) F56S, W60S, F62T, and any one of Y10C, Y10F, and Y10W; iii) F56S, W60S, F62T, and R12C; iv) F56S, W60S, F62T, and any one of D98C, D98F, and D98W; v) F56S, W60S, F62T, and any one of M99C, M99F, and M99W; or 【Table 2】 g) The antigen-binding molecule of claim 1, comprising any one of the amino acid sequences of SEQ ID NOs: 4 to 30.

3. LD1 comprises the B2M domain and HD1 comprises the EA3 domain, or LD1 comprises the EA3 domain and HD1 comprises the B2M domain, and the B2M domain and the EA3 domain bind to each other to form a dimer; The EA3 domain is a) the amino acid sequence of LHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW (SEQ ID NO: 33); b) an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 33; c) an amino acid sequence having 1, 2, 3, 4, 5, or 6 single amino acid substitutions relative to the sequence of SEQ ID NO: 33 at one or more amino acid positions 13, 23, 53, 55, 59, and 63 of SEQ ID NO: 33; d) an amino acid sequence having 1, 2, 3, 4, 5, or 6 single amino acid substitutions relative to the sequence of SEQ ID NO: 33 at one or more of amino acid positions 13, 23, 53, 55, 59, and 63 of SEQ ID NO: 33, wherein each of the single amino acid substitutions is independently selected from the group consisting of A, C, and L; e) the amino acid sequence of the human EA3 sequence set forth in SEQ ID NO: 33, but containing 1, 2, 3, 4, 5, or 6 single amino acid substitutions at positions H192, R202, E232, R234, D238, or Q242, with positions numbered according to the numbering of the EA3 amino acids in the table below: 【Table 3】 f) the amino acid sequence of the human EA3 sequence shown in SEQ ID NO: 33, but comprising a substitution or set of substitutions selected from the following, with positions numbered according to the numbering of said EA3 amino acids in the table below: i) H192C, ii) R202A or R202C; iii) E232C, iv) R234A, R234L, or R234C; v) D238C, vi) Q242A or Q242L, vii) R234A and Q242A; viii) R234A and Q242L; ix) R234A and Q242A; x) R234L and Q242L, or 【Table 4】 g) The antigen-binding molecule of claim 1 or 2, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 35 to 46.

4. LD1 comprises the first ICAM-1 D1 domain, HD1 comprises the second ICAM-1 D1 domain, and the first ICAM-1 D1 domain and the second ICAM-1 D1 domain bind to each other to form a dimer; Each of the first ICAM-1 D1 domain and the second ICAM-1 D1 domain comprises: a) the amino acid sequence of QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVY (SEQ ID NO: 49); b) an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 49; c) an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single amino acid substitutions relative to the sequence of SEQ ID NO: 49 at one or more amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67, and 78 of SEQ ID NO: 49, and optionally further comprising a C-terminal cysteine amino acid addition; d) an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single amino acid substitutions relative to the sequence of SEQ ID NO: 49 at one or more amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67, and 78 of SEQ ID NO: 49, wherein each of said single amino acid substitutions is independently selected from the group consisting of V, T, F, W, A, K, E, C, and R, and optionally further comprising a C-terminal cysteine amino acid addition; e) the amino acid sequence of human ICAM-1 D1 sequence as set forth in SEQ ID NO:49, but comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 single amino acid substitutions at positions T2, I10, R13, L18, T20, T23, E34, P38, L42, R49, V51, E53, P63, S67, or T78, wherein the positions are numbered according to the numbering of said ICAM-1 D1 amino acids in the table below, and optionally further comprising a C-terminal cysteine amino acid addition (84C), wherein the positions are numbered according to the numbering of said ICAM-1 D1 amino acids in the table below; 【Table 5】 f) an amino acid sequence of the human ICAM-1D1 sequence set forth in SEQ ID NO:49, but containing a set of substitutions selected from the following, with positions numbered according to the numbering of said ICAM-1 D1 amino acids in the table below: i) E34K, ii) T2V, I10T, T23A, E34K, P38T, P63V, S67A, and T78A; iii) T2V, I10T, R13C, T23A, E34K, P38T, R49E, P63V, S67A, T78A, and 84C; iv) T2V, I10T, R13C, T23A, E34K, P38T, E53R, P63V, S67A, T78A, and 84C; v) T2V, I10T, R13C, L18F, T23A, E34K, P38T, P63V, S67A, T78A, and 84C; vi) T2V, I10T, R13C, L18A, T20A, T23A, E34K, P38T, L42A, V51A, P63V, S67A, T78A, and 84C; vii) T2V, I10T, R13C, L18W, T23A, E34K, P38T, P63V, S67A, T78A, and 84C; or 【Table 6】 g) The antigen-binding molecule of claim 1, comprising a different amino acid sequence selected from the group consisting of the amino acid sequences set forth in any one of SEQ ID NOs: 51 to 58.

5. The antigen-binding molecule of any one of claims 1 to 4, comprising a first light chain elbow region between VL1 and LD1.

6. The antigen-binding molecule of any one of claims 1 to 5, comprising a first heavy chain elbow region between VH1 and HD1.

7. a first light chain polypeptide comprising, in amino- to carboxy-terminal order, VL1-LE1-LD1, where LE1 is a first light chain elbow region; a first light chain polypeptide comprising, in amino-terminal to carboxy-terminal order, VH1-HE1-HD1, where HE1 is a first heavy chain elbow region; The antigen-binding molecule of any one of claims 1 to 6, wherein LE1 connects the carboxy terminus of the VL1 to the amino terminus of the LD1, and HE1 connects the carboxy terminus of the VH1 to the amino terminus of the HD1.

8. The antigen-binding molecule of claim 7, wherein LE1 or HE1 is an amino acid sequence of 3 to 25 amino acids in length.

9. LE1 or HE1 is RTV, GGS, RTVGGS (SEQ ID NO: 59), RTVGGSRTV (SEQ ID NO: 60), AST, ASTK (SEQ ID NO: 61), ASTKG (SEQ ID NO: 62), ASTKGG (SEQ ID NO: 63), ASTKGGS (SEQ ID NO: 64), ASTKGGGS (SEQ ID NO: 65), ASTKGGGGGS (SEQ ID NO: 66), ASTKGGGGSG (SEQ ID NO: 67), ASTKGGGGSGG (SEQ ID NO: 68), ASTKGGGGSGGS (SEQ ID NO: 69) ASTKGGGGSGGGGS (SEQ ID NO: 70), ASTKGGGGSGGGGS (SEQ ID NO: 71), RTVA (SEQ ID NO: 72), RTVAG (SEQ ID NO: 73), RTVAGG (SEQ ID NO: 74), RTVAGGS (SEQ ID NO: 75), RTVAGGGGS (SEQ ID NO: 76), RTVAGGGGS (SEQ ID NO: 77), RTVAGGGGSG (SEQ ID NO: 78), RTVAGGGGSGG (SEQ ID NO: 79), RTVAGGGGSGGGS (SEQ ID NO: 80), RTVAGGGGSGGGS (SEQ ID NO: 81), RTVAGGGGSGGGGGS (SEQ ID NO: 82), GGGGSGGGGGS (SEQ ID NO: 83), GGGGSGGGGSGGGGGGS (SEQ ID NO: 84), GGGGSGGGGGSGGGGGSGGGGGS (SEQ ID NO: 85), and GGGGSGGGGGSGGGGGSGGGGGSGGGGGS (SEQ ID NO: 86).

10. a) LD1 is the B2M domain and LE1 is an amino acid sequence selected from the group consisting of RTV, GGS, RTVGGS (SEQ ID NO:59), RTVGGSRTV (SEQ ID NO:60), RTVA (SEQ ID NO:72), RTVAG (SEQ ID NO:73), RTVAGG (SEQ ID NO:74), RTVAGGS (SEQ ID NO:75), RTVAGGGGS (SEQ ID NO:76), RTVAGGGGS (SEQ ID NO:77), RTVAGGGGGSG (SEQ ID NO:78), RTVAGGGGSGG (SEQ ID NO:79), RTVAGGGGSGGGS (SEQ ID NO:80), RTVAGGGGSGGGS (SEQ ID NO:81), and RTVAGGGGSGGGGGS (SEQ ID NO:82); b) HD1 is the EA3 domain and HE1 is an amino acid sequence selected from the group consisting of GGS, AST, ASTK (SEQ ID NO: 61), ASTKG (SEQ ID NO: 62), ASTKGG (SEQ ID NO: 63), ASTKGGS (SEQ ID NO: 64), ASTKGGGS (SEQ ID NO: 65), ASTKGGGGS (SEQ ID NO: 66), ASTKGGGGSG (SEQ ID NO: 67), ASTKGGGGSGG (SEQ ID NO: 68), ASTKGGGGSGGS (SEQ ID NO: 69), ASTKGGGGSGGGS (SEQ ID NO: 70), or ASTKGGGGSGGGGS (SEQ ID NO: 71); or c) LD1 or HD1 is the first ICAM-1 D1 domain or the second ICAM-1 D1 domain, and LE1 or HE1 is an amino acid sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 83), GGGGSGGGGGSGGGGS (SEQ ID NO: 84), GGGGSGGGGSGGGGGSGGGGGS (SEQ ID NO: 85), and GGGGSGGGGSGGGGGSGGGGGSGGGG (SEQ ID NO: 86).

11. The antigen-binding molecule of any one of claims 1 to 10, comprising a first light chain spacer region fused to the C-terminus of the LD1.

12. The antigen-binding molecule of any one of claims 1 to 11, comprising a first heavy chain spacer region fused to the C-terminus of HD1.

13. a first light chain polypeptide comprising, in amino- to carboxy-terminal order, VL1-LD1-LS1, where LS1 is a first light chain spacer region; The antigen-binding molecule of any one of claims 1 to 12, comprising: a first heavy chain polypeptide comprising, in order from the amino terminus to the carboxy terminus, VH1-HD1-HS1, where HS1 is a first heavy chain spacer region.

14. The antigen-binding molecule of claim 13, wherein HS1 or LS1 is an amino acid sequence of 2 to 9 amino acids in length.

15. HS1 or LS1 is an amino acid sequence selected from the group consisting of EPKSS (SEQ ID NO: 87), SG, EPKSC (SEQ ID NO: 88), GGSGECSG (SEQ ID NO: 89), GGGSGECSG (SEQ ID NO: 90), GGSGESSG (SEQ ID NO: 91), and GGGSGESSG (SEQ ID NO: 92). The antigen-binding molecule of claim 13 or 14.

16. The antigen-binding molecule of any one of claims 12 to 15, wherein the heavy chain spacer region further comprises a hinge region.

17. The antigen-binding molecule of any one of claims 1 to 16, wherein the first light chain polypeptide or the first heavy chain polypeptide further comprises a C-terminal tag, and optionally the C-terminal tag is a 6xHis tag (SEQ ID NO: 93), a Strep-tag II tag, or a human influenza hemagglutinin tag.

18. The antigen-binding molecule of any one of claims 1 to 17, wherein the first heavy chain polypeptide further comprises heavy chain constant domain 2 (CH2).

19. The antigen-binding molecule of any one of claims 1 to 18, wherein the first heavy chain polypeptide further comprises heavy chain constant domain 3 (CH3).

20. The antigen-binding molecule of any one of claims 1 to 19, which does not contain a dimerization sequence of heavy chain constant domain 1 (CH1) or light chain constant domain (CL).

21. Immunoglobulin, Fab, Fab', F(ab') ２ , an antibody, a biparatope antibody, a bispecific antibody, a triparatope antibody, a trispecific antibody, a tetraparatope antibody, a tetraspecific antibody, a multiparatope antibody, a multispecific antibody, or any fragment of the antigen-binding molecule that binds to a target antigen.

22. The antigen-binding molecule according to any one of claims 1 to 21, further comprising a second light chain polypeptide comprising a second light chain variable domain (VL2) and a second heavy chain polypeptide comprising a second heavy chain variable domain (VH2), wherein VL2 and VH2 form a second paratope.

23. The antigen-binding molecule of claim 22 , wherein the first paratope and the second paratope bind to different antigens.

24. The antigen-binding molecule of claim 22 or 23, further comprising a third light chain polypeptide comprising a third light chain variable domain (VL3) and a third heavy chain polypeptide comprising a third heavy chain variable domain (VH3), wherein VL3 and VH3 form a third paratope.

25. The antigen-binding molecule of claim 24 , wherein the first paratope, the second paratope, and the third paratope bind to different antigens.

26. The antigen-binding molecule of claim 24 or 25, further comprising a fourth light chain polypeptide comprising a fourth light chain variable domain (VL4) and a fourth heavy chain polypeptide comprising a fourth heavy chain variable domain (VH4), wherein VL4 and VH4 form a fourth paratope.

27. The antigen-binding molecule of claim 26, wherein the first paratope, the second paratope, the third paratope, and the fourth paratope bind to different antigens.

28. The antigen-binding molecule of any one of claims 1 to 27, wherein the first paratope specifically binds to a first tumor-associated antigen (TAA1).

29. 24. The antigen-binding molecule of claim 22 or 23, wherein the first paratope specifically binds to a first tumor-associated antigen (TAA1), and the second paratope specifically binds to a second tumor-associated antigen (TAA2).

30. The first paratope specifically binds to a first tumor-associated antigen (TAA1), the second paratope specifically binds to a second tumor-associated antigen (TAA2), and the third paratope specifically binds to a third tumor-associated antigen (TAA3). The antigen-binding molecule of claim 24 or 25.

31. The first paratope specifically binds to a first tumor-associated antigen (TAA1), the second paratope specifically binds to a second tumor-associated antigen (TAA2), the third paratope specifically binds to a third tumor-associated antigen (TAA3), and the fourth paratope specifically binds to a fourth tumor-associated antigen (TAA4). The antigen-binding molecule of claim 26 or 27.

32. An isolated polynucleotide encoding the antigen-binding molecule of any one of claims 1 to 31, wherein optionally the isolated polynucleotide sequence comprises a nucleotide sequence selected from SEQ ID NOs: 3 and 263 to 343, or a nucleotide sequence having at least 85% sequence identity to any of SEQ ID NOs: 3 and 263 to 343.

33. 33. A vector comprising the isolated polynucleotide of claim 32.

34. A host cell comprising the vector of claim 33.

35. A method for producing an antigen-binding molecule, comprising: i) culturing the host cell of claim 34 under suitable conditions such that the antigen-binding molecule is expressed by the host cell; ii) isolating the antigen-binding molecule.

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