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

Abstract

Antigen binding molecules are described that are engineered to replace the light chain constant domain (CL) and the heavy chain constant domain 1 (CH1) with an HLA class I histocompatible antigen alpha-E alpha-3 (HLA-E) / beta-2 microglobulin (B2M) or intercellular adhesion molecule 1 domain 1 (ICAM-1D1) / ICAM-1D1 dimerization domain.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 389,814, filed on July 15, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on July 12, 2023, is named 253505_000341_SL, and is 391,390 bytes in size. Technical Field

[0005] Provided herein are antigen binding molecules comprising one or more antigen-binding polypeptides, which form a variable region in conjunction with a target antigen, and replace an antibody light chain constant domain (CL) and an antibody heavy chain constant domain 1 (CH1) with a dimerization domain as described herein. Replacing the dimerization interface between the light chain constant domain and the heavy chain constant domain 1 provides a means for selectively assembling homologous chains in a multiparatopic antigen binding molecule. Background Art

[0006] Methods for producing bispecific heterodimeric antibodies with modified heavy chain IgG constant regions to promote efficient formation of heavy chain heterodimer pairs and specific pairing of the heavy and light chain arms have been described (WO 2018 / 237192A1). Summary of the Invention

[0007] In one aspect, provided herein are antigen binding molecules comprising one or more antigen binding polypeptides, each of which comprises a variable domain that binds to a target antigen and a dimerization domain as described herein in place of a light chain constant domain (CL) or a heavy chain constant domain 1 (CH1).

[0008] In specific embodiments, the antigen binding molecules bind to one or more target antigens. In specific embodiments, the antigen binding molecules as described herein comprise two or more polypeptides, each comprising a variable domain, wherein the variable domains form a paratope that binds to one or more target antigens.

[0009] In specific embodiments, antigen binding molecules as described herein comprise two, three, four, five, six, seven or eight polypeptides, optionally wherein each of the two, three, four, five, six, seven or eight polypeptides comprises a dimerization domain, and a variable domain (in combination with a homologous variable domain) that is bound to a target antigen as described herein. In specific embodiments, a dimerization domain is bound to another dimerization domain to form a dimer (particularly, a heterodimer, e.g., six polypeptides can form up to three dimers with each other).

[0010] In a specific embodiment, the polypeptide of the antigen binding molecule as described herein comprises a light chain polypeptide having a light chain variable domain and a light chain dimerization domain, and a heavy chain polypeptide having a heavy chain variable domain and a heavy chain dimerization domain. In a specific embodiment, the light chain variable domain and the heavy chain variable domain constitute the variable region that binds to the target antigen.

[0011] In a specific embodiment of the antigen binding molecules as 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) chain, a lambda (λ) chain, a sigma (σ) chain, an iota (ι) chain, or one or more fragments thereof. In a specific embodiment of the antigen binding molecules as 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) chain, a delta (δ) chain, an alpha (α) chain, a mu (μ) chain, an epsilon (ε) chain, or one or more fragments thereof.

[0012] In one aspect, an antigen binding molecule as described herein comprises a first light chain polypeptide comprising, in amino-terminal to carboxyl-terminal order: 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, in amino-terminal to carboxyl-terminal order: 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 beta-2 microglobulin (B2M) domain and HD1 comprises an HLA-E In some embodiments, the present invention relates to an ICAM-1D1 domain comprising a first ICAM-1D1 domain and a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer; or ii) LD1 comprises a first ICAM-1D1 domain and HD1 comprises a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with 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; and ii) the first ICAM-1D1 domain is different from the second ICAM-1D1 domain, and VL1 and VH1 form a first paratope.

[0013] In one aspect, the antigen binding molecule as described herein comprises a first light chain polypeptide comprising, in amino-terminal to carboxyl-terminal order: a first light chain variable domain; a first light chain elbow region comprising 1) 1 to 8 consecutive amino acids selected from amino acid positions 108-115 of a human immunoglobulin kappa constant domain according to EU or Kabat numbering, or 2) an amino acid sequence of at least 3 amino acids in length; and a first light chain dimerization domain selected from an HLA-E A light chain polypeptide comprising: an A3 (EA3) domain, a β2 microglobulin (B2M) domain, or a first ICAM-1D1 domain; and a first heavy chain polypeptide comprising, in amino-terminal to carboxyl-terminal order: a first heavy chain variable domain; a first heavy chain elbow region comprising 1) 1 to 8 consecutive amino acids selected from amino acid positions 118-125 of human IgG1 according to EU numbering or amino acid positions 114-121 of human IgG1 according to Kabat numbering, or 2) an amino acid sequence of at least 3 amino acids in length; and a first heavy chain dimerization domain, wherein i) the first light chain dimerization domain comprises a B2M structure; In some embodiments, the first light chain dimerization domain comprises a first ICAM-1D1 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 associate with each other to form a dimer; or ii) the first light chain dimerization domain comprises a first ICAM-1D1 domain and the first heavy chain dimerization domain comprises a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with 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-1D1 domain is different from the second ICAM-1D1 domain; and the first light chain variable domain and the first heavy chain variable domain form a first paratope.

[0014] In one aspect, the antigen binding molecule as described herein comprises a dimer of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, in amino-terminal to carboxyl-terminal order, (i) a first immunoglobulin fragment that does not comprise a dimerization sequence of a CH1 or CL domain, and (ii) a first dimerization domain that comprises an HLA-E A3 (EA3) domain, a beta-2 microglobulin (B2M) domain, or a first ICAM-1D1 domain, and the second polypeptide comprises, in amino-terminal to carboxyl-terminal order, (i) a second immunoglobulin fragment that does not comprise a dimerization sequence of a CH1 or CL domain, and (ii) a second dimerization domain that comprises an EA3 domain, a B2M domain, or a second ICAM-1D1 domain, wherein i) the first dimerization domain comprises a B2M domain and the second dimerization domain comprises an EA3 domain, or the first and second dimerization domains comprise a B2M domain. wherein the first immunoglobulin fragment comprises a first immunoglobulin variable domain and the second immunoglobulin fragment comprises a second immunoglobulin variable domain, wherein the first and second immunoglobulin variable domains form a first paratope.

[0015] In one aspect, the present invention provides a composition comprising a plurality of species of polypeptides, wherein at least one first species of polypeptides comprises a first polypeptide comprising, in amino-terminal to carboxyl-terminal order: a first immunoglobulin fragment that does not comprise a dimerization sequence of a CH1 or CL domain, and a first dimerization domain comprising an HLA-E A3 (EA3) domain, a beta-2 microglobulin (B2M) domain, or a first ICAM-1D1 domain; and at least one second species of polypeptides comprises a second polypeptide comprising, in amino-terminal to carboxyl-terminal order: a second immunoglobulin fragment that does not comprise a dimerization sequence of a CH1 or CL domain, and a first dimerization domain comprising an HLA-E A3 (EA3) domain, a beta-2 microglobulin (B2M) domain, or a first ICAM-1D1 domain. The first dimerization domain comprises the B2M domain and the second dimerization domain comprises the EA3 domain, or the first dimerization domain comprises the EA3 domain and the second dimerization domain comprises the B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer; or (b) the first dimerization domain comprises the first ICAM-1D1 domain and the second dimerization domain comprises the second ICAM-1D1 domain, wherein the first ICAM-1D1 domain comprises the EA3 domain, or the first dimerization domain comprises the EA3 domain and the second dimerization domain comprises the B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer; The CAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer; (a) at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain or the EA3 domain, respectively, and (b) the first ICAM-1D1 domain is different from the second ICAM-1D1 domain; the first immunoglobulin fragment comprises a first immunoglobulin variable domain and the second immunoglobulin fragment comprises a second immunoglobulin variable domain, wherein the first immunoglobulin variable domain and the second immunoglobulin variable domain form a first paratope.

[0016] In one aspect, provided herein is a composition comprising a plurality of species of polypeptides, wherein each species of polypeptides comprises means for binding to a first target antigen; and means for dimerization, wherein in at least one species of polypeptides, the means for binding comprises a first means for binding to the first target antigen, and the first means for dimerization comprises: HLA-E wherein the first and second means for dimerization bind to each other to form a dimer, and at least one of the EA3 domains or the B2M domains is different from the wild-type B2M domain (SEQ ID NO: 2) or the wild-type EA3 domain (SEQ ID NO: 3). NO:33), the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain, and the first means and the second means for binding to the first target antigen form a first paratope.

[0017] In one aspect, provided herein is a method of producing a multiparatopic antibody in a single host cell, the method comprising providing one or more polynucleotides encoding a first light chain polypeptide comprising a first light chain variable domain; and a first light chain dimerization domain, the first light chain dimerization domain being an HLA-E A3 (EA3) domain, a beta-2 microglobulin (B2M) domain, or a first ICAM-1D1 domain; a first heavy chain comprising a first heavy chain variable domain; and a first heavy chain dimerization domain, the first heavy chain dimerization domain being an EA3 domain, a B2M domain, or a second ICAM-1D1 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 (ii) the first light chain dimerization domain comprises an EA3 domain and the first heavy chain dimerization domain comprises an EA3 domain; The first heavy chain dimerization domain comprises a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer; or (ii) the first light chain dimerization domain comprises a first ICAM-1D1 domain and the first heavy chain dimerization domain comprises a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with 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 a wild-type EA3 domain (SEQ ID NO:33), 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, and linkage to a second antibody heavy chain; the second antibody heavy chain, the second antibody heavy chain comprising the second heavy chain variable domain; a second heavy chain dimerization domain, the second heavy chain dimerization domain being not an EA3 domain, a B2M domain, or an ICAM-1 domain, or being a first heavy chain constant domain (CH1), and wherein the second heavy chain dimerization domain dimerizes with the second light chain dimerization domain; and linkage to the first antibody heavy chain, and a second antibody light chain, the second antibody light chain comprising the second light chain variable domain;and a second light chain dimerization domain, the second light chain dimerization domain being not an EA3 domain, a B2M domain, or an ICAM-1 domain, or being a light chain constant domain (CL), and wherein the second light chain dimerization domain dimerizes with the second heavy chain dimerization domain, delivering the one or more polynucleotide sequences to a host cell, culturing the host cell under conditions permissive for expression of the multiparatopic antibody, thereby producing the multiparatopic antibody, and 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 chain dimerization domain dimerize together, the second light chain dimerization domain and the second heavy chain dimerization domain dimerize together, the first heavy chain and the second heavy chain are linked together, 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: 3), respectively. NO:33), ii) the first ICAM-1D1 domain is different from the second ICAM-1D1 domain.;

[0018] In a specific embodiment, the first light chain dimerization domain includes a β2 microglobulin (B2M) domain, and the first heavy chain dimerization domain includes an HLA-E A3 (EA3) domain. In a specific embodiment, the first light chain dimerization domain includes an EA3 domain, and the first heavy chain dimerization domain includes a B2M domain. In a specific embodiment, the B2M domain and the EA3 domain are combined with each other to form a dimer. In a specific embodiment, 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). In a specific embodiment, B2M is different from the wild-type B2M domain (SEQ ID NO: 2). In a specific embodiment, the EA3 domain is different from the wild-type EA3 domain (SEQ ID NO: 33).

[0019] In a specific embodiment, the first light chain dimerization domain comprises a first ICAM-1D1 domain and the first heavy chain dimerization domain comprises a second ICAM-1D1 domain. In a specific embodiment, the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer. In a specific embodiment, the first ICAM-1D1 domain is different from the second ICAM-1D1 domain.

[0020] In specific embodiments of the antigen binding molecules as described herein, the first light chain dimerization domain includes a B2M domain and the first heavy chain dimerization domain includes an EA3 domain, or the first light chain dimerization domain includes an EA3 domain and the first heavy chain dimerization domain includes a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer, and the B2M domain comprises the amino acid sequence RTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 2), or consists of the amino acid sequence.

[0021] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain includes a B2M domain and the first heavy chain dimerization domain includes an EA3 domain, or the first light chain dimerization domain includes an EA3 domain and the first heavy chain dimerization domain includes a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer, and the B2M domain comprises an amino acid sequence having at least 91% sequence identity with SEQ ID NO:2, or consists of the amino acid sequence.

[0022] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain includes a B2M domain and the first heavy chain dimerization domain includes an EA3 domain, or the first light chain dimerization domain includes an EA3 domain and the first heavy chain dimerization domain includes a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer, and the B2M domain comprises an amino acid sequence having one, two, three, four, five, six, seven or eight single amino acid substitutions to the sequence of SEQ ID NO: 2 at one or more of amino acid positions 4, 8, 10, 54, 58, 60, 96 and 97 of SEQ ID NO: 2, or consists of the amino acid sequence.

[0023] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain includes a B2M domain and the first heavy chain dimerization domain includes an EA3 domain, or the first light chain dimerization domain includes an EA3 domain and the first heavy chain dimerization domain includes a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer, and the B2M domain comprises an amino acid sequence having one, two, three, four, five, six, seven or eight single amino acid substitutions to the sequence SEQ ID NO: 2 at one or more of amino acid positions 4, 8, 10, 54, 58, 60, 96 and 97 of SEQ ID NO: 2, or consists of this amino acid sequence, wherein each of these single amino acid substitutions is independently selected from the group consisting of F, W, C, S and T.

[0024] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain comprises an amino acid sequence that is the human B2M sequence shown in SEQ ID NO: 2 but has a set of substitutions of F56S, W60S and F62T (wherein positions are numbered according to the B2M amino acid numbering in Table 1), optionally further having one, two, three, four or five single amino acid substitutions at positions K6, Y10, R12, D98 or M99 (wherein positions are numbered according to the B2M amino acid numbering in Table 1), or consists of this amino acid sequence.

[0025] In a specific embodiment of the antigen binding molecule as 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 associate with each other to form a dimer, and the B2M domain comprises the sequence of SEQ ID NO: NO:2 shows the human B2M sequence but has a group of substituted amino acid sequences selected from the following (wherein positions are numbered according to the B2M amino acid numbering in Table 1), or consists of the amino acid sequence: 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.

[0026] In specific embodiments of the antigen binding molecules as described herein, the first light chain dimerization domain includes a B2M domain and the first heavy chain dimerization domain includes an EA3 domain, or the first light chain dimerization domain includes an EA3 domain and the first heavy chain dimerization domain includes a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer, and the B2M domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-30, or consists of the amino acid sequence.

[0027] In specific embodiments of the antigen binding molecules as described herein, the first light chain dimerization domain includes a B2M domain and the first heavy chain dimerization domain includes an EA3 domain, or the first light chain dimerization domain includes an EA3 domain and the first heavy chain dimerization domain includes a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer, and wherein the EA3 domain comprises the amino acid sequence LHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDT ELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW (SEQ ID NO: 33), or consists of the amino acid sequence.

[0028] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain includes a B2M domain and the first heavy chain dimerization domain includes an EA3 domain, or the first light chain dimerization domain includes an EA3 domain and the first heavy chain dimerization domain includes a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer, and wherein the EA3 domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:33, or consists of the amino acid sequence.

[0029] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain includes a B2M domain and the first heavy chain dimerization domain includes an EA3 domain, or the first light chain dimerization domain includes an EA3 domain and the first heavy chain dimerization domain includes a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer, and wherein the EA3 domain comprises an amino acid sequence having one, two, three, four, five or six single amino acid substitutions 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, or consists of the amino acid sequence.

[0030] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain includes a B2M domain and the first heavy chain dimerization domain includes an EA3 domain, or the first light chain dimerization domain includes an EA3 domain and the first heavy chain dimerization domain includes a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer, and wherein the EA3 domain comprises an amino acid sequence having one, two, three, four, five or six single amino acid substitutions to the sequence SEQ ID NO: 33 at one or more of amino acid positions 13, 23, 53, 55, 59 and 63 of SEQ ID NO: 33, or consists of this amino acid sequence, wherein each of these single amino acid substitutions is independently selected from the group consisting of A, C and L.

[0031] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain includes a B2M domain and the first heavy chain dimerization domain includes an EA3 domain, or the first light chain dimerization domain includes an EA3 domain and the first heavy chain dimerization domain includes a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer, and wherein the EA3 domain comprises an amino acid sequence that is the human EA3 sequence shown in SEQ ID NO: 33 but has one, two, three, four, five or six single amino acid substitutions at positions H192, R202, E232, R234, D238 or Q242 (wherein the positions are numbered according to the EA3 amino acid numbering in Table 2), or consists of such an amino acid sequence.

[0032] In a specific embodiment of the antigen binding molecule as 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 associate with each other to form a dimer, and wherein the EA3 domain comprises a sequence represented by SEQ ID NO: The human EA3 sequence shown in NO:33 but having an amino acid sequence selected from the following (wherein positions are numbered according to the EA3 amino acid numbering in Table 2), or consisting of the amino acid sequence: H192C; R202A or R202C; E232C; R234A, R234L or R234C; D238C; Q242A or Q242L; R234A and Q242A; R234A and Q242L; R234A and Q242A; or R234L and Q242L.

[0033] In specific embodiments of the antigen binding molecules as described herein, the first light chain dimerization domain includes a B2M domain and the first heavy chain dimerization domain includes an EA3 domain, or the first light chain dimerization domain includes an EA3 domain and the first heavy chain dimerization domain includes a B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer, and wherein the EA3 domain comprises an amino acid sequence of any one of SEQ ID NOs: 32-46, or consists of the amino acid sequence.

[0034] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain comprises a first ICAM-1D1 domain and the first heavy chain dimerization domain comprises a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer, and wherein the first ICAM-1D1 domain or the second ICAM-1D1 domain comprises or consists of the amino acid sequence QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKV YELSNVQEDSQPMCYSNCPDGQSTAKTFLTVY (SEQ ID NO: 49).

[0035] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain comprises a first ICAM-1D1 domain and the first heavy chain dimerization domain comprises a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer, and wherein the first ICAM-1D1 domain or the second ICAM-1D1 domain comprises or consists of an amino acid sequence that has at least 81% sequence identity to SEQ ID NO:49.

[0036] In a specific embodiment of the antigen binding molecule as described herein, 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, wherein the first ICAM-1 D1 domain and the second ICAM-1 D1 domain associate with each other to form a dimer, and wherein 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 of SEQ ID NO: 49 at one or more of amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67 and 78 of SEQ ID NO: 49, optionally further adding a cysteine ​​amino acid at the C-terminus.

[0037] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain comprises a first ICAM-1D1 domain and the first heavy chain dimerization domain comprises a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer, and wherein the first ICAM-1D1 domain or the second ICAM-1D1 domain comprises a sequence of SEQ ID NO: 49 at one or more of amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67, and 78. : 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 single amino acid substitutions of NO: 49, optionally further adding a cysteine ​​amino acid at the C-terminus, or consisting of an amino acid sequence, wherein each of the single amino acid substitutions is independently selected from the group consisting of V, T, F, W, A, K, E, C and R.

[0038] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain comprises a first ICAM-1D1 domain and the first heavy chain dimerization domain comprises a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer, and wherein the first ICAM-1D1 domain or the second ICAM-1D1 domain comprises a sequence represented by SEQ ID NO: The amino acid sequence of human ICAM-1D1 as shown in NO:49 but having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirty-three, fourteen, or fifteen single amino acid substitutions at positions T2, I10, R13, L18, T20, T23, E34, P38, L42, R49, V51, E53, P63, S67, or T78 (wherein positions are numbered according to the ICAM-1D1 amino acid numbering in Table 3), optionally further comprising the addition of a cysteine ​​amino acid (84C) at the C-terminus (wherein positions are numbered according to the ICAM-1D1 amino acid numbering in Table 3).

[0039] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain comprises a first ICAM-1D1 domain and the first heavy chain dimerization domain comprises a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer, and wherein the first ICAM-1D1 domain or the second ICAM-1D1 domain comprises a sequence represented by SEQ ID NO: The human ICAM-1D1 sequence shown in NO:49 but having or consisting of a set of substituted amino acid sequences selected from the group consisting of E34K; T2V, I10T, T23A, E34K, P38T, P63V, S67A and T78A; T2V, I10T, R13C, T23A, E34K, P38T, R49E, P63V, S67A, T78A and B4C; T2V, I10T, R13C, T23A, E34K, P38T, R49E, P63V, S67A, T78A and B4C; 38T, E53R, P63V, S67A, T78A and 84C; T2V, I10T, R13C, L18F, 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.

[0040] In a specific embodiment of the antigen binding molecule as described herein, the first light chain dimerization domain comprises a first ICAM-1D1 domain and the first heavy chain dimerization domain comprises a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer, and wherein the first ICAM-1D1 domain or the second ICAM-1D1 domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 48-58.

[0041] In a specific embodiment of the antigen binding molecules as 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 a specific embodiment, 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 a specific embodiment, the elbow region comprises an amino acid sequence having a length of at least 3 amino acids. In a specific embodiment, the elbow region comprises an amino acid sequence having a length of 3 to 25 amino acids, or consists of the amino acid sequence.

[0042] In specific embodiments of the antigen binding molecules as described herein, the 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); RTVAGG (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); RTVAGGGGSGGGGS (SEQ ID NO: 82); GGGGSGGGGS (SEQ ID NO: 83); GGGGSGGGGSGGGGS (SEQ ID NO: 84); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:85); and GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:86).

[0043] In the specific embodiments of antigen binding molecules as described herein, the polypeptide of antigen binding molecules includes the spacer fused to the C-terminal of its dimerization domain. In specific embodiments, light chain polypeptide (e.g., the first light chain polypeptide) includes the spacer fused to the C-terminal of its dimerization domain. In specific embodiments, heavy chain polypeptide (e.g., the first heavy chain polypeptide) includes the spacer fused to the C-terminal of its dimerization domain. In specific embodiments, spacer includes an amino acid sequence with a length of at least 2 amino acids. In specific embodiments, spacer includes an amino acid sequence with a length of 2 to 9 amino acids, or consists of the amino acid sequence.

[0044] In specific embodiments of the antigen binding molecules as described herein, the spacer 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); GGSGESSG (SEQ ID NO:91); and GGGSGESSG (SEQ ID NO:92).

[0045] In specific embodiments, the spacer region further comprises a hinge region.

[0046] In specific embodiments of the antigen binding molecules as described herein, the polypeptide of the antigen binding molecule further comprises a C-terminal tag, optionally wherein the C-terminal tag is a 6x His tag (SEQ ID NO: 93), a streptavidin tag (e.g., a Strep-tag II tag), or a human influenza hemagglutinin tag.

[0047] In specific embodiments of the antigen binding molecules as described herein, the antigen binding molecule is an immunoglobulin, a Fab, Fab', F(ab')2, an antibody, a biparatopic antibody, a bispecific antibody, a triparatopic antibody, a trispecific antibody, a tetraparatopic antibody, a tetraspecific antibody, a multiparatopic antibody, a multispecific antibody, or any fragment of the antigen binding molecule that binds to the target antigen.

[0048] In a specific embodiment, an antigen binding molecule as described herein further comprises a second light chain polypeptide (LC2) having a second light chain variable domain (VL2) and a second heavy chain polypeptide (HC2) having a second heavy chain variable domain (VH2), wherein VL2 and VH2 form a second paratope. In a specific embodiment, the first paratope and the second paratope bind to different antigens.

[0049] In a specific embodiment, an antigen binding molecule as described herein further comprises a third light chain polypeptide (LC3) having a third light chain variable domain (VL3) and a third heavy chain polypeptide (HC3) having a third heavy chain variable domain (VH3), wherein VL3 and VH3 form a third paratope. In a specific embodiment, the first paratope, the second paratope, and the third paratope bind to different antigens.

[0050] In a specific embodiment, an antigen binding molecule as described herein further comprises a fourth light chain polypeptide (LC4) having a fourth light chain variable domain (VL4) and a fourth heavy chain polypeptide (HC4) having a fourth heavy chain variable domain (VH4), wherein VL4 and VH4 form a fourth paratope. In a specific embodiment, the first paratope, the second paratope, the third paratope, and the fourth paratope bind to different antigens.

[0051] In specific 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).

[0052] In another aspect, provided herein is an isolated polynucleotide encoding an antigen binding molecule as described herein.

[0053] In another aspect, provided herein is a vector comprising an isolated polynucleotide encoding an antigen binding molecule as described herein.

[0054] In another aspect, provided herein is a host cell containing a vector comprising an isolated polynucleotide encoding an antigen binding molecule as described herein.

[0055] In another aspect, provided herein is a pharmaceutical composition comprising an antigen binding molecule as described herein, an isolated polynucleotide as described herein, or a host cell as described herein, and a pharmaceutically acceptable excipient.

[0056] In another aspect, provided herein is a method for producing an antigen binding molecule as described herein, comprising: culturing a host cell as described herein under suitable conditions such that the antigen binding molecule is expressed by the host cell; and isolating the antigen binding molecule.

[0057] 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 as described herein.

[0058] 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 as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figures 1A to 1C Depicted is the CH1-CL replacement strategy as described herein. Figure 1A Shown are generalized bispecific antibodies (bsAbs) characterized by two unique variable domains (hashed lines and grey dots), each with its own unique VH and VL located N-terminally to a CH or CL domain native to both the light and heavy chain constant regions. Figure 1B Shown is the replacement of the CH1 and CL domains of one arm of a native bsAb in a “pseudo-Fab” (pFab) design, where the CH1 and CL domains are replaced by alternative Ig domains (horizontal lines). Figure 1C The CH1 and CL replacement strategy is shown to be extended to multispecific antibodies, where both CH1 domains and the CL domain are replaced by different pFab regions (horizontal lines for one pFab, vertical lines for the other pFab). Standard antibody constant domains are shown as white ovals.

[0060] Figures 2A to 2C Depicted is the structure of the HLA-E A3 (EA3) domain / beta-2 microglobulin (B2M) domain heterodimer compared to the structure of the native CH1-CL heterodimer. Figure 2A The Fab structure from RCSB PDB ID: 5TZ2 is shown. The heavy chain is shown in dark grey, while the light chain is shown in light grey. The distance from heavy chain VH residue S112 (Kabat) to CH1 residue S119 (EU) is The distance from VL I106 (Kabat) to CL K111 (EU) is Figure 2B The EA3 / B2M domain heterodimer is shown overlaid on the Fab structure. The distances between the variable domain residues indicated above and the N-termini of the aligned EA3 subunits (residue L201) and B2M (residue R23) are respectively and Figure 2C The ICAM-1D1 domains are shown overlaid on the Fab structure. The distances between the variable domain residues indicated above and the N-terminus of the aligned ICAM-1D1 homodimer (residue Q1) are and

[0061] Figures 3A to 3BDepicted are sequence alignments and percent identities of HLA-E A3 (EA3) and beta-2 microglobulin (B2M) wild-type sequences with the human IgG1 G1m CH1 domain, human IgG1 kappa light chain constant domain, and human IgG1 lambda light chain constant domain. Figure 3A Sequence alignment of the CH1 domain and the kappa and lambda constant domains with EA3 and B2M is shown. Figure 3B As shown, EA3 shows higher identity to the CH1 domain than to the CL domain, while B2M shows higher identity to the CL domain. In a specific "pseudo-Fab" domain, EA3 replaces CH1, while B2M replaces CL. The figure discloses SEQ ID NOs: 98-99, 368-369, and 2, respectively, in order of appearance.

[0062] Figures 4A to 4B Depicted is the design of a B2M(SST) variant as described herein. Figure 4B The structure of HLA-E A3 (EA3) is shown, indicating the hydrophobic interactions between the B2M domain and α1 / α2 of the EA3 domain. Figure 4B Shown is a sequence alignment of human B2M and B2M(SST). The figure discloses SEQ ID NOs: 2 and 4, respectively, in order of appearance.

[0063] Figure 5 Depicts in vitro analysis of the ability of HLA-E A3 (EA3) / beta-2 microglobulin (B2M) pseudo Fab (pFab) to bind to the RSV-F glycoprotein. In the absence of engineered salt bridges or disulfide bonds, EA3 / B2M pseudo Fabs HLPPB117 and HLPPB270 show weaker binding to the RSV-F glycoprotein than to the B23B173 native Fab control. Wild-type EA3 / B2M dimers lacking anti-RSV-F variable domains were used as negative controls.

[0064] Figures 6A to 6B Depicted are intact LC-MS molecular weight analyses of the HLA-E A3 (EA3) / beta-2 microglobulin (B2M) pseudo-Fab HLPPB17 and a native Fab control (B23B173). Figure 6A The presence of each light and heavy chain arm of the EA3 / B2M pFab is shown. Figure 6B The presence of intact B23B173 is shown.

[0065] 7A to 7D Depicted is a comparison of expression and solubility levels of engineered pseudo-Fabs (pFabs) comprising electrostatic mutations present in the HLA-E A3 (EA3) domain / beta-2 microglobulin (B2M) domain by reducing SDS-PAGE. Figure 7AResults are shown for pFabs HLPPW10, HLPPW13, HLPPW14, HLPPW15, HLPPW16, HLPPW17, and HLPPW18. Figure 7B The results for pFabs HLPPW19, HLPPW20, HLPPW21, HLPPW22, HLPPW23, HLPPW24, HLPPW25, and HLPPW26 are shown. Figure 7C The results for pFabs HLPPW27, HLPPW28, HLPPW29, HLPPW30, HLPPW32, HLPPW33, and HLPPW34 are shown. Figure 7D Results are shown for pFabs HLPPW35, HLPPW36, HLPPW37, HLPPW38, HLPPW40, and HLPPW41. 7A to 7D In the figure, lane S is the pure CHO supernatant, and lane P is the resuspended CHO pellet fraction.

[0066] Figures 8A to 8D Depicted are the comparison of expression levels of engineered HLA-E A3 (EA3) / beta-2 microglobulin (B2M) pseudo-Fabs (pFabs) HLPPW42, HLPPW43, HLPPW54, HLPPW55, HLPPW56, HLPPW57, HLPPW48, HLPPW49, HLPPW50, HLPPW51, HLPPW52, HLPPW53, HLPPW42, HLPPB17, HLPPB270 and normal Fab HLPPW58, and analysis of disulfide bond integrity by SDS-PAGE. Figure 8A Shown are expression levels detected by luminescence using anti-HIS capture and anti-HA / anti-StrepII detection. Figures 8B to 8D Shown are the results of reducing (R) and non-reducing (N) SDS-PAGE. Figures 8B to 8D Lane R is the resuspended CHO pellet fraction.

[0067] Figures 9A to 9B Depicted is an ELISA analysis of the ability of engineered HLA-E A3 (EA3) / beta-2 microglobulin (B2M) pseudoFab (pFab) from pure CHO supernatant to bind to RSV-F glycoprotein. Figure 9A The luminescence signals of HLPPW42, HLPPW43, HLPPW54, HLPPW55, HLPPW56, HLPPW57, HLPPW48, HLPPW49, HLPPW50, HLPPW51, HLPPW52, HLPPW53, HLPPW42, HLPPB17, and HLPPB270 are shown compared to the normal Fab HLPPW58. Figure 9BCalculated EC50 and 95% confidence interval values ​​are shown.

[0068] FIG. 10A to FIG. 10B Depicted are ELISA binding of purified disulfide engineered HLA-E A3 (EA3) / beta-2 microglobulin (B2M) pseudoFab (pFab) to RSV glycoprotein. Figure 10A Shown are the luminescence signals from HLPPW42, HLPPW43, HLPPW54, HLPPW55, HLPPW56, HLPPW57, HLPPW48, HLPPW49, HLPPW50, HLPPW51, HLPPW52, and HLPPW53 compared to the luminescence signal from normal Fab HLPPW58. Figure 10B Calculated EC50 and maximum signal values ​​are shown.

[0069] 11A to 11D Depicted is the size exclusion chromatography (SEC) profile of the HLA-E A3 (EA3) / beta-2 microglobulin (B2M) disulfide variant. The results shown belong to HLPPB17 ( Figure 11A )、HLPPW57( Figure 11B )、HLPPW49( Figure 11C ) and HLPPW53( Figure 11D ), which appear as monodisperse material. Gel filtration standards are overlaid with dashed lines. Signals are expressed relative to the maximum signal for each sample.

[0070] Figure 12 depicts mass spectrometry analysis of HLA-E A3 (EA3) / beta-2 microglobulin (B2M) disulfide variants. Results shown are for HLPPB17 ( Figure 12A )、HLPPW57( Figure 12B )、HLPPW49( Figure 12C ) and HLPPW53( Figure 12D ).

[0071] 13A to 13B Depicted is stability analysis by thermal melting as measured using nanoscale differential scanning fluorimetry (NanoDSF). Figure 13A Melting curves and first derivatives are shown. Figure 13B Melting onset temperature (Ton), melting temperature 1 (Tm1), melting temperature 2 (Tm2), melting temperature 3 (Tm3) and aggregation onset temperature (Tagg) are shown. Two unique disulfide "pin" pairs were identified with stabilities comparable to the Fab control.

[0072] Figure 14Depicted is the non-reducing PAGE of the HLPPW57-derived HLA-E A3 (EA3) / beta-2 microglobulin (B2M) pseudo-Fab variants HLPPW59, HLPPW60, HLPPW61, HLPPW62, HLPPW63, HLPPW64, HLPPW65, HLPPW66, HLPPW67, HLPPW68, HLPPW69, and HLPPW70.

[0073] Figures 15A to 15C Depicted are size exclusion chromatography (SEC) profiles of selected HLA-E A3 (EA3) / beta-2 microglobulin (B2M) elbow variants. Figures 15A to 15C Results are shown for HLPPW59, HLPPW60, and HLPPW63, which appear as monodisperse material. Gel filtration standards are overlaid in light grey. Signals are expressed relative to the maximum signal for each sample.

[0074] Figure 16 Depicted is ELISA binding of purified disulfide engineered HLA-E A3 (EA3) / beta-2 microglobulin (B2M) pseudo-Fab to RSV glycoprotein. Shown are the luminescent signals of HLPPW59, HLPPW60, HLPPW61, HLPPW62, HLPPW63, HLPPW64, HLPPW65, HLPPW66, HLPPW67, HLPPW68, HLPPW69, and HLPPW70 compared to HLPPB271 and HLPPW58.

[0075] 17A to 17D Depicted is a stability analysis by thermal melting, measured using nanoscale differential scanning fluorimetry (NanoDSF), used to evaluate the impact of stability on the identification of two unique disulfide "pin" pairs of interest whose stabilities were comparable to a Fab control. Figure 17A The melting curves of HLPPW60, HLPPW61, HLPPW62, HLPPW63, HLPPW64, HLPPW65, HLPPW66, HLPPW67, HLPPW68, HLPPW69 and HLPPW70 are shown. Figure 17B The first derivatives of these species are shown. Figure 17C The melting curve is shown. Figure 17D The first derivatives of HLPPB271 and HLPPW58 are shown.

[0076] 18A to 18BA comparison of purified ICAM-1D1 / ICAM-1D1 dimers is depicted to confirm the presence of both chains of the heterodimer. Western blot analysis was performed on purified ICAM-1D1 / ICAM-1D1 dimers HLPPB5, HLPPB6, HLPPB9, and HLPPB10. HLPPB17 and B23B173 were used as HLA-E A3 (EA3) / β-2 microglobulin (B2M) heterodimer and native Fab controls, respectively.

[0077] 19A to 19D Depicted are the complete LC-MS molecular weight analyses of the ICAM-1D1 / ICAM-1D1 pseudo-Fab variants HLPPB5, HLPPB6, HLPPB9, and HLPPB10.

[0078] Figure 20 Depicted is an ELISA analysis of the ability of engineered ICAM-1D1 / ICAM-1D1 pseudo-Fabs to bind to RSV-F glycoprotein from pure CHO supernatant. Results are shown for HLPPB6 and HLPPB10 compared to a B23B173 native Fab control.

[0079] Figures 21A to 21B Depicted are stability analysis by thermal melting as measured using Nanoscale Differential Scanning Fluorometry (NanoDSF) for bispecific antibodies HLPPB421, HLPPB423, HLPPB425, and HLPPB426. Figure 21A Melting curves and first derivatives are shown. Figure 21B The melting onset temperature (Ton), melting temperature 1 (Tm1), melting temperature 2 (Tm2), melting temperature 3 (Tm3), and aggregation onset temperature (Tagg) are shown.

[0080] Figure 22 Depicted are non-reducing (NR) and reducing (R) PAGEs of purified bispecific antibodies HLPPB423, HLPPB425, HLPPB421, and HLPPB426, each consisting of an HLPPW59-derived pseudo-Fab in one arm and a standard IgG Fab in the other. All samples were normalized to total protein A280 and loaded equally. The ladder is indicated by (L), and molecular weights are given in kD.

[0081] Figure 23Depicted are size exclusion chromatography (SEC) profiles of purified bispecific antibodies HLPPB423, HLPPB425, HLPPB421, and HLPPB426, each composed of a pseudo-Fab derived from HLPPW59 on one arm and a standard IgG Fab on the other. Gel filtration standards are overlaid in light gray. Signals are expressed relative to the maximum signal for each sample.

[0082] Figures 24A to 24C Biolayer interferometry (BLI) binding profiles of purified bispecific antibodies are depicted. Figure 24A Shown are the BLI binding patterns of human epidermal growth factor receptor 2 (HER2) targeting HLPPB423, HLPPB425, HLPPB421, and HLPPB426 to HER2. Figure 24B Shown are the BLI binding profiles of mesenchymal epithelial transition (MET) targeting antibodies HLPPB423 and HLPPB425 to MET. Figure 24C Shown are the BLI binding profiles of cluster of differentiation 3 (CD3) targeting antibodies HLPPB421 and HLPPB426 to CD3. DETAILED DESCRIPTION

[0083] Provided herein are antigen binding molecules comprising one or more polypeptides (e.g., antigen binding polypeptides) having antibody variable domains in conjunction with target antigens, and replacing light chain constant domain (CL) or heavy chain constant domain 1 (CH1) with a dimerization domain as described herein. In a specific embodiment, the antigen binding molecules are bound to one or more target antigens. In a specific embodiment, the antigen binding molecules comprise two or more polypeptides, wherein these polypeptides form a paratope that is bound to one or more target antigens. In a specific embodiment, a pair of polypeptides each comprise a variable domain and a dimerization domain, wherein the variable domains form a paratope that is bound to the target antigen.

[0084] 7.1 Definitions

[0085] The methods disclosed herein may be more readily understood with reference to the following detailed description in conjunction with the accompanying drawings, which form a part of this disclosure. It should be understood that the methods disclosed herein are not limited to the specific methods described and / or illustrated herein, and that the terminology used herein is for the purpose of describing specific embodiments by way of example only and is not intended to be limiting.

[0086] All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein.

[0087] When a list is provided, it is understood that each individual element of the list and each combination of the list is a separate embodiment unless otherwise indicated. For example, a list of embodiments presented as "A, B, or C" will be understood to include embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0088] 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 so forth.

[0089] The transition terms "comprising," "consisting essentially of," and "consisting of" are intended to suggest their recognized meanings in patent parlance; that is, (i) "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude 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 equivalent) are also provided, as are those embodiments described independently with "consisting of" and "consisting essentially of."

[0090] "About" means within an acceptable error range for the 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 determination, result, or embodiment, unless expressly indicated otherwise in the embodiments or elsewhere in the specification, "about" means within one standard deviation or up to 5%, whichever is greater, as is customary in the art.

[0091] "Antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" 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 (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγRs) expressed on the effector cells.

[0092] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells are eliminated by internalization by phagocytes (such as macrophages or dendritic cells).

[0093] "Antigen" refers to any molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portion thereof, or combination thereof) capable of mediating an immune response. Exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells.

[0094] "Antigen-binding fragment" or "antigen-binding domain" refers to the portion of a protein that binds to an antigen. Antigen-binding fragments can be synthetic, enzymatically obtainable, or genetically engineered polypeptides and include portions of immunoglobulins that bind to an antigen, such as 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, humped VH domains, VHH domains, minimal recognition units consisting of amino acid residues that mimic the CDRs of an antibody (such as the FR3-CDR3-FR4 portion, HCDR1, HCDR2 and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3), alternative scaffolds that bind to an antigen, and multispecific proteins comprising antigen-binding fragments. Antigen binding fragments (such as VH and VL) can be linked together via synthetic linkers to form various types of single antibody designs, wherein in those cases where the VH and VL domains are expressed by separate single chains, the VH / VL domains can be paired intramolecularly or intermolecularly to form monovalent antigen binding domains, such as single-chain Fv (scFv) or diabodies. Antigen binding fragments can also be conjugated to other antibodies, proteins, antigen binding fragments, or alternative scaffolds, which can be monospecific or multispecific to engineer bispecific and multispecific proteins.

[0095] "Antibody" refers broadly to and includes immunoglobulin molecules, specifically monoclonal antibodies (including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies), antigen-binding fragments, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), dimeric, tetrameric or multimeric antibodies, single-chain antibodies, domain antibodies, and any other modified configuration of immunoglobulin molecules that contain an antigen-binding site with the desired specificity. "Full-length antibodies" are composed of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed of the 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 hypervariable regions, which are called complementarity determining regions (CDRs) and are interspersed with framework regions (FRs). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes based on the amino acid sequence of the heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The light chains of antibodies from any vertebrate species can be assigned to one of two distinct classes, kappa and lambda, based on the amino acid sequence of their constant domains.

[0096] "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 have cross-reactivity to other related antigens, for example, to the same antigen from another species (homologous), such as humans or monkeys, e.g., cynomolgus (cynomolgus, cyno) or chimpanzees (Pan troglodytes), or may bind to an epitope shared between two or more different antigens.

[0097] "Complement-dependent cytotoxicity" or "CDC" refers to a mechanism of cell death induction in which the Fc effector domain of a target-binding protein binds and activates the complement component C1q, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0098] "Complementarity determining regions" (CDRs) are regions of antibodies that bind to antigens. There are three CDRs in VH (HCDR1, HCDR2, HCDR3) and three CDRs in VL (LCDR1, LCDR2, LCDR3). CDRs can be defined using various delineations, 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 delineations and variable region numbering is described (see, e.g., Lefranc et al. (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-670; International Immunogenetics (IMGT) database; Web resource, http: / / www_imgt_org). Available programs (such as abYsis from UCL Business PLC) can be used to delineate CDRs. Unless otherwise expressly stated in the specification, as used herein, the terms "CDR," "HCDR1," "HCDR2," "HCDR3," "LCDR1," "LCDR2," and "LCDR3" include CDRs defined by any of the above methods (Kabat, Chothia, IMGT, or AbM).

[0099] "Reduce," "decrease," or "reduce" generally refers to the ability of the test molecule to mediate a reduced response (i.e., a downstream effect) when compared to a response mediated by a control or vehicle. Exemplary responses include enhanced binding of a protein to its antigen or receptor, enhanced binding to an FcγR, or enhanced Fc effector function, such as enhanced ADCC, CDC, and / or ADCP. A decrease can be a statistically significant difference in the response measured between the test molecule and a control (or vehicle), or a decrease in the measured response, such as a decrease of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30-fold or more, such as 500, 600, 700, 800, 900, or 1000-fold or more.

[0100] "Enhance," "promote," or "increase" generally refers to the ability of the test molecule to mediate a stronger response (i.e., a downstream effect) when compared to the response mediated by a control or vehicle. Exemplary responses are enhanced binding of a protein to its antigen or receptor, enhanced binding to an 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 a control (or vehicle), or an increase in the measured response, such as an increase of about 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more, such as 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more.

[0101] An "expression vector" refers to a vector that can be used in a biological system or a reconstructed biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0102] "Heterologous" refers to a polypeptide or polynucleotide comprising two or more polypeptides or two or more polynucleotides that are not found in the same relationship to each other in nature.

[0103] "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.

[0104] "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.

[0105] "Human antibody" refers to an antibody that is optimized to have a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody comprises a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is obtained by using a system of human germline immunoglobulins or rearranged immunoglobulin genes, the human antibody comprises a heavy chain variable region and a light chain variable region "derived from" a sequence of human origin. Such exemplary systems are human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals, such as mice or rats carrying human immunoglobulin loci. Because of the differences between the systems for obtaining human antibodies and human immunoglobulin loci, the introduction of somatic mutations or the intentional introduction of substitutions into the framework or CDR or both, "human antibodies" typically comprise amino acid differences compared to immunoglobulins expressed in humans. Typically, a "human antibody" has an amino acid sequence that 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 to an amino acid sequence encoded by a human germline immunoglobulin gene or a rearranged immunoglobulin gene. In some cases, a "human antibody" may comprise a consensus framework sequence derived from human framework sequence analysis (e.g., as described in Knappik et al., (2000) J Mol Biol 296:57-86), or a synthetic HCDR3 bound to a human immunoglobulin gene library displayed on phage (e.g., as described in Shi et al., (2010) J Mol Biol 397:385-396 and International Patent Publication No. WO2009 / 085462). The definition of "human antibody" does not include antibodies in which at least one CDR is derived from a non-human species.

[0106] "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. A humanized antibody may comprise substitutions in the framework such that the framework may not be an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0107] "Modulate" refers to the ability of a test molecule to increase or decrease an enhanced or decreased response (ie, a downstream effect) when compared to the response mediated by a control or vehicle.

[0108] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules, i.e., the individual antibodies comprising the population are identical, differing 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, methionine oxidation, or asparagine or glutamine deamidation. Monoclonal antibodies generally 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, monovalent, bivalent, or multivalent.

[0109] "Multispecific" refers to a molecule that binds to two or more different antigens or two or more different epitopes within the same antigen. A multispecific molecule may have cross-reactivity to other related antigens, for example, to the same antigen from another species (homologous), such as humans or monkeys, e.g., cynomolgus (cyno) or chimpanzees, or may bind to an epitope shared between two or more different antigens.

[0110] "Polynucleotide" refers to a molecule comprising a chain of nucleotides covalently linked by a phosphate sugar backbone or other equivalent covalent chemical bonds. cDNA is a typical example of a polynucleotide.

[0111] "Protein" or "polypeptide" are used interchangeably herein and refer to a molecule comprising one or more polypeptides, each comprising at least two amino acid residues linked by a peptide bond. A protein can be a monomer or a protein complex of two or more subunits, which can be identical or different. Small polypeptides of less than 50 amino acids can be referred to as "peptides." A protein can be a heterologous fusion protein, a glycoprotein, or a protein modified by a post-translational modification such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation, or biotinylation.

[0112] "Recombinant" refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules that are prepared, expressed, created, or isolated by recombinant means.

[0113] "Specifically binds," "specifically binds," or "binding" refers to binding of a protein to an antigen or an epitope within an antigen with a greater affinity than for other antigens. Typically, a protein (such as the antigen binding proteins described herein) binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (K D ) is about 1×10 -6 M or smaller, approximately 1×10 -7 M or smaller, about 5×10 -8 M or smaller, approximately 1×10-8 M or smaller, approximately 1×10 -9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller or about 1×10 -12 M or smaller, usually K D Than its K binding to nonspecific antigens (e.g., BSA, casein) D Small, at most 1 / 100 of the latter.

[0114] "Subject" includes any human or non-human animal. "Non-human animals" include 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" are used interchangeably herein.

[0115] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual.

[0116] "Treating" or "treating" a disease or condition means achieving one or more of the following: reducing the severity and / or duration of the condition, inhibiting worsening of symptoms characteristic of the condition being treated, limiting or preventing recurrence of the condition in a subject previously suffering from the condition, or limiting or preventing recurrence of symptoms of the condition in a subject previously experiencing symptoms of the condition.

[0117] "Trispecific" refers to a molecule (such as an antibody) that specifically binds to three different antigens or three different epitopes within the same antigen. Trispecific molecules may have cross-reactivity to other related antigens, for example, to the same antigen from another species (homologous), such as humans or monkeys, e.g., cynomolgus (cynomolgus, cyno) or chimpanzees (Pan troglodytes), or may bind to epitopes shared between three or more different antigens.

[0118] "Variant," "mutant," or "altered" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications (eg, one or more substitutions, insertions, or deletions).

[0119] Unless specifically stated otherwise, throughout the specification, amino acid residues of antibody constant regions are numbered according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0120] 7.2 Antigen-binding polypeptides

[0121] In some aspects, the antigen binding molecules include polypeptides that form a paratope that is bound to one or more target antigens. In a specific embodiment, the polypeptide of the antigen binding molecules is an antigen binding polypeptide. In a specific embodiment, the polypeptide of the antigen binding molecules includes a variable domain that is bound to the target antigen. In a specific embodiment, the polypeptide of the antigen binding molecules includes at least one dimerization domain as described herein. In a specific embodiment, at least one dimerization domain is combined with another dimerization domain of the antigen binding polypeptide as described herein to form a dimer. In a preferred embodiment, the polypeptide of the antigen binding molecules includes a variable domain and at least one dimerization domain.

[0122] In a specific embodiment, the antigen binding molecules comprise two, three, four, five, six, seven or eight polypeptides. In a specific embodiment, each of the two, three, four, five, six, seven or eight polypeptides comprises a variable domain that is bound to the target antigen. In a specific embodiment, each of the two, three, four, five, six, seven or eight polypeptides comprises at least one dimerization domain. In a specific embodiment, each of the two, three, four, five, six, seven or eight polypeptides comprises a variable domain that is bound to the target antigen and at least one dimerization domain. In a specific embodiment, the dimerization domain of the antigen binding polypeptide is bound to another dimerization domain of another antigen binding polypeptide to form a dimer (particularly, a heterodimer, for example, six polypeptides can form up to three dimers with each other). In a preferred embodiment, the antigen binding molecules include two polypeptides, wherein each polypeptide includes a variable domain and at least one dimerization domain, wherein the variable domains form a variable region that is attached to the target antigen, and the dimerization domains are combined with each other to form a dimer, preferably a heterodimer. In a preferred embodiment, the antigen binding molecules include four polypeptides, wherein each polypeptide includes a variable domain and at least one dimerization domain, wherein the variable domains form two variable regions that are each attached to the target antigen, and the dimerization domains are combined with each other to form two dimers. In a preferred embodiment, the antigen binding molecules include six polypeptides, wherein each polypeptide includes a variable domain and at least one dimerization domain, wherein the variable domains form three variable regions that are each attached to the target antigen, and the dimerization domains are combined with each other to form three dimers.

[0123] In a specific embodiment, the polypeptide of the antigen binding molecule includes a light chain polypeptide containing a variable domain, and a heavy chain polypeptide containing a variable domain. In a specific embodiment, the variable domains of the light chain and the variable domains of the heavy chain form a variable region comprising a paratope that is bound to the target antigen. In a specific embodiment, the light chain polypeptide is the first light chain polypeptide (LC1), the second light chain polypeptide (LC2), the third light chain polypeptide (LC3), the fourth light chain polypeptide (LC4), or another light chain polypeptide (e.g., LC5+) of the antigen binding molecule. In a specific embodiment, the heavy chain polypeptide is the first heavy chain polypeptide (HC1), the second heavy chain polypeptide (HC2), the third heavy chain polypeptide (HC3), the fourth heavy chain polypeptide (HC4), or another heavy chain polypeptide (e.g., HC5+) of the antigen binding molecule.

[0124] In a specific embodiment, the light chain polypeptide comprises an immunoglobulin or antibody light chain, or one or more fragments thereof. In a specific embodiment, the light chain polypeptide comprises an immunoglobulin or antibody kappa chain, lambda chain, sigma chain, iota chain, or one or more fragments thereof. In a specific embodiment, the light chain polypeptide is a complete immunoglobulin light chain comprising a light chain variable domain (VL) and a light chain constant domain (CL). In a preferred embodiment, the light chain constant domain is completely or partially replaced by a dimerization domain as described herein. In a specific embodiment, the light chain polypeptide can, but need not, contain an immunoglobulin light chain constant region or a fragment thereof. In a specific embodiment, the light chain polypeptide is not a complete immunoglobulin light chain. In a specific embodiment, the light chain polypeptide does not comprise an immunoglobulin light chain constant domain (CL) and a fragment thereof. In a specific embodiment, the light chain polypeptide does not comprise a dimerization sequence of a light chain constant domain. In a specific embodiment, the dimerization sequence of the light chain constant domain binds to the heavy chain constant domain to form a dimer. In a specific embodiment, the dimerization sequence of the light chain constant domain mediates dimerization between the light chain and the heavy chain. In a specific embodiment, the light chain polypeptide comprises a fragment of a constant light chain domain (CL). In a specific embodiment, the light chain polypeptide comprises 1 to 8 consecutive amino acids selected from amino acid positions 108-115 of a human immunoglobulin kappa constant domain according to EU numbering. In a specific embodiment, the light chain polypeptide comprises 1 to 8 consecutive amino acids selected from amino acid positions 108-115 of a human immunoglobulin kappa constant domain according to Kabat numbering.

[0125] In a specific embodiment, the heavy chain polypeptide comprises an immunoglobulin or antibody heavy chain, or one or more fragments thereof. In a specific embodiment, the heavy chain polypeptide comprises an immunoglobulin or antibody gamma chain, delta chain, alpha chain, μ chain, epsilon chain, or one or more fragments thereof. In a specific embodiment, the heavy chain polypeptide is a complete immunoglobulin heavy chain, which comprises a heavy chain variable domain (VH), a heavy chain constant domain 1 (CH1), a hinge region, a heavy chain constant domain 2 (CH2), and a heavy chain constant domain 3 (CH3). In a preferred embodiment, the heavy chain constant domain 1 (CH1) is completely or partially replaced by a dimerization domain as described herein. In a specific embodiment, the heavy chain polypeptide may, but need not, contain an immunoglobulin heavy chain constant domain 1 (CH1) or a fragment thereof. In a specific embodiment, the heavy chain polypeptide may, but need not, contain an immunoglobulin heavy chain constant domain 2 (CH2) or a fragment thereof. In a specific embodiment, the heavy chain polypeptide may, but need not, contain an immunoglobulin heavy chain constant domain 3 (CH3) or a fragment thereof. In a specific embodiment, the heavy chain polypeptide may, but need not, contain an immunoglobulin heavy chain constant domain 2 (CH2) or a fragment thereof and an immunoglobulin heavy chain constant domain 3 (CH3). In a specific embodiment, the heavy chain polypeptide is not a complete immunoglobulin heavy chain. In a specific embodiment, the heavy chain polypeptide does not include an immunoglobulin heavy chain constant domain 1 (CH1) and a fragment thereof. In a specific embodiment, the heavy chain polypeptide does not include a dimerization sequence of a heavy chain constant domain 1 (CH1). In a specific embodiment, the dimerization sequence of the heavy chain constant domain 1 (CH1) is bound to a light chain constant domain (CL) to form a dimer. In a specific embodiment, the dimerization sequence of the heavy chain constant domain mediates dimerization between the heavy chain and the light chain. In a specific embodiment, the heavy chain polypeptide includes a fragment of a heavy chain constant domain (CH1). In a specific embodiment, the heavy chain polypeptide includes 1 to 8 consecutive amino acids selected from amino acid positions 118-125 of human IgG1 according to EU numbering. In a specific embodiment, the light chain polypeptide comprises 1 to 8 contiguous amino acids selected from amino acid positions 114-121 of human IgGl according to Kabat numbering.

[0126] In specific embodiments, the light chain polypeptide is a first light chain polypeptide comprising, from amino-terminus to carboxyl-terminus, VL1-LD1, wherein VL1 is a first light chain variable domain, and LD1 is a first light chain dimerization domain. In specific embodiments, the heavy chain polypeptide is a first heavy chain polypeptide comprising, from amino-terminus to carboxyl-terminus, VH1-HD1, wherein VH1 is a first heavy chain variable domain, and HD1 is a first heavy chain dimerization domain. In specific embodiments, VL1 and VH1 form a first paratope that binds to a first target antigen. In specific embodiments, VL1 and VH1 form a first variable region that comprises a first paratope that binds to a first target antigen. In specific embodiments, LD1 comprises a beta-2 microglobulin (B2M) domain, and HD1 comprises an HLA-EA3 (EA3) domain. In specific embodiments, LD1 comprises an HLA-EA3 (EA3) domain, and HD1 comprises a beta-2 microglobulin (B2M) domain. In specific embodiments, the beta-2 microglobulin (B2M) domain and the HLA-E A3 (EA3) domain associate with each other to form a dimer. In specific 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 specific embodiments, the B2M domain is different from the wild-type B2M domain (SEQ ID NO: 2). In specific embodiments, the EA3 domain is different from the wild-type EA3 domain (SEQ ID NO: 33). In specific embodiments, at least one of the B2M domain or the EA3 domain is different from the wild-type B2M domain (SEQ ID NO: 2) and the wild-type EA3 domain (SEQ ID NO: 33), respectively. In specific embodiments, LD1 comprises a first ICAM-1D1 domain and HD1 comprises a second ICAM-1D1 domain. In specific embodiments, the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer. In specific embodiments, the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain.

[0127] In specific embodiments, VH1 and / or VL1 are derived from any class of immunoglobulin antibodies (including IgM, IgG, IgD, IgA, and IgE), and any isotype antibody (including IgG1, IgG2, IgG3, and IgG4, as well as IgA1 and IgA2). In specific embodiments, VH1 and / or VL1 are derived from IgG antibodies, such as IgG1 antibodies, IgG2 antibodies, or IgG4 antibodies (e.g., IgG4 null antibodies, and variants of IgG4 antibodies).

[0128] In a specific embodiment, VH1 is derived from an immunoglobulin or antibody γ chain, δ chain, α chain, μ chain or ε heavy chain. In a specific embodiment, VL1 is derived from an immunoglobulin or antibody κ chain, λ chain, σ chain or ι light chain.

[0129] In specific embodiments, VH1 and / or VL1 are derived from a human antibody, a humanized antibody, or an antibody from a non-human animal, such as a mouse antibody, a rat antibody, a camel antibody, a llama antibody, or a chimeric antibody thereof.

[0130] In a specific embodiment, the first light chain polypeptide comprises an elbow region between VL1 and LD1. In a specific embodiment, the first light chain polypeptide comprises, in amino-terminal to carboxyl-terminal order: VL1-LE1-LD1, wherein 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.

[0131] In a specific embodiment, the first light chain polypeptide comprises a first light chain spacer fused to the C-terminus of LD1. In a specific embodiment, the first light chain polypeptide comprises, from amino terminus to carboxyl terminus, VL1-LD1-LS1, wherein VL1 is a first light chain variable domain, LD1 is a first light chain dimerization domain, and LS1 is a first light chain spacer.

[0132] In a specific embodiment, the first light chain polypeptide comprises, in order from amino terminus to carboxyl terminus: VL1-LE1-LD1-LS1, wherein 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.

[0133] In a specific embodiment, the first heavy chain polypeptide comprises an elbow region between VH1 and HD1. In a specific embodiment, the first heavy chain polypeptide comprises, in amino-terminal to carboxyl-terminal order: VH1-HE1-HD1, wherein 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.

[0134] In a specific embodiment, the first heavy chain polypeptide comprises a first heavy chain spacer fused to the C-terminus of HD1. In a specific embodiment, the first heavy chain polypeptide comprises, in amino-terminal to carboxyl-terminal order: VH1-HD1-HS1, wherein VH1 is a first heavy chain variable domain, HD1 is a first heavy chain dimerization domain, and HS1 is a first heavy chain spacer.

[0135] In a specific embodiment, the first heavy chain polypeptide comprises, in order from amino terminus to carboxyl terminus: VH1-HE1-HD1-HS1, wherein 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.

[0136] The light chain polypeptides as described herein can be the first, second, third or fourth light chain polypeptides of the antigen binding molecule, each of which is intended to be specified as a first light chain polypeptide as described herein, but renumbered accordingly (e.g., VLn, LEn, LDn and LSn, wherein 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 as described herein can be the first, second, third or fourth heavy chain polypeptides of the antigen binding molecule, each of which is intended to be specified as a first heavy chain polypeptide as described herein, but renumbered accordingly (e.g., VHn, HEn, HDn and HSn, wherein 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).

[0137] In a specific embodiment, VHn and / or VLn (wherein n is 1 for the first light chain polypeptide, 2 for the second light chain polypeptide, 3 for the third light chain polypeptide, or 4 for the fourth light chain polypeptide) are derived from any class of immunoglobulin antibodies (including IgM, IgG, IgD, IgA, and IgE), and any isotype antibody (including IgG1, IgG2, IgG3, and IgG4, as well as IgA1 and IgA2). In a specific embodiment, VHn and / or VLn are derived from an IgG antibody, such as an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody (e.g., an IgG4 null antibody, and a variant of an IgG4 antibody).

[0138] In a specific embodiment, VHn (wherein 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 γ chain, δ chain, α chain, μ chain or ε heavy chain. In a specific embodiment, VLn (wherein 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 κ chain, λ chain, σ chain or ι light chain.

[0139] In a specific embodiment, VHn and / or VLn (wherein 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 from a non-human animal, such as a mouse antibody, a rat antibody, a camel antibody, a llama antibody, or a chimeric antibody thereof.

[0140] In a specific embodiment, the first heavy chain polypeptide further comprises an immunoglobulin constant domain. In a specific embodiment, the first heavy chain polypeptide further comprises a heavy chain constant domain 2 (CH2) of an antibody or fragment thereof, optionally wherein the CH2 domain is fused to the C-terminus of the first heavy chain dimerization domain or the first heavy chain spacer. In a specific embodiment, the first heavy chain polypeptide further comprises a heavy chain constant domain 3 (CH3) of an antibody or fragment thereof, optionally wherein the CH3 domain is fused to the C-terminus of the first heavy chain dimerization domain, the C-terminus of the first heavy chain spacer, or the C-terminus of the CH2 domain (when present). In a specific embodiment, the first heavy chain polypeptide further comprises a heavy chain fourth constant domain (CH4) of an antibody or fragment thereof, optionally wherein the CH4 domain is fused to the C-terminus of the first heavy chain dimerization domain, the C-terminus of the first heavy chain spacer, the C-terminus of the CH2 domain (when present), or the C-terminus of the CH3 domain (when present).

[0141] In specific embodiments, CH2 and / or CH3 are derived from any class of immunoglobulin antibodies (including IgM, IgG, IgD, IgA, and IgE), and any isotype antibody (including IgG1, IgG2, IgG3, and IgG4, as well as IgA1 and IgA2). In specific embodiments, CH2 and / or CH3 are derived from IgG antibodies, such as IgG1 antibodies, IgG2 antibodies, or IgG4 antibodies (e.g., IgG4 null antibodies, and variants of IgG4 antibodies).

[0142] In specific embodiments, CH2 and / or CH3 are derived from an immunoglobulin or antibody gamma chain, delta chain, alpha chain, mu chain, or epsilon heavy chain.

[0143] In specific embodiments, CH2 and / or CH3 are derived from a human antibody, a humanized antibody, or an antibody from a non-human animal, such as a mouse antibody, a rat antibody, a camel antibody, a llama antibody, or a chimeric antibody thereof.

[0144] 7.3 Dimerization Domain

[0145] In some aspects, the dimerization domain of the polypeptide of the antigen binding molecule forms a dimer with another dimerization domain of another polypeptide of the antigen binding molecule. In a specific embodiment, the polypeptide of the antigen binding molecule as described herein comprises a tool for dimerization with another polypeptide of the antigen binding molecule. In a specific embodiment, the means for dimerization is a dimerization domain as described herein.

[0146] In a specific embodiment, the dimerization domain combines with another dimerization domain to form a dimer. In a specific embodiment, the light chain dimerization domain completely or partially replaces the light chain constant domain (CL). In a specific embodiment, the light chain dimerization domain completely or partially replaces the dimerization sequence of the light chain constant domain (CL). In a specific embodiment, the heavy chain dimerization domain completely or partially replaces the heavy chain constant domain 1 (CH1). In a specific embodiment, the heavy chain dimerization domain completely or partially replaces the dimerization sequence of the heavy chain constant domain 1 (CH1).

[0147] 7.3.1 Beta-2 Microglobulin (B2M) Domain

[0148] In a specific embodiment, the dimerization domain as described herein includes a beta-2 microglobulin (B2M) domain. In a specific embodiment, the B2M domain is a human B2M domain. In a specific embodiment, the B2M domain is a wild-type human B2M domain, or a fragment thereof. In a specific embodiment, the B2M domain comprises the amino acid sequence B2M or a fragment thereof (e.g., a mature B2M lacking a signal sequence) shown in UniProt accession number P61769, or consists of the amino acid sequence or a fragment thereof. The amino acid sequence of B2M shown in UniProt accession number P61769 is: MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGF HPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYAC RVNHVTLSQPKIVKWDRDM (SEQ ID NO: 1). In a specific embodiment, the B2M domain comprises or consists of the amino acid sequence RTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHS DLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 2).

[0149] In a specific embodiment, the B2M domain is a variant B2M domain comprising an amino acid sequence that is different from the wild-type B2M domain SEQ ID NO: 2. In a specific embodiment, the variant B2M domain has at least one amino acid substitution, deletion, or insertion relative to the wild-type B2M domain at at least one position numbered according to the B2M amino acid numbering in Table 1 below. In a specific embodiment, the variant B2M domain has at least one amino acid substitution, deletion, or insertion relative to the wild-type B2M domain at at least one position numbered according to the amino acid sequence SEQ ID NO: 2. In a specific embodiment, the variant B2M domain differs from the wild-type B2M domain SEQ ID NO: 1 or SEQ ID NO: 2 in that one, two, three, four, five, six, seven, or eight amino acids are substituted, deleted, or inserted.

[0150] Table 1: B2M amino acid numbering

[0151]

[0152]

[0153] In specific 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.

[0154] In a specific embodiment, the B2M domain comprises or consists of an amino acid sequence having one, two, three, four, five, six, seven, or eight single amino acid substitutions to the sequence of SEQ ID NO: 2 at one or more of amino acid positions 4, 8, 10, 54, 58, 60, 96, and 97 of SEQ ID NO: 2. In a specific embodiment, the B2M domain comprises or consists of an amino acid sequence having one, two, three, four, five, six, seven, or eight single amino acid substitutions to the sequence of SEQ ID NO: 2 at one or more of amino acid positions 4, 8, 10, 54, 58, 60, 96, and 97 of SEQ ID NO: 2, wherein each of these single amino acid substitutions is independently selected from the group consisting of F, W, C, S, and T.

[0155] In a specific embodiment, the B2M domain comprises or consists of an amino acid sequence that is the human B2M sequence set forth in SEQ ID NO: 2 but having a set of substitutions F56S, W60S, and F62T (wherein positions are numbered according to the B2M amino acid numbering in Table 1), optionally further having one, two, three, four, or five single amino acid substitutions at positions K6, Y10, R12, D98, or M99 (wherein positions are numbered according to the B2M amino acid numbering in Table 1). In a specific embodiment, the B2M domain comprises or consists of an amino acid sequence that is the human B2M sequence set forth in SEQ ID NO:2 but having a set of substitutions selected from the group consisting of: 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.

[0156] In a specific embodiment, the B2M domain comprises the amino acid sequence of any one of SEQ ID NOs: 2-30, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 2, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 4, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 5, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 6, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 7, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 8, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 9, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 10, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 11, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 12, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 13, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 14, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 15, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 16, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 17, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 18, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 19, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 20. In a specific embodiment, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 21. In a specific embodiment, the B2M domain comprises or consists of the amino acid sequence of SEQ ID NO: 22.In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 23, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 24, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 25, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 26, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 27, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 28, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 29, or consists of the amino acid sequence. In a specific embodiment, the B2M domain comprises the amino acid sequence of SEQ ID NO: 30, or consists of the amino acid sequence.

[0157] 7.3.2 HLA-E A3 (EA3) Domain

[0158] In a specific embodiment, the dimerization domain as described herein comprises an HLA class I histocompatibility antigen alpha chain-E alpha-3 domain (referred to herein as HLA-E A3 or EA3). In a specific embodiment, the EA3 domain is a human EA3 domain. In a specific embodiment, the EA3 domain is a human wild-type EA3 domain, or a fragment thereof. In a specific embodiment, the EA3 domain comprises or consists of the amino acid sequence EA3 set forth in UniProt accession number P13747 or a fragment thereof. The amino acid sequence of the complete HLA class I histocompatibility antigen alpha chain-E (HLA-E) containing the EA3 domain, shown as UniProt accession number P13747, is: MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNED LRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL (SEQ ID NO: 31). The amino acid sequence of EA3 shown in UniProt accession number P13747 (e.g., positions 204-295 of SEQ ID NO: 31) is: EPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW (SEQ ID NO: 32). In a specific embodiment, the EA3 domain comprises or consists of the amino acid sequence LHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDT ELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW (SEQ ID NO: 33).

[0159] In specific 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 specific embodiments, the variant EA3 domain has at least one amino acid substitution, deletion, or insertion relative to the wild-type EA3 domain at at least one position numbered according to the EA3 amino acid numbering in Table 2 below. In specific embodiments, the variant EA3 domain has at least one amino acid substitution, deletion, or insertion relative to the wild-type EA3 domain at at least one position numbered according to the amino acid sequence of SEQ ID NO: 33. In specific 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 in that one, two, three, four, five, or six amino acids are substituted, deleted, or inserted.

[0160] Table 2: EA3 amino acid numbering

[0161]

[0162] In specific embodiments, the EA3 domain comprises, or consists of, an amino acid sequence that has 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 preferred embodiments, the EA3 domain comprises an amino acid sequence that has at least 93% sequence identity to SEQ ID NO: 33.

[0163] In a specific embodiment, the EA3 domain comprises or consists of an amino acid sequence having one, two, three, four, five or six single amino acid substitutions 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. In a specific embodiment, the EA3 domain comprises or consists of an amino acid sequence having one, two, three, four, five or six single amino acid substitutions 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 these single amino acid substitutions is independently selected from the group consisting of A, C and L.

[0164] In a specific embodiment, the EA3 domain comprises or consists of an amino acid sequence that is the human EA3 sequence set forth in SEQ ID NO:33 but has one, two, three, four, five or six single amino acid substitutions at positions H192, R202, E232, R234, D238 or Q242 (wherein positions are numbered according to the EA3 amino acid numbering in Table 2). In a specific embodiment, the EA3 domain comprises or consists of an amino acid sequence that is the human EA3 sequence shown in SEQ ID NO:33 but has a set of substitutions selected from the group consisting of: 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.

[0165] In a specific embodiment, the EA3 domain comprises the amino acid sequence of any one of SEQ ID NOs: 32-33 and 35-46, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO: 32, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO: 33, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO: 35, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO: 36, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO: 37, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO: 38, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO: 39, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO: 40, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO:41, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO:42, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO:43, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO:44, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO:45, or consists of the amino acid sequence. In a specific embodiment, the EA3 domain comprises the amino acid sequence of SEQ ID NO:46, or consists of the amino acid sequence.

[0166] 7.3.3 ICAM-1 D1 Domain

[0167] In specific embodiments, the dimerization domain as described herein comprises or consists of the intercellular adhesion molecule 1 domain 1 (ICAM-1D1). In specific embodiments, the ICAM-1D1 domain is a human ICAM-1D1 domain. In specific embodiments, the ICAM-1D1 domain is a wild-type human ICAM-1D1 domain, or a fragment thereof. In specific embodiments, the ICAM-1D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 49.

[0168] In a specific embodiment, the ICAM-1 D1 domain comprises or consists of the amino acid sequence ICAM-1 D1 set forth in UniProt Accession No. P05362, or a fragment thereof. The amino acid sequence of intercellular adhesion molecule 1 (ICAM-1) comprising the ICAM-1 D1 domain set forth in UniProt Accession No. P05362 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: GGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQP MCYSNCPDGQSTA (SEQ ID NO: 48). In a specific embodiment, the ICAM-1 D1 domain comprises or consists of the amino acid sequence QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKV YELSNVQEDSQPMCYSNCPDGQSTAKTFLTVY (SEQ ID NO: 49).

[0169] In a specific embodiment, 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 a specific embodiment, the variant ICAM-1 D1 domain has at least one amino acid substitution, deletion or insertion relative 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 a specific embodiment, the variant ICAM-1 D1 domain has at least one amino acid substitution, deletion or insertion relative 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 specific 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 a substitution, deletion or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.

[0170] Table 3: ICAM-1 D1 amino acid number

[0171]

[0172] In specific embodiments, the ICAM-1 D1 domain comprises, or consists of, an amino acid sequence that has 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 that has at least 81% sequence identity to SEQ ID NO: 49.

[0173] In a specific embodiment, 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 of SEQ ID NO: 49 at one or more of amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67 and 78 of SEQ ID NO: 49, optionally further comprising the addition of a cysteine ​​amino acid at the C-terminus. In a specific embodiment, 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 to the sequence of SEQ ID NO: 49 at one or more of amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67 and 78 of SEQ ID NO: 49, optionally further adding a cysteine ​​amino acid at the C-terminus, wherein each of these single amino acid substitutions is independently selected from the group consisting of V, T, F, W, A, K, E, C and R.

[0174] In a specific embodiment, 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 having 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 positions are numbered according to the ICAM-1 D1 amino acid numbering in Table 3), optionally further comprising the addition of a cysteine ​​amino acid (84C) at the C-terminus (wherein positions are numbered according to the ICAM-1 D1 amino acid numbering in Table 3). In a specific embodiment, the ICAM-1 D1 domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:49 The human ICAM-1D1 sequence shown in NO:49 but having or consisting of a set of substituted amino acid sequences selected from the group consisting of the following, wherein positions are numbered according to the ICAM-1D1 amino acid numbering in Table 3: i) E34K; ii) T2V, I10T, T23A, E34K, P38T, P63V, S67A, and T78A; iii) T2V, I10T, R13C, T23A, E34K, P38T, R49E, P63V, S67A, T78A, and B4C; iv) T2V, I10T, R13C, T23A, E34K, P38T, E53R , P63V, S67A, T78A, and 84C; R234A, R234L, and R234C; 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.

[0175] In specific 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 specific embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 48. In specific embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 49. In specific embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 51. In specific embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 52. In specific embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 53. In specific embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 54. In specific embodiments, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 55. In a specific embodiment, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 56. In a specific embodiment, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 57. In a specific embodiment, the ICAM-1 D1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 58.

[0176] 7.4 Elbow Area

[0177] In some aspects, the elbow area of ​​the polypeptide of antigen binding molecules is connected to its variable domains to its dimerization domain.In specific embodiments, the light chain polypeptides or heavy chain polypeptides of antigen binding molecules include elbow area.In specific embodiments, elbow area is between the variable domains and dimerization domain of light chain, or between the variable domains and dimerization domain of heavy chain polypeptide.In specific embodiments, light chain polypeptides (for example, the first, second, third, fourth or other light chain polypeptides) include light chain elbow area (for example, respectively the first, second, third, fourth or other light chain elbow area).In specific embodiments, heavy chain polypeptides (for example, the first, second, third, fourth or other heavy chain polypeptides) include heavy chain elbow area (for example, respectively the first, second, third, fourth or other heavy chain elbow area).

[0178] In specific embodiments, the light chain elbow region comprises or consists of an amino acid sequence having a length of 3 to 25 amino acids. In some embodiments, the elbow region is at least three amino acids in length. In a specific embodiment, 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); ASTKGGGGSG (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); 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); RTVAGGGGSGGGGS (SEQ ID NO:81); RTVAGGGGSGGGGS (SEQ ID NO:82); GGGGSGGGS (SEQ ID NO:83); GGGGSGGGGSGGGGS (SEQ ID NO:84); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:85); and GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:86).

[0179] In a specific embodiment, 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).

[0180] In a specific embodiment, 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).

[0181] In a specific embodiment, the light chain dimerization domain is the 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); GGGGSGGGGSGGGGS (SEQ ID NO:84); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:85); and GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:86). In specific 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); GGGGSGGGGSGGGGS (SEQ ID NO:84); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:85); and GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:86).

[0182] 7.5 Spacers and Labels

[0183] In some aspects, the spacer of the polypeptide of the antigen binding molecule is fused to the C-terminus of its dimerization domain. In a specific embodiment, the light chain spacer is fused to the C-terminus of the light chain dimerization domain of the antigen binding molecule as described herein. In a specific embodiment, the heavy chain spacer is fused to the C-terminus of the heavy chain dimerization domain of the antigen binding molecule as described herein.

[0184] In a specific embodiment, the light chain spacer comprises a hinge region. In a specific embodiment, the light chain spacer is connected to the hinge region. In a specific embodiment, the heavy chain spacer comprises a hinge region. In a specific embodiment, the heavy chain spacer is connected to the hinge region. In a specific embodiment, the hinge region is an antibody hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region).

[0185] In a specific embodiment, the light chain spacer or the heavy chain spacer comprises or consists of an amino acid sequence having a length of 2 to 9 amino acids. In a specific embodiment, the light chain spacer or the heavy chain spacer 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); GGSGESSG (SEQ ID NO: 91); and GGGSGESSG (SEQ ID NO: 92).

[0186] In a specific embodiment, the spacer of the polypeptide of the antigen binding molecule is connected to another part (e.g., another protein). In a specific embodiment, the spacer of the polypeptide of the antigen binding molecule is connected to a C-terminal tag, optionally wherein the C-terminal tag is an affinity tag or a purification tag. In a specific embodiment, the C-terminal tag is a 6x His tag (SEQ ID NO: 93), a streptavidin tag (e.g., a Strep-tag II tag), or a human influenza hemagglutinin tag. In a specific embodiment, the 6x His tag is a 6x polyhistidine (i.e., His-His-His-His-His-His (SEQ ID NO: 93)). In a specific embodiment, the C-terminal tag comprises the amino acid sequence HHHHHH (SEQ ID NO: 93), or consists of the amino acid sequence. In a specific embodiment, the C-terminal tag comprises the amino acid sequence WSHPQFEK (SEQ ID NO: 94), or consists of the amino acid sequence. In a specific embodiment, the C-terminal tag comprises the amino acid sequence YPYDVPDYA (SEQ ID NO: 95), or consists of the amino acid sequence.

[0187] 7.6 Target Antigen

[0188] In a specific embodiment, the paratope of antigen binding molecules as described herein is bound to the target antigen epitope. In a specific embodiment, the paratope is formed by the variable region of antigen binding molecules as described herein, preferably by the light chain variable domain and the heavy chain variable domain of antigen binding molecules as described herein. In a specific embodiment, the paratope is formed by two variable domains (e.g., light chain variable domain and heavy chain variable domain) of antigen binding molecules as described herein.

[0189] In a specific embodiment, the target antigen is an antigen associated with a disease or condition in a subject. In a specific embodiment, the target antigen is an antigen associated with a cancer in a subject. In a specific embodiment, the target antigen is an antigen specific for a cancer in a subject. In a specific embodiment, the target antigen is an antigen of respiratory syncytial virus (RSV).

[0190] In a specific embodiment, the target antigen is a tumor-associated antigen (TAA), which is an antigen that is overexpressed in a subject's tumor cells (preferably cancerous tumor cells) relative to a subject's non-tumor cells. In a specific embodiment, the first paratope of an antigen binding molecule as described herein binds to a first tumor-associated antigen (TAA1), the second paratope of the antigen binding molecule (if present) binds to a second tumor-associated antigen (TAA2), the third paratope of the antigen binding molecule (if present) binds to a third tumor-associated antigen (TAA3), and the fourth paratope of the antigen binding molecule (if present) binds to a fourth tumor-associated antigen (TAA4).

[0191] In specific embodiments, the target antigen is a tumor-specific antigen (TSA), which is unique to tumor cells or expressed only on tumor cells of a subject. In specific embodiments, the first paratope of an antigen binding molecule as described herein binds to a first tumor-specific antigen (TSA1), the second paratope of the antigen binding molecule (if present) binds to a second tumor-specific antigen (TSA2), the third paratope of the antigen binding molecule (if present) binds to a third tumor-specific antigen (TSA3), and the fourth paratope of the antigen binding molecule (if present) binds to a fourth tumor-specific antigen (TAA4).

[0192] 7.7 Variable Regions, Variable Domains, and Paratopes

[0193] In one aspect, the antigen binding molecules of the present invention are bound to the target antigen (that is, the paratope of the antigen binding molecules is bound to the epitope of the target antigen). In a specific embodiment, the antigen binding molecules as described herein include one or more variable regions, and the variable region forms one or more paratopes that are bound to one or more target antigens. In a specific embodiment, the variable region includes two variable domains (for example, two variable domains from two antigen-binding polypeptides in conjunction with the target antigen as described herein). In a specific embodiment, the variable region includes the light chain variable domain and the heavy chain variable domain of the antibody that is bound to the target antigen. In a specific embodiment, the variable region includes the light chain variable domain and the heavy chain variable domain of an arm of the antibody that is bound to the target antigen.

[0194] In a specific embodiment, the polypeptide of an antigen binding molecule as described herein comprises a tool for binding to a target antigen. In a specific embodiment, the tool for binding is a variable domain (e.g., a light chain variable domain or a heavy chain variable domain). In a specific embodiment, the tool for binding to two polypeptide chains of an antigen binding molecule as described herein forms a paratope.

[0195] In a specific embodiment, the variable region of an antigen binding molecule as described herein forms a paratope that is bound to a target antigen (e.g., a target antigen epitope). In a specific embodiment, the variable domains of the two polypeptide chains of an antigen binding molecule as described herein form a paratope that is bound to a target antigen (e.g., a target antigen epitope). In a specific embodiment, light chain complementary determining region 1 (LCDR1), light chain complementary determining region 2 (LCDR2), light chain complementary determining region 3 (LCDR3), heavy chain complementary determining region 1 (HCDR1), heavy chain complementary determining region 2 (HCDR2) and heavy chain complementary determining region 3 (HCDR3) form a paratope that is bound to a target antigen (e.g., a target antigen epitope).

[0196] In specific embodiments, the paratope as described herein binds to a target antigen. In specific embodiments, the paratope as described herein binds to a target antigen epitope. In specific embodiments, different paratopes (e.g., the first paratope, the second paratope, the third paratope, or the fourth paratope) of the same antigen binding molecule bind to different target antigens, respectively.

[0197] In a specific embodiment, the antigen binding molecule comprises two variable regions that bind to two epitopes. In a specific embodiment, the antigen binding molecule comprises two variable regions that bind to two target antigens. In a specific embodiment, the antigen binding molecule comprises two variable regions that bind to two different epitopes, optionally wherein the two different epitopes are from different target antigens.

[0198] In a specific embodiment, the antigen binding molecule comprises three variable regions that bind to three epitopes. In a specific embodiment, the antigen binding molecule comprises three variable regions that bind to three target antigens. In a specific embodiment, the antigen binding molecule comprises three variable regions that bind to three different epitopes, optionally wherein each of the three different epitopes is from a different target antigen.

[0199] In a specific embodiment, the antigen binding molecule comprises four variable regions that bind to four epitopes. In a specific embodiment, the antigen binding molecule comprises four variable regions that bind to four target antigens. In a specific embodiment, the antigen binding molecule comprises four variable regions that bind to four different epitopes, optionally wherein each of the four different epitopes is from a different target antigen.

[0200] In a specific embodiment, the variable domain of the polypeptide as described herein comprises an antibody light chain variable domain or a fragment thereof. In a specific embodiment, the variable domain or fragment thereof comprises an antibody light chain framework region 1 (LFR1), light chain complementary determining region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementary determining region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementary determining region 3 (LCDR3), light chain framework region 4 (LFR4), or any combination thereof that binds to a target antigen or forms a paratope that binds to a target antigen.

[0201] In a specific embodiment, the variable domain of the polypeptide as described herein comprises an antibody heavy chain variable domain or a fragment thereof. In a specific embodiment, the variable domain or fragment thereof comprises an antibody heavy chain framework region 1 (HFR1) that binds to a target antigen or forms a paratope that binds to a target antigen, heavy chain complementary determining region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementary determining region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementary determining region 3 (HCDR3), heavy chain framework region 4 (HFR4), or the amino acid sequence of any combination thereof.

[0202] In a specific embodiment, antigen binding molecules as described herein are bound to respiratory syncytial virus (RSV). In a specific embodiment, the polypeptide of antigen binding molecules as described herein includes a light chain variable domain (VL), and the light chain variable domain includes the amino acid sequence SEQ ID NO:97 or consists of the amino acid sequence. In a specific embodiment, the polypeptide of antigen binding molecules as described herein includes a heavy chain variable domain (VH), and the heavy chain variable domain includes the amino acid sequence SEQ ID NO:96 or consists of the amino acid sequence. In a specific embodiment, antigen binding molecules as described herein include a light chain variable domain (VL) and a heavy chain variable domain (VH), and the light chain variable domain includes the amino acid sequence SEQ ID NO:97 or consists of the amino acid sequence, and the heavy chain variable domain includes the amino acid sequence SEQ ID NO:96 or consists of the amino acid sequence.

[0203] In a specific embodiment, the antigen binding molecules as described herein bind to human epidermal growth factor receptor 2 (HER2). In a specific embodiment, the polypeptide of the antigen binding molecules as described herein comprises a light chain variable domain (VL) comprising or consisting of the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIK (SEQ ID NO: 362). In a specific embodiment, the polypeptide of an antigen binding molecule as described herein comprises a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 363). In a specific embodiment, an antigen binding molecule as described herein comprises a light chain variable domain (VL) and a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence SEQ ID NO: 362, and the heavy chain variable domain comprising or consisting of the amino acid sequence SEQ ID NO: 363.

[0204] In specific embodiments, antigen binding molecules as described herein are bound to mesenchymal epithelial transition factor (MET).In specific embodiments, the polypeptide of antigen binding molecules as described herein includes light chain variable domain (VL), and the light chain variable domain includes amino acid sequence DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKA PKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYA YPWTFGQGTKVEIK (SEQ ID NO:364) or consists of the amino acid sequence. In a specific embodiment, the polypeptide of an antigen binding molecule as described herein comprises a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEW VGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS (SEQ ID NO: 365). In a specific embodiment, an antigen binding molecule as described herein comprises a light chain variable domain (VL) and a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence SEQ ID NO: 364, and the heavy chain variable domain comprising or consisting of the amino acid sequence SEQ ID NO: 365.

[0205] In specific embodiments, the antigen binding molecules as described herein bind to cluster of differentiation 3 (CD3).In specific embodiments, the polypeptide of the antigen binding molecules as described herein comprises a light chain variable domain (VL) comprising or consisting of the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFG QGTKVEIK (SEQ ID NO: 366). In a specific embodiment, the polypeptide of an antigen binding molecule as described herein comprises a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO: 367). In a specific embodiment, an antigen binding molecule as described herein comprises a light chain variable domain (VL) and a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence SEQ ID NO: 366, and the heavy chain variable domain comprises or consists of the amino acid sequence SEQ ID NO: 367.

[0206] In a specific embodiment, antigen binding molecules as described herein are bound to both HER2 and MET. In a specific embodiment, the polypeptide of antigen binding molecules as described herein includes a light chain variable domain (VL1), and the light chain variable domain includes the amino acid sequence SEQ ID NO:362 or is composed of the amino acid sequence. In a specific embodiment, the polypeptide of antigen binding molecules as described herein includes the first heavy chain variable domain (VH1), and the first heavy chain variable domain includes the amino acid sequence SEQ ID NO:363 or is composed of the amino acid sequence. In a specific embodiment, antigen binding molecules as described herein include the first light chain variable domain (VL) and the first heavy chain variable domain (VH), the first light chain variable domain includes the amino acid sequence SEQ ID NO:362 or is composed of the amino acid sequence, the first heavy chain variable domain includes the amino acid sequence SEQ ID NO:363 or is composed of the amino acid sequence. In a specific embodiment, the polypeptide of antigen binding molecules as described herein includes the second light chain variable domain (VL2), and the second light chain variable domain includes the amino acid sequence SEQ ID NO:364 or is composed of the amino acid sequence. In a specific embodiment, the polypeptide of the antigen binding molecule as described herein comprises a second heavy chain variable domain (VH2), the second heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 365. In a specific embodiment, the antigen binding molecule as described herein comprises a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2), the second light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 364, and the second heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 365.

[0207] In a specific embodiment, antigen binding molecules as described herein are bound to both HER2 and CD3. In a specific embodiment, the polypeptide of antigen binding molecules as described herein includes a light chain variable domain (VL), and the light chain variable domain includes the amino acid sequence SEQ ID NO:362 or is composed of the amino acid sequence. In a specific embodiment, the polypeptide of antigen binding molecules as described herein includes a heavy chain variable domain (VH), and the heavy chain variable domain includes the amino acid sequence SEQ ID NO:363 or is composed of the amino acid sequence. In a specific embodiment, antigen binding molecules as described herein include a light chain variable domain (VL) and a heavy chain variable domain (VH), and the light chain variable domain includes the amino acid sequence SEQ ID NO:362 or is composed of the amino acid sequence, and the heavy chain variable domain includes the amino acid sequence SEQ ID NO:363 or is composed of the amino acid sequence. In a specific embodiment, the polypeptide of antigen binding molecules as described herein includes a second light chain variable domain (VL2), and the second light chain variable domain includes the amino acid sequence SEQ ID NO:366 or is composed of the amino acid sequence. In a specific embodiment, the polypeptide of the antigen binding molecule as described herein comprises a second heavy chain variable domain (VH2), the second heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 367. In a specific embodiment, the antigen binding molecule as described herein comprises a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2), the second light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 366, and the second heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 367.

[0208] 7.8 Antigen Binding Molecules

[0209] In one aspect, the antigen binding molecules of the present invention comprise a dimer of two polypeptides, each of which comprises a variable domain and a dimerization domain as described herein, wherein 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 the heavy chain constant domain 1 (CH1) provides a means for selectively assembling homologous antibody chains. For example, one arm of a biparatopic antibody (e.g., a light chain polypeptide and a heavy chain polypeptide) is modified with a B2M / EA3 or ICAM-1D1 / ICAM-1D1 dimerization domain as described herein, and the other arm is unmodified. In another example, one arm of a triparatopic antibody (e.g., a light chain polypeptide and a heavy chain polypeptide) is modified with a B2M / EA3 dimerization domain as described herein, and one arm (e.g., a light chain polypeptide and a heavy chain polypeptide) is modified with an ICAM-1D1 / ICAM-1D1 dimerization domain as described herein, and the other arm is unmodified.

[0210] In a specific embodiment, the antigen binding molecule is an immunoglobulin or an antigen binding fragment thereof. In a specific embodiment, the antigen binding molecule is an antibody or an antigen binding fragment thereof. In a specific embodiment, the antigen binding molecule is an immunoglobulin fragment or an antibody fragment comprising a variable region and at least one constant domain and binding to a target antigen. In a specific embodiment, the immunoglobulin or antibody fragment is Fab, Fab', F(ab')2 or a bispecific Fab. In a specific embodiment, 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 a specific embodiment, the antigen binding molecule is a complete immunoglobulin or an antibody. In a specific embodiment, the antigen binding molecule is not a complete immunoglobulin or an antibody. In a specific embodiment, the antigen binding molecule is any fragment of an antigen binding molecule that is bound to a target antigen as described herein.

[0211] 7.8.1 Fab-based Antigen Binding Molecules

[0212] In a specific embodiment, the antigen binding molecule is Fab. In a specific embodiment, Fab comprises an antibody fragment having a variable region that binds to a target antigen and comprises a light chain and a heavy chain fragment bridged by a disulfide bond. In a specific embodiment, the antigen binding molecule is a pseudo-Fab (pFab). In a specific embodiment, pFab comprises a variable region that binds to a target antigen and two dimerization domains as described herein. In a specific embodiment, the antigen binding molecule is Fab'. In a specific embodiment, Fab' comprises an antibody fragment having a single variable region that binds to a target antigen, comprising Fab and an additional portion of a heavy chain connected by a hinge region. In a specific embodiment, the antigen binding molecule is a pseudo-Fab' (pFab'). In a specific embodiment, pFab' comprises a variable region that binds to a target antigen, two dimerization domains as described herein, and a heavy chain hinge region. In a specific embodiment, the antigen binding molecule is F(ab')2. In a specific embodiment, F(ab')2 comprises two Fab' molecules joined by an interchain disulfide bond in the hinge region of the heavy chain. In a specific embodiment, the Fab' molecules of F(ab')2 can be directed against the same or different epitopes. In a specific embodiment, the antigen binding molecule is a pseudo F(ab')2 (pF(ab')2). In a specific embodiment, pF(ab')2 comprises two variable regions each binding to a target antigen, two antigen binding domains, two or more dimerization domains as described herein, and a heavy chain hinge region. In a specific embodiment, the antigen binding molecule is a bispecific Fab. In a specific embodiment, the bispecific Fab comprises a Fab molecule having two variable regions each binding to a target antigen, wherein each variable region can be directed against a different epitope.

[0213] Various techniques have been developed to produce antibody fragments. Traditionally, these fragments are derived via proteolysis of intact antibodies (see, for example, Morimoto et al., 1992, J. Biochem. Biophys., Methods, Vol. 24: pp. 107-117; and Brennan et al., 1985, Science, Vol. 229: pp. 81-83). However, these fragments can now be produced directly by recombinant host cells. Fab-like antibody fragments can be expressed in and secreted from Escherichia coli (E. coli) or yeast cells, allowing large quantities of these fragments to be easily produced. Antibody fragments can be isolated from the above-mentioned antibody phage libraries. According to another method, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. Fab and F(ab')2 fragments containing salvage receptor binding epitope residues with extended in vivo half-life are described in, for example, U.S. Patent No. 5,869,046. Other techniques for producing antibody fragments are apparent to those skilled in the art. Antibody fragments may also be "linear antibodies," as described, for example, in the references cited above. Such linear antibodies may be monospecific or multispecific, such as bispecific.

[0214] The antibodies provided herein include but are not limited to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, molecules containing an antigen binding site that is bound to a target antigen. The immunoglobulin molecules provided herein can be any class (for example, IgG, IgE, IgM, IgD, and IgA) or any subclass (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules containing an antigen binding site that is bound to a target antigen. In a specific embodiment, the antibody provided herein is an IgG antibody, such as an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody (for example, a variant of an IgG4 inactive body and an IgG4 antibody). In a preferred embodiment, the IgG antibody is an IgG1 antibody.

[0215] In a specific embodiment, the antigen binding molecule is a Fab, Fab', F(ab')2 or bispecific Fab comprising a variable region derived from an antibody specific for one or more target antigens. In a specific embodiment, the antigen binding molecule comprises a variable region derived from an antibody specific for one or more target antigens and a dimerization domain as described herein.

[0216] 7.8.2 Antibody-based Antigen Binding Molecules

[0217] The antigen-binding molecules of the present disclosure can be derived from antibodies by fully or partially replacing the dimerization interface between the light chain constant domain and the heavy chain constant domain 1 (CH1) of the antibody.

[0218] 7.8.2.1 Monoclonal Antibodies

[0219] In specific embodiments, the antigen binding molecules provided herein comprise monoclonal antibodies or fragments thereof. Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., 1975, Nature 256:495-97, or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent number 4,816,567).

[0220] In the hybridoma method, with the target antigen as described above, mice or other suitable host animals (such as hamsters) are immunized to stimulate the lymphocytes that produce or can produce antibodies, and these antibodies will specifically bind to the target antigen epitope for immunity. Alternatively, lymphocytes can be immunized in vitro. After immunity, lymphocytes are isolated, and then lymphocytes are fused to myeloma cell lines using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, 1986, pp. 59-103).

[0221] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that, in specific 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 phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of cells lacking HGPRT.

[0222] In one embodiment, the present invention relates to a fusion partner myeloma cell line that is fused to a stable high level of antibody production, supported by selected antibody-producing cells, and sensitive to the selective culture medium selected for the unfused parental cells. An exemplary myeloma cell line is a mouse myeloma cell line, such as SP-2 and derivatives, such as the X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, VA), and those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA). In addition, human myeloma cell lines and mouse-human heteromyeloma cell lines have 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).

[0223] The culture medium in which the hybridoma cells are grown 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 by in vitro binding assays (such as RIA or ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis described in Munson et al., 1980, Anal. Biochem., 107:220-39.

[0224] Once hybridoma cells producing antibodies of desired specificity, affinity and / or activity are identified, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 culture media. In addition, for example, by intraperitoneal injection of hybridoma cells into mice, these cells can be grown as ascites tumors in animals in vivo.

[0225] 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 affinity chromatography (e.g., using protein A or protein G-agarose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and the like.

[0226] The DNA encoding the monoclonal antibody is easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells can be used as the source of such DNA. After separation, the DNA can be placed in expression vectors, which are then transfected into host cells such as Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody proteins, to achieve the synthesis of the monoclonal antibody in recombinant host cells. Review articles on the recombinant expression of the DNA encoding the antibody in bacteria include Skerra et al., 1993, Curr. Opinion in Immunol. 5: 256-62 and Plückthun, 1992, Immunol. Revs., 130: 151-88.

[0227] In another embodiment, monoclonal antibodies or antibody fragments 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 phage display methods, functional antibody domains are displayed on the surface of phage particles, which carry polynucleotide sequences encoding the functional antibody domains. Examples of phage display methods that can be used to prepare the antibodies described herein include those disclosed 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., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT Application No. PCT / GB91 / 01134; International Publication Nos. 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. Patent 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.

[0228] In principle, synthetic antibody clones are selected by screening a phage library containing phage, which displays various fragments of the antibody variable region (Fv) fused to the phage coat protein. This type of phage library is screened for the target antigen of expectation. The clone expressing the Fv fragment that can be combined with the desired antigen is adsorbed onto the antigen, and therefore separated from the non-binding clones in the library. The binding clone is then eluted from the antigen, and can be further enriched by additional antigen adsorption / elution cycles.

[0229] The 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 they are each fused to a constant domain and interact non-covalently) as described, for example, in Winter et al., 1994, Ann. Rev. Immunol. 12:433-55.

[0230] The repertoire of VH genes and VL genes can be cloned separately by PCR and randomly recombined in a phage library, and then the antigen binding clones in the phage library can be searched, as described in Winter et al. (supra). The library from the immune source can provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the original repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self antigens and self antigens without any immunization, as described in Griffiths et al., 1993, EMBO J 12:725-34. Finally, the original library can also be prepared synthetically by cloning unrearranged V-gene segments from stem cells, and then using PCR primers containing random sequences to encode highly variable CDR3 regions and complete rearrangement in vitro, as described in, for example, Hoogenboom and Winter, 1992, J.Mol.Biol.227:381-88.

[0231] The screening of the library can be achieved by various techniques known in the art. For example, the target antigen can be used for coating the hole of the adsorption plate, expressed on the host cell attached to the adsorption plate or for cell sorting, conjugated to biotin to capture the beads coated with Strep-tag II, or for panning display library in any other method. Can be as Bass et al., 1990, Proteins 8: 309-14 and WO 92 / 09690 by using long washing and monovalent phage display, and as Marks et al., 1992, Biotechnol. 10: 779-83 by using low antigen coating density, promote the selection of antibodies with slow dissociation kinetics (for example, good binding affinity).

[0232] Antibodies can be obtained by designing an appropriate antigen screening program to select phage clones of interest, and then using the VH sequences and / or VL sequences (e.g., Fv sequences) or various CDR sequences from the VH sequences and VL sequences of the phage clones of interest and appropriate constant region (e.g., Fc) sequences as described in Kabat et al. (supra) to construct full-length antibody clones.

[0233] The antibodies described herein may also, for example, include chimeric antibodies. Chimeric antibodies are molecules in which different parts 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, 1985, Science 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.

[0234] 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, for example, culture medium, ascites fluid, serum, cell lysates, synthetic reaction materials, etc., by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. In specific embodiments, the isolated or purified antibody is substantially free of cellular material or other proteins from the cell or tissue source from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0235] 7.8.2.2 Humanized Antibodies

[0236] In a specific embodiment, the antigen binding molecules provided herein comprise humanized antibodies (e.g., deimmunized or compounded human antibodies) or fragments thereof. In a specific embodiment, the humanized antibodies comprise human framework sequences and / or human constant region sequences. In a specific embodiment, the humanized antibodies can 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., variants of IgG4, and IgG4 ineffective antibodies). In a specific embodiment, the humanized antibodies comprise kappa or lambda light chain constant sequences.

[0237] Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 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 techniques disclosed in, for example, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO93 / 17105, Tan et al., J. Immunol. Vol. 169: 1119-1125, 2002; Caldas et al., Protein Eng., Vol. 13 No. 5: 353-360, 2000; Morea et al., Methods, Vol. 20 No. 3: 267-279, 2000; Baca et al., J. Biol. Chem. Vol. 272 ​​No. 16: 10678-10684, 1997; Roguska et al., Protein Eng., Vol. 9, No. 10: pp. 895-904, 1996; Couto et al., Cancer Res., Vol. 55 (Suppl. 23): 5973s-5977s, 1995; Couto et al., Cancer Res., Vol. 55, No. 8: pp. 1717-1722, 1995; Sandhu JS, Gene, Vol. 150, No. 2: pp. 409-410, 1994; and Pedersen et al., J. Mol. Biol. Vol. 235, No. 3: pp. 959-973, 1994. See also U.S. Patent Publication No. US2005 / 0042664 A1 (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0238] Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are typically taken from the "import" variable domain. Humanization can be performed, for example, by replacing the corresponding sequence of a human antibody with a hypervariable region sequence according to Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-36).

[0239] In a specific embodiment, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of six CDRs of a parental non-human (e.g., rodent) antibody are grafted onto a human antibody framework. For example, Padlan et al. determined that only about one-third of the residues in the CDRs actually contact the antigen, and referred to these residues as "specificity determining residues" or SDRs (Padlan et al., 1995, FASEB J. 9: 133-39). In the SDR grafting technique, only SDR residues are grafted onto a human antibody framework (see, e.g., Kashmiri et al., 2005, Methods 36: 25-34).

[0240] The selection of human variable domains (both light and heavy chains) for preparing humanized antibodies is important for reducing antigenicity. For example, according to the so-called "best fit" method, the sequence of the variable domains of non-human (e.g., rodent) antibodies is screened for the entire library of known human variable domain sequences. The human sequence closest to rodents can be selected as the human framework of humanized antibodies (Sims et al., 1993, J.Immunol.151:2296-308; and Chothia et al., 1987, J.Mol.Biol.196:901-17). Another method uses a specific framework of the consensus sequence of all human antibodies derived from a specific light chain or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al., 1992, Proc.Natl.Acad.Sci.USA 89:4285-89; and Presta et al., 1993, J.Immunol.151:2623-32). In a specific case, the framework is derived from the consensus sequence of the most abundant human subclasses, VL6 subgroup I (VL6I) ​​and VH subgroup III (VHIII).In another approach, human germline genes are used as a source of framework regions.

[0241] In an alternative paradigm based on CDR comparison, called superhumanization, FR homology is irrelevant. The method involves comparing a non-human sequence with a functional human germline genomic library. Genes that encode the same or closely related canonical structures as the murine sequence are then selected. Next, among the genes that share a canonical structure with the non-human antibody, genes with the highest homology within the CDR are selected as FR donors. Finally, non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., 2002, J. Immunol. 169: 1119-25).

[0242] It is also generally desired that the antibody be humanized while retaining its affinity for the antigen and other favorable biological properties. To achieve this goal, according to a method, humanized antibodies are prepared using a three-dimensional model of the parent and humanized sequence by the analytical process of the parent sequence and various conceptually humanized products. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. 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). Examination of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, for example, analysis of residues that influence the ability of the candidate immunoglobulin to bind to its target antigen. In this way, FR residues can be selected and combined from 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 substantially most involved in influencing antigen binding.

[0243] Another method for antibody humanization is based on a measure of antibody humanness called human chain content (HSC). This method compares the mouse sequence with the repertoire of human germline genes, and the differences are scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measurement to generate a variety of different humanized variants (Lazar et al., 2007, Mol. Immunol., 44: 1986-98).

[0244] In addition to the above methods, empirical methods can also be used to generate and select humanized antibodies. These methods include methods based on generating a large gene library of humanized variants and using enrichment technology or high-throughput screening technology to select the best clone. Antibody variants can be separated from phage, ribosome and yeast display libraries, and separated by bacterial colony screening (see, for example, Hoogenboom, 2005, Nat.Biotechnol.23:1105-16; Dufner et al., 2006, TrendsBiotechnol.24:523-29; Feldhaus et al., 2003, Nat.Biotechnol.21:163-70; and Schlapschy et al., 2004, Protein Eng.Des.Sel., 17:847-60).

[0245] In the FR library approach, a collection of residue variants is introduced at specific positions in the FRs, and the libraries are subsequently screened to select the FRs that best support the transplanted CDRs. The residues to be replaced may 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: 487-99), or some or all of the "Vernier" residues from the more limited target residue group identified by the likes of Baca (1997, J. Biol. Chem. 272: 10678-84).

[0246] In FR shuffling, the complete FR is combined with the non-human CDR, rather than generating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., 2005, Methods 36:43-60). The library can be screened for binding in a two-step approach: first, humanizing the VL, then humanizing the VH. Alternatively, a one-step FR shuffling approach can be used. This approach has been shown to be more effective than two-step screening because 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).

[0247] The "human source transformation" method is based on the minimum specific determinant (MSD) necessary for experimental identification, and is based on replacing the non-human fragment sequence into the human FR library and evaluating the combination. It starts from the CDR3 district of non-human VH and VL chains, and gradually replaces other districts of non-human antibodies with human FR, including CDR1 and CDR2 of both VH and VL. The method generally results in epitope retention and identification of antibodies with multiple subclasses of different human V-segment CDRs. Human source transformation allows the separation of antibodies 91% to 96% homologous to human germline gene antibodies (see, for example, Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0248] "Human engineering" methods involve changing non-human antibodies or antibody fragments such as mice or chimeric antibodies or antibody fragments by specifically changing the amino acid sequence of the antibody, so as to produce modified antibodies with reduced immunogenicity in the human race, which still retain the desired binding properties of the original non-human antibody. Generally speaking, this technology involves classifying the amino acid residues of non-human (e.g., mouse) antibodies as "low-risk" residues, "medium-risk" residues or "high-risk" residues. Classification is performed using an overall risk / reward calculation that assesses the predicted benefit of a specific substitution (e.g., for the immunogenicity in humans) for the risk of affecting the folding of the resulting antibody. The specific human amino acid residues that will be substituted at a given position (e.g., low or medium risk) of a non-human (e.g., mouse) antibody sequence can be selected by comparing the amino acid sequence from the variable region of the non-human antibody with the corresponding region of a specific or shared human antibody sequence. The amino acid residue at the low or medium risk position in the non-human sequence can replace the corresponding residue in the human antibody sequence according to the comparison. Techniques for making human engineered proteins are described in more detail in Studnicka et al., 1994, Protein Engineering 7:805-14; US Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619; and PCT Publication WO 93 / 11794.

[0249] Composite human antibodies can be used, for example TM Composite human antibodies are produced using the technology of Antitope Ltd. (Cambridge, United Kingdom). To produce composite human antibodies, variable region sequences are designed from fragments of multiple human antibody variable region sequences in a manner that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibodies. Such antibodies can include human constant region sequences, for example, human light chain constant region and / or heavy chain constant region.

[0250] In a specific embodiment, the antigen binding molecule comprises a deimmunized antibody whose T cell epitope has been removed. Methods for preparing 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 variable regions and human constant regions depleted of T cell epitopes. In short, the VH and VL of the cloned antibody are then tested in a T cell proliferation assay to identify overlapping peptides derived from the VH and VL of the antibody to identify T cell epitopes. T cell epitopes are identified via computer methods to identify peptides that bind to human MHC class II. Mutations are introduced into VH and VL to eliminate binding to human MHC class II. Mutated VH and VL are then utilized to generate deimmunized antibodies.

[0251] 7.8.2.3 Human Antibodies

[0252] In a specific embodiment, the antigen binding molecules provided herein comprise fully human anti-human antibodies or fragments thereof. Full human antibodies can be produced 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 phage display library of human origin with known human constant domain sequences. Alternatively, the human monoclonal antibodies of the present disclosure can be prepared by a hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the preparation of human monoclonal antibodies have been described, for example, by Kozbor, 1984, J. Immunol. 133: 3001-05; Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987); and Boerner et al., 1991, J. Immunol. 147: 86-95.

[0253] It is also possible to produce transgenic animals (e.g., mice) that, upon immunization, are capable of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. Transgenic mice expressing a repertoire of human antibodies have been used to generate 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; Brüggemann 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).

[0254] Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies to the target antigen (e.g., such B lymphocytes can be recovered from an individual or can have been 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 U.S. Pat. No. 5,750,373).

[0255] Gene shuffling can also be used to derive human antibodies from non-human (e.g., rodent) antibodies, wherein the human antibodies have affinity and specificity similar to the starting non-human antibodies. According to this method, which is also referred to as "epitope imprinting" or "guided selection," the heavy chain or light chain variable region of the non-human antibody fragment obtained by phage display technology as described herein is replaced by a human V domain gene library to produce a non-human chain / human chain scFv or Fab chimera population. Non-human chain / human chain chimeric scFv or Fab separation is caused by antigen selection, wherein the human chain recovers the antigen binding site (e.g., epitope guidance selection of human chain partners (for which imprinting)) that is destroyed after removing the corresponding non-human chain in the primary phage display clone. When the process is repeated to replace the remaining non-human chains, human antibodies are obtained (see, e.g., PCT WO 93 / 06213; and Osbourn et al., 2005, Methods 36: 61-68). Unlike traditional humanization of non-human antibodies by CDR grafting, this technology provides fully human antibodies that do not have FR or CDR residues of non-human origin. Examples of guided selection for humanizing mouse 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 highly expressed on hepatocellular carcinoma (see, e.g., Bao et al., 2005, Cancer Biol. Ther., 4: 1374-80).

[0256] A potential disadvantage of the guided selection method is that the reorganization of one antibody chain while keeping the other antibody chain constant may lead to epitope drift. In order to maintain the epitope recognized by the non-human antibody, CDR retention can be applied (see, for example, Klimka et al., 2000, Br. J. Cancer. 83: 252-60; and Beiboer et al., 2000, J. Mol. Biol. 296: 833-49). In this method, the non-human VH CDR3 is usually retained because this CDR may be located in 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 of the non-human antibody as well as the VH CDR2, VL CDR2 and VL CDR1 can be retained.

[0257] 7.8.2.4 Multispecific Antibodies

[0258] Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificity for at least two different antigens. In a specific embodiment, the multispecific antibodies provided herein are bispecific antibodies. In a specific embodiment, the bispecific antibodies are mouse chimeric human antibodies or humanized antibodies. In a specific embodiment, one binding specificity is directed against one target and / or target antigen, while another binding specificity is directed against another target and / or target antigen. In a specific embodiment, the bispecific antibodies can be bound 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).

[0259] Methods for preparing multispecific antibodies are known in the art, such as by co-expressing two immunoglobulin heavy chain-light chain pairs, wherein the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For more details on generating multispecific antibodies (e.g., bispecific antibodies), see, e.g., Bispecific Antibodies (Kontermann, ed., 2011).

[0260] 7.8.2.5 Fc Engineering

[0261] It may be necessary to modify the antibodies provided herein by Fc engineering. In specific embodiments, modifications to the Fc region of an antibody cause the effector functions of the antibody to be reduced or eliminated. In specific embodiments, the effector function is ADCC, ADCP, and / or CDC. In specific 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 into the Fc region of the antibody.

[0262] In a specific embodiment, modification of the Fc region of an antibody causes an enhancement of the effector function of the antibody. In a specific embodiment, the effector function is ADCC, ADCP and / or CDC. In a specific embodiment, 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 into the Fc region of the antibody.

[0263] In a specific embodiment, the antibody can be engineered using the knob-in-hole (KiH) technique. The "knob-in-hole" technique may include: one or more mutations selected from Y349C, T366S, L368A, and Y407V in the CH3 domain of the "hole" arm Fc region; and mutations S354C and / or T366W in the CH3 domain of the "knob" arm Fc region. These mutations can promote heteromultimer formation. The "knob-in-hole" technique has been described in U.S. Patent Nos. 5,731,168 and 8,216,805, the entire contents of which are incorporated herein by reference.

[0264] In a specific embodiment, one or both Fc regions of an antibody can be engineered to include RF mutations. "RF mutation" generally refers to a mutation of amino acid HY to RF in the CH3 domain of the Fc region, such as mutations H435R and Y436F in the CH3 domain, as described by Jendeberg, L. et al. (1997, J. Immunological Meth., 201: 25-34). RF mutations are described as being beneficial for purification purposes because they eliminate binding to protein A. In some embodiments, one Fc region of an antibody includes RF mutations, and the other Fc region does not include RF mutations.

[0265] In a specific embodiment, 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:

[0266] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:360)

[0267] In some embodiments, the antigen binding molecules described herein comprise a "hole" arm comprising mutations T366S, L368A, and Y407V in the CH3 domain of the Fc region. The "hole" arm may also comprise an RF mutation. In one embodiment, the "hole" arm comprises or consists of the following amino acid sequence:

[0268] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLSLSPGK (SEQ ID NO: 361)

[0269] To extend the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into an antibody (particularly an antibody fragment), for example, as described in U.S. Patent No. 5,739,277. The term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for extending the serum half-life of the IgG molecule in vivo.

[0270] 7.8.2.6 Antibody Variants

[0271] In specific embodiments, it is envisioned that the amino acid sequence modifications of the antibodies or antigen-binding fragments provided herein are modified. 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. Therefore, in addition to the antibodies described herein, it is contemplated that antibody variants may be prepared. For example, antibody variants may be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or polypeptide. It will be appreciated by those skilled in the art that amino acid changes may alter the post-translational processes of the antibody, such as changing the number or position of glycosylation sites or changing membrane anchoring characteristics.

[0272] In specific embodiments, the antibodies provided herein are chemically modified, for example, by covalently attaching any type of molecule to the antibody. Antibody derivatives can include, for example, antibodies chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting groups / blocking groups, proteolytic cleavage, connection with cellular ligands or other proteins, etc. Many chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, preparation, metabolic synthesis of tunicamycin, etc. In addition, the antibody can contain one or more atypical amino acids.

[0273] Variant can be the replacement, deletion or insertion of one or more codons encoding an antibody or polypeptide, which causes the amino acid sequence to change compared to a native sequence antibody or polypeptide. Amino acid substitution can be the result of replacing another amino acid with an amino acid having similar structural properties and / or chemical properties, such as replacing leucine with serine, for example, conservative amino acid substitution. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that cause amino acid substitutions. Insertion or deletion can optionally be within the range of about 1 to 5 amino acids. In a specific embodiment, replacement, deletion or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, substitution is a conservative amino acid substitution carried out at one or more predicted non-essential amino acid residues. Allowed variants can be determined by systematically inserting, deleting or replacing the amino acids in the sequence and testing the resulting variants for the activity exhibited by the full-length or mature native sequence.

[0274] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of an antibody to an enzyme (e.g., for antibody-directed enzyme prodrug therapy) or a polypeptide to extend the serum half-life of the antibody.

[0275] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similarly charged side chain. Families of amino acid residues with similarly charged side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-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 the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed, and the activity of the protein can be determined.

[0276] The basic modification in the biological properties of the antibody is achieved by selecting substitutions that have a significantly different effect on maintaining (a) the structure of the polypeptide backbone in the region of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain. Alternatively, conservative (e.g., within a group of amino acids with similar properties and / or side chains) substitutions can be made so as to maintain or not significantly change these properties. Amino acids can be grouped according to the similarity of their side chain properties (see, e.g., Lehninger, Biochemistry 73-75 (2nd 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).

[0277] Alternatively, naturally occurring residues can be divided into several 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 affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0278] Non-conservative substitutions entail exchanging a member of one of these classes for another. Such substituted residues may also be introduced into conservative substitution sites or into remaining (non-conservative) sites. Thus, in one embodiment, the antibody or antigen-binding fragment thereof that binds to the 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 below. 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., the antibodies described in Section 7 below.

[0279] Can use method known in the art to change, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning and PCR mutagenesis.Can carry out site-directed mutagenesis (see, for example, Carter, 1986, Biochem J.237:1-7 to cloned DNA; With Zoller et al., 1982, Nucl.Acids Res., 10:6487-500), cassette mutagenesis (see, for example, Wells et al., 1985, Gene34:315-23) or other known techniques, to produce antigen binding molecule variant DNA.

[0280] Any cysteine ​​residue that is not involved in maintaining the correct conformation of the antibodies provided herein can also be substituted, for example, with another amino acid such as alanine or serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds can be added to the antibody to improve its stability (e.g., when the antibody is an antibody fragment such as an Fv fragment).

[0281] In a specific embodiment, the antibody molecule of the present disclosure is a "deimmunization" antibody. A "deimmunization" antibody is an antibody derived from a humanized antibody or a chimeric antibody, and compared with the corresponding original non-deimmunized antibody, the "deimmunization" antibody has one or more changes in its amino acid sequence, and these changes cause the immunogenicity of the antibody to decrease. One of the methods for producing such antibody mutants includes identifying and removing the T cell epitopes of the antibody molecule. In the first step, the immunogenicity of the antibody molecule can be measured by several methods, for example, by in vitro determination of T cell epitopes as known in the art or by predicting such epitopes via computer simulation. Once the key residues of the T cell epitope function are identified, mutations can be performed to remove immunogenicity and retain antibody activity. For review, see, for example, Jones et al., 2009, Methods in Molecular Biology 525:405-23.

[0282] 7.8.2.7 In vitro affinity maturation

[0283] In a specific embodiment, the antibody variants with improved characteristics (such as affinity, stability or expression level) compared with the parent antibody can be prepared by in vitro affinity maturation. Like natural prototype, in vitro affinity maturation is based on the principle of mutation and selection. Antibody library is displayed on the surface of organism (for example, phage, bacterium, yeast or mammalian cell) or associates (for example, covalently or non-covalently) with its encoding mRNA or DNA. The affinity of the antibody displayed is selected to allow separation of organisms or complexes carrying the genetic information of the encoding antibody. Two or three rounds of mutation and selection using display methods such as phage display to produce antibody fragments with affinity in the low nanomolar range are usually produced. The antibody of affinity maturation can have nanomolar or even picomolar affinity to the target antigen.

[0284] Phage display is a common method for displaying and selecting antibodies. Antibodies are displayed on the surface of Fd or M13 phage as fusions with phage coat proteins. Selection involves exposure to an antigen to allow the phage-displayed antibodies to bind to their target, a process known as "panning." Phage that bind to the antigen are recovered and used to infect bacteria to produce phage for further rounds of selection. For review, see, for example, Hoogenboom, 2002, Methods. Mol. Biol. 178: 1-37; and Bradbury and Marks, 2004, J. Immunol. Methods 290: 29-49.

[0285] In a yeast display system (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 adhesion subunit of the yeast lectin protein Aga2p, which is attached to the yeast cell wall via a disulfide bond bound to Aga1p. Displaying the protein via Aga2p protrudes from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall. Libraries are screened using magnetic separation and flow cytometry to select antibodies with improved affinity or stability. Binding to a soluble antigen of interest is determined by labeling yeast with biotinylated antigen and a secondary reagent conjugated to a fluorophore, such as Strep-tag II. Surface-expressed variants of the antibody can be measured by immunofluorescence labeling of hemagglutinin or c-Myc epitope tags flanked by scFvs. It has been shown that expression is correlated with the stability of the displayed protein, thus allowing the selection of antibodies with improved stability and affinity (see, e.g., Shusta et al., 1999, J. Mol. Biol. 292:949-56). Another advantage of yeast display is that the displayed protein is folded in the endoplasmic reticulum of eukaryotic yeast cells using endoplasmic reticulum chaperones and quality control mechanisms. Once maturation is complete, the antibody affinity can be conveniently "titrated" while being displayed on the yeast surface, thereby eliminating the need for expression and purification of each clone. A theoretical limitation of yeast surface display is that the size of the functional library may be smaller than with other display methods; however, recent methods use the mating system of yeast cells to generate libraries estimated to be 10 14 The combinatorial diversity of genes is shown in Table 1 (see, e.g., U.S. Patent Publication No. 2003 / 0186374; and Blaise et al., 2004, Gene 342:211-18).

[0286] In ribosome display, antibody-ribosome-mRNA (ARM) complex is generated for selection in a cell-free system. The DNA library encoding the specific antibody library is fused to the spacer gene lacking a stop codon. When translated, the spacer is still attached to the peptidyl tRNA and occupies the ribosomal channel, and therefore allows the target protein to protrude from the ribosome and fold. The resulting mRNA, ribosome and protein complex can be combined with a surface-bound ligand, thereby allowing the antibody and its encoding mRNA to be separated simultaneously by affinity capture with the ligand. The ribosome-bound mRNA is then reverse transcribed back to cDNA, which can then be mutagenized and used for the next round of selection (see, for example, Fukuda et al., 2006, Nucleic Acids Res. 34: e127). In mRNA display, puromycin is used as an adapter molecule to establish a covalent bond between the antibody and the mRNA (Wilson et al., 2001, Proc. Natl. Acad. Sci. USA 98: 3750-55).

[0287] Because these methods are performed entirely in vitro, they offer two major advantages over other selection techniques. First, the diversity of the library is not limited by the efficiency of bacterial cell transformation, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, after each selection round, random mutations can be easily introduced, for example, by non-proofreading polymerases, because the library does not have to be transformed after any diversification step. In specific embodiments, mammalian display systems can be used.

[0288] Diversity can also be introduced into the CDR of the antibody library in a targeted manner or by random introduction. The former method includes sequentially targeting all CDRs of the antibody via high-level or low-level mutagenesis, or targeting the separation hotspots of somatic hypermutations (see, for example, Ho et al., 2005, J.Biol.Chem.280:607-17) or residues suspected of affecting affinity based on experimental basis or structural reasons. Diversity can also be introduced by replacing naturally diverse regions via DNA shuffling or similar techniques (see, for example, 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 residues (see, e.g., Bond et al., 2005, J. Mol. Biol. 348: 699-709), employ loop deletions and insertions in the CDRs, or employ hybridization-based diversification (see, e.g., U.S. Patent Publication No. 2004 / 0005709). Additional methods for generating CDR diversity are disclosed, e.g., in U.S. Patent No. 7,985,840. Additional methods that can be used to generate antibody libraries and / or antibody affinity maturation are disclosed, e.g., in U.S. Patent Nos. 8,685,897 and 8,603,930, and U.S. Publication Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0289] Screening of the library can be achieved by various techniques known in the art. For example, the antibody can be immobilized on a solid support, column, needle, or cellulose / poly(vinylidene fluoride) membrane / other filter, expressed on host cells attached to an adsorption plate or for cell sorting, or conjugated to biotin for capture with Strep-tag II-coated beads, or used in any other method for panning display libraries.

[0290] For review of in vitro affinity maturation methods, see, eg, Hoogenboom, 2005, Nature Biotechnology 23: 1105-16; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia 4: 39-51; and references therein.

[0291] 7.8.2.8 Antibody Modification

[0292] Covalent modification of the antibody bound to the target antigen is included within the scope of the present disclosure. Covalent modification includes reacting the targeted amino acid residue of the antibody with an organic derivatizing agent that can react with the selected side chain or N- or C-terminal residue of the antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues into corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, hydroxyphosphorylation of seryl or threonyl residues, methylation of the α-amino group of lysine, arginine and histidine side chains (see, for example, Creighton, Proteins:Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0293] Other types of covalent modifications of the antibodies provided herein that are included within the scope of the present 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 (e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes) in a manner described, e.g., in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0294] 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)).

[0295] Also provided herein are fusion proteins comprising an antigen binding molecule provided herein that binds to a target antigen and a heterologous polypeptide.

[0296] Also provided herein are sets of antigen binding molecules that bind to one or more target antigens. In specific embodiments, the antigen binding domain sets have different association rates, different dissociation rates, different affinities for the target antigens, and / or different specificities for the target antigens. In some embodiments, these sets comprise about 10, 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, about 1000 or more antibodies, or consist of the above number of antibodies. The antigen binding domain sets can be used in assays, such as ELISA, in, for example, 96-well or 384-well plates.

[0297] 7.8.2.9 Immunoconjugates

[0298] The present disclosure also provides conjugates comprising any of the antibodies or antigen-binding fragments thereof of the present disclosure covalently bound to one or more non-antibody agents via a synthetic linker.

[0299] In specific embodiments, the antibodies provided herein are conjugated or recombinantly fused to therapeutic agents (e.g., cytotoxic agents) or diagnostic molecules or detectable molecules. The conjugated or recombinantly fused antibodies can be used, for example, to treat or prevent a disease or condition. The conjugated or recombinantly fused antibodies can be used, for example, to monitor or prognose the onset, development, progression, and / or severity of a disease or condition.

[0300] Such diagnosis and detection can be achieved, for example, by coupling the antibody to a detectable substance, which includes but is not limited to 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, dichlorotriazinylaminofluorescein, dansyl chloride or phycoerythrin; luminescent materials such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin or aequorin; chemiluminescent materials such as, but not limited to, acridinium ester-based compounds or HALOTAG; radioactive materials such as, but not limited to, iodine (131I, 125I, 123I and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In and 111In), technetium (99Tc), Thallium (201Ti), gallium (68Ga and 67Ga), palladium (103Pd), 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; positron-emitting metals using various positron emission tomography techniques; and non-radioactive paramagnetic metal ions.

[0301] Also provided herein are antibodies that are recombinantly fused or chemically conjugated (covalently or non-covalently) to a heterologous protein or polypeptide (or a fragment thereof, e.g., to 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 generate a fusion protein, 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 fused to the antibody can be used to target the antibody to a specific cell type.

[0302] In addition, the antibodies provided herein can be fused to a marker or "tag" sequence (such as a peptide) to facilitate purification. In a specific embodiment, the marker or tag amino acid sequence is a hexahistidine peptide (SEQ ID NO: 93), such as the tag provided in the pQE vector (see, for example, QIAGEN, Inc.), 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 a convenient method for fusion protein purification. Other peptide tags used for purification include, but are not limited to, hemagglutinin ("HA") tags, which correspond to epitopes derived from influenza hemagglutinin proteins (Wilson et al., 1984, Cell 37: 767-78) and "FLAG" tags.

[0303] Methods for fusing or conjugating moieties, including polypeptides, to antibodies are known (see, 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., 2nd 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 Prospective of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy, in Monoclonal Antibodies for Cancer Detection and Therapy, pp. 303-316 (Baldwin et al., ed., 1985); Thorpe et al., 1982, Immunol. Rev. 62:119-58; U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP 307,434, EP 367,166, EP 394,827; PCT Publications WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, and WO 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).

[0304] 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 employed to alter the activity of an antibody as provided herein, including, for example, antibodies with higher affinity and lower off-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). Can carry out random mutagenesis to change antibody or encoded antibody before recombination by error-prone PCR, random nucleotide insertion or other methods.The polynucleotide encoding antibody provided herein can be recombined with one or more components, motifs, sections, parts, domains, fragments etc. of one or more heterologous molecules.

[0305] The antibodies provided herein can also be conjugated to a second antibody to form an antibody heteroconjugate as described, for example, in US Patent No. 4,676,980.

[0306] Antibodies as provided herein can also be attached to a solid support, which is particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0307] The linker can be a "cleavable linker" that facilitates release of the conjugate in the cell, but 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) designed to evade multidrug transporter-mediated resistance, disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers comprising amino acids such as valine and / or citrulline, such as 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 (see, e.g., Kovtun et al., 2010, Cancer Res. 70: 2528-37).

[0308] Conjugates of antibodies and agents can be prepared using a variety of bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMCC, Sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate). The present disclosure also contemplates that conjugates of antibodies and agents can be prepared using any suitable method as disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2nd ed., 2008)).

[0309] Conventional conjugation strategies for antibodies and reagents have been based on random conjugation chemistry, involving the ε-amino groups of Lys residues or the thiol groups of Cys residues, which result in heterogeneous conjugates. Recently developed technologies allow for site-specific conjugation with antibodies, resulting in uniform loading and avoiding conjugate subpopulations with altered antigen binding or pharmacokinetics. These technologies include "thiomab" engineering including cysteine ​​substitutions at positions on heavy and light chains, which provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly nor alter antigen binding (see, e.g., Junutula et al., 2008, J. Immunol. Meth. 332: 41-52; and Junutula et al., 2008, Nature Biotechnol. 26: 925-32). In another approach, selenocysteine ​​was co-translationally inserted into the antibody sequence by recoding the stop codon UGA from stop to selenocysteine ​​insertion, thereby 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).

[0310] 7.9 Polynucleotides

[0311] In a specific embodiment, the present disclosure encompasses polynucleotides (interchangeably referred to herein as nucleic acids) encoding antigen binding molecules or fragments thereof as described herein. The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that only comprise the coding sequence of a polypeptide and polynucleotides that comprise additional coding and / or non-coding sequences. The polynucleotides of the present disclosure can be in RNA form or DNA form. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single-stranded, can be a coding strand or a non-coding (antisense) strand.

[0312] In a specific embodiment, the polynucleotide comprises a coding sequence for a polypeptide fused in the same reading frame to a polynucleotide that facilitates, for example, expression and secretion of the polypeptide from a host cell (e.g., a leader sequence that acts as a secretory sequence for controlling transport of the polypeptide). The polypeptide may have a leader sequence that is cleaved by the host cell to form a "mature" form of the polypeptide.

[0313] In a specific embodiment, the polynucleotide comprises a coding sequence for a polypeptide fused to a marker or tag sequence in the same reading frame. For example, in a specific embodiment, the marker sequence is a hexahistidine tag (SEQ ID NO: 93) provided by a vector, which allows for efficient purification of the polypeptide fused to the marker in the presence of a bacterial host. In a specific embodiment, the marker is used in conjunction with other affinity tags.

[0314] The present disclosure also relates to variants of the polynucleotides described herein, wherein the variants encode, for example, fragments, analogs, and / or derivatives of the polypeptides. In specific embodiments, the present disclosure provides a polynucleotide 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 specific 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.

[0315] As used herein, the phrase "a polynucleotide having a nucleotide sequence that is 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 include up to five point mutations per every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides in 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 those terminal positions, either interspersed individually among the nucleotides of the reference sequence or in one or more contiguous groups within the reference sequence.

[0316] Polynucleotide variants can contain changes in coding regions, non-coding regions or both. In specific embodiments, polynucleotide variants contain changes that produce silent substitutions, additions or deletions but do not change the characteristics or activity of the encoded polypeptide. In specific embodiments, polynucleotide variants include silent substitutions that do not cause the amino acid sequence of the polypeptide to change (due to the degeneracy of the genetic code). Polynucleotide variants can be produced for a variety of reasons, for example, in order to optimize the codon expression of a specific host (that is, the codons in human mRNA are changed to preferred codons for bacterial hosts such as Escherichia coli). In specific embodiments, polynucleotide variants include at least one silent mutation in the non-coding region or the coding region of the sequence.

[0317] In a specific embodiment, polynucleotide variants are generated to regulate or alter the expression (or expression level) of an encoded polypeptide. In a specific embodiment, polynucleotide variants are generated to increase the expression of an encoded polypeptide. In a specific embodiment, polynucleotide variants are generated to reduce the expression of an encoded polypeptide. In a specific embodiment, polynucleotide variants increase the expression of an encoded polypeptide compared to the parent polynucleotide sequence. In a specific embodiment, polynucleotide variants decrease the expression of an encoded polypeptide compared to the parent polynucleotide sequence.

[0318] In specific embodiments, the present disclosure provides a polynucleotide 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 specific embodiments at least about 96%, 97%, 98% or 99% identical to the polynucleotides listed in the sequence listing provided herein (e.g., SEQ ID NOs: 3 and 263-343).

[0319] In specific embodiments, the present disclosure provides a polynucleotide 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 specific embodiments at least about 96%, 97%, 98% or 99% identical to a polynucleotide selected from the polynucleotides provided herein (e.g., SEQ ID NOs: 3 and 263-343).

[0320] In specific embodiments, the polynucleotide is isolated. In specific embodiments, the polynucleotide is substantially pure.

[0321] Also provided are vectors and cells comprising the polynucleotides described herein. In specific embodiments, the expression vector comprises a polynucleotide molecule. In specific embodiments, the host cell comprises an expression vector comprising a polynucleotide molecule. In specific embodiments, the host cell comprises one or more expression vectors comprising a polynucleotide molecule. In specific embodiments, the host cell comprises a polynucleotide molecule as described herein. In specific embodiments, the host cell comprises one or more polynucleotide molecules as described herein.

[0322] 7.10 Methods or Processes for Preparing Antigen-Binding Molecules

[0323] In yet another aspect, provided herein is a method or process for preparing antigen binding molecules as described herein. Although the antigen binding molecules described below are antibodies (for example, comprising the biparatopic antibodies of dimerization domain as described herein), the following content should be understood to be also applicable to other antigen binding molecules of the present invention, unless it is understood to be inapplicable from context.

[0324] The recombinant expression of the antigen binding molecules provided herein can be achieved by constructing an expression vector containing one or more polynucleotides encoding antigen binding molecules. Once the polynucleotides encoding the antigen binding molecules provided herein, the heavy chain or light chain of antigen binding molecules, or a fragment thereof (such as but not necessarily containing heavy chain and / or light chain variable domains) have been obtained, technology well known in the art can be used to produce the vector for the production of the antigen binding molecules by recombinant DNA technology. Therefore, this paper describes a method for preparing protein by expressing a polynucleotide containing the nucleotide sequence encoding the antigen binding molecules. Methods well known to those skilled in the art can be used to construct an expression vector containing the antigen binding molecules coding sequence and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic technology, and genetic recombination in vivo. Replicable vectors are also provided, which comprise the nucleotide sequence of the antigen binding molecules, the heavy chain or light chain of the antigen binding molecules, the heavy chain or light chain of the antigen binding molecules, or a fragment thereof, or heavy chain or light chain CDR that is operably connected to a promoter. Such vectors can include nucleotide sequences encoding the constant regions of antigen binding molecules (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464), and the variable domains of the antibody can be cloned into such vectors to express the entire heavy chain, the entire light chain, or both the entire heavy chain and the entire light chain. In a specific embodiment, the antigen binding molecule comprises one or more antigen binding polypeptides having an antibody variable domain and, instead of a light chain constant domain (CL) or a heavy chain constant domain 1 (CH1), having a dimerization domain as described herein.

[0325] By conventional techniques, the expression vector is transferred to a host cell, and then the transfected cell is cultivated by conventional techniques to produce the antigen binding molecules provided herein. Therefore, this paper also provides a host cell containing the polynucleotides of the antigen binding molecules or fragments thereof provided herein that are operably connected to the coding of a heterologous promoter, or its heavy chain or light chain or a fragment of a heavy chain or light chain. The host cell can be any type of cell, for example, a primary cell, a cell in culture, or a cell from a cell line. In a specific embodiment, the host cell is a cell transfected with the nucleic acid molecules (for example, a vector) provided herein. In a specific embodiment, the host cell is an offspring or potential offspring of a cell transfected with the nucleic acid molecules (for example, a vector) provided herein. In a specific embodiment, the host cell is a cell transfected with the nucleic acid molecules (for example, a vector) provided herein. In a specific embodiment, the host cell is a cell transfected with the nucleic acid molecules (for example, a vector) provided herein. In a specific embodiment, the vector encoding the heavy chain and the light chain can be co-expressed in the host cell to express a complete immunoglobulin molecule. In a specific embodiment, the vector encoding each of the heavy chain and the light chain can be co-expressed in the host cell to express a complete immunoglobulin molecule. In specific embodiments for expressing six-chain antigen binding molecules (e.g., triparatopic antigen binding molecules), vectors encoding each of the heavy and light chains can be co-expressed in a host cell to express a complete immunoglobulin molecule, as described in detail below. In specific embodiments for expressing eight-chain antigen binding molecules (e.g., tetraparatopic antigen binding molecules), vectors encoding each of the heavy and light chains can be co-expressed in a host cell to express a complete immunoglobulin molecule, as described in detail below. In specific embodiments, multiparatopic antigen binding molecules as described herein are produced in a single host cell.

[0326] A variety of host expression vector systems can be utilized to express the antigen binding molecules provided herein (see, for example, U.S. Patent number 5,807,715). Such host expression system represents a vehicle by which a coding sequence of interest can be produced and subsequently purified, and also represents a cell that can in situ express the antigen binding molecules provided herein when transformed or transfected with an appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antigen binding molecule coding sequences, such as bacteria (e.g., Escherichia coli and Bacillus subtilis (B.subtilis)); yeast transformed with a recombinant yeast expression vector containing an antigen binding molecule coding sequence (e.g., Pichia pastoris (Saccharomyces Pichia); insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antigen-binding molecule coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antigen-binding molecule coding sequence; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) having a recombinant expression construct containing a mammalian cell-derived protein. The promoter of the genome (for example, metallothionein promoter) or the promoter derived from mammalian virus (for example, adenovirus late promoter; vaccinia virus 7.5K promoter). Bacterial cells (such as Escherichia coli) or eukaryotic cells, especially cells for expressing complete recombinant antigen-binding molecules, can be used to express recombinant antigen-binding molecules. For example, mammalian cells (such as Chinese hamster ovary cells (CHO)) combined with vectors (such as major intermediate early gene promoter elements from human cytomegalovirus) are effective expression systems for antigen-binding molecules (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio / Technology 8:2). In a specific embodiment, the antigen binding molecules provided herein are produced in CHO cells. In a specific embodiment, the expression of the nucleotide sequence encoding the antigen binding molecules provided herein that immunospecifically bind to the target antigen is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter. In a specific embodiment, the expression of the nucleotide sequence encoding the antigen binding molecules provided herein that immunospecifically bind to the target antigen is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0327] In bacterial systems, according to the purposes of the expressed antigen binding molecules, many expression vectors can be advantageously selected. For example, when a large amount of such antigen binding molecules are to be produced, in order to generate the pharmaceutical composition of antigen binding molecules, it is instructing the carrier for the expression of the high-level fusion protein product that is easy to purify may be ideal. Such carriers include but are not limited to Escherichia coli expression vector pUR278 (Ruther et al., 1983, EMBO 12: 1791), wherein the antigen binding molecule coding sequence and the lac Z coding region can be individually connected to the carrier in the same frame to produce fusion protein; pIN carrier (Inouye and Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke and Schuster, 1989, J.Biol.Chem. 24: 5503-5509); etc. pGEX carriers can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to the matrix glutathione agarose beads, followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0328] In insect systems, the California californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in fall armyworm (Spodoptera frugiperda) cells. The antigen binding molecule coding sequence can be individually cloned into the non-essential region (e.g., the polyhedrin gene) of the virus and placed under the control of the AcNPV promoter (e.g., the polyhedrin promoter).

[0329] In mammalian host cells, many viral-based expression systems can be utilized. When adenovirus is used as an expression vector, the antigen binding molecules coding sequence of interest can be connected to adenovirus transcription / translation control complex, for example, late promoter and triplet leader sequence. Then, the chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. The non-essential region (for example, E1 or E3 region) of the viral genome will be inserted alive and can express the recombinant virus of the antibody molecule in the infected host (for example, referring to Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). For the effective translation of the inserted antigen binding molecules coding sequence, specific start signals may also be needed. These signals include ATG start codon and adjacent sequences. In addition, the start codon must be identical with the reading frame of the desired coding sequence, to ensure the translation of the whole insert. These exogenous translation control signals and start codons can be both natural and synthetic multiple sources. Expression efficiency can be enhanced by including appropriate transcription enhancer elements, transcription terminators, and the like (see, eg, Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0330] In addition, the expression of the sequence inserted can be selected or the host cell strain of modifying and processing gene product in the ad hoc manner desired. Such modification (e.g., glycosylation) and processing (e.g., cracking) of protein product may be very important for the function of protein. Different host cells have the characteristic and specific mechanisms for post-translational processing and modification of protein and gene product. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of expressed foreign proteins. For this reason, eukaryotic host cells with the cellular mechanisms of suitable processing, glycosylation and phosphorylation of the primary transcript of 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, BT20 and T47D, NS0 (mouse myeloma cell line that does not endogenously produce any immunoglobulin chain), CRL7030 and HsS78Bst cells. In specific embodiments, the fully human antigen binding molecules provided herein are produced in mammalian cells (such as CHO cells).

[0331] For the long-term, high-yield production of recombinant proteins, stable expression can be utilized.For example, the cell line of the antigen binding molecules of stable expression can be engineered.Do not use the expression vector containing viral replication origin, but use the DNA and selective marker controlled by appropriate expression control elements (for example, promoter, enhancer, sequence, transcription terminator, polyadenylation site etc.) to transform host cells.After foreign DNA is introduced, engineered cells can be allowed to grow 1-2 days in enrichment medium, then switched to selection medium. The selective marker in the recombinant plasmid gives selection resistance, and allows cells to be stably integrated into its chromosome by plasmid and grow into colonies, which can then be cloned and expanded into cell line. This method can be advantageously used for the cell line of the antigen binding molecules of engineered expression. This type of engineered cell line may be particularly useful in screening and evaluating the compositions that directly or indirectly interact with antigen binding molecules.

[0332] A number of selection systems are available, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska and Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes, which can be used for tk- cells, hgprt- cells, or aprt- cells, respectively. In addition, antimetabolite resistance can be used as the basis for selection 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 and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol., Vol. 32: pp. 573-596; Mulligan, 1993, Science, Vol. 260: pp. 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., 1984, Gene 30: 147).Methods generally known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clones, 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, the entireties of which are incorporated herein by reference.

[0333] The expression level of antigen-binding molecules can be increased by vector amplification (for 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, Volume 3 (Academic Press, New York, 1987)). When the marker in the vector system expressing the antigen-binding molecule is amplifiable, the increase in the inhibitor level present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antigen-binding molecule gene, the output of the antigen-binding molecule will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3: 257).

[0334] Host cell can be co-transfected with two or more expression vectors provided herein.Two or more vectors can contain identical selective markers, which enable to express, for example, heavy chain polypeptides and light chain polypeptides of antigen binding molecules equally. Alternatively, a single vector encoding and capable of expressing the different component polypeptides (for example, heavy chain polypeptides and light chain polypeptides of antigen binding molecules) of the present disclosure can be used. The encoding sequence can include cDNA or genomic DNA.

[0335] Once the antigen binding molecules disclosed 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 by chromatography (e.g., ion exchange chromatography, affinity chromatography (particularly affinity chromatography for specific antigens followed by protein A) and fractionated column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. Additionally, the antigen binding molecules disclosed herein can be fused to heterologous amino acid sequences described herein or otherwise known in the art to facilitate purification.

[0336] 7.11 Pharmaceutical Compositions

[0337] In one aspect, the disclosure also provides a pharmaceutical composition comprising at least one antigen binding molecule of the disclosure. In a specific embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the antigen binding molecules provided herein, and a pharmaceutically acceptable excipient. In a specific embodiment, the antigen binding molecules are separated. In a specific embodiment, the antigen binding molecules are purified. Any antigen binding molecules provided herein are envisioned for use in pharmaceutical compositions.

[0338] Pharmaceutical compositions containing antigen binding molecules or fragments thereof are prepared by mixing the protein having the desired degree of purity with optional physiologically acceptable excipients for storage in the form of aqueous solutions or lyophilized or other dry forms (see, e.g., Remington, Remington's Pharmaceutical Sciences (18th ed., 1980)).

[0339] The antigen-binding molecules of the present disclosure can be formulated in any suitable form for delivery to target cells / tissues, for example, as microcapsules or macroemulsions (Remington, supra; Park et al., 2005, Molecules 10:146-61; Malik et al., 2007, Curr. Drug. Deliv., 4:141-51), as sustained-release formulations (Putney and Burke, 1998, Nature Biotechnol. 16:153-57), or in liposomes (Maclean et al., 1997, Int. J. Oncol. 11:325-32; Kontermann, 2006, Curr. Opin. Mol. Ther., 8:39-45).

[0340] The antigen binding molecules provided herein can also be embedded in microcapsules, which are prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or embedded in coarse emulsions. Such techniques are disclosed, for example, in Remington (supra).

[0341] Various compositions and delivery systems are known and can be used with antigen binding molecules as described herein, including but not limited to encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antigen binding molecules / receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262: 4429-32), constructing the nucleic acid as part of a retroviral or other vector, etc. In another embodiment, the composition can be provided as a controlled 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 a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof as described herein) or a composition 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., 1985, Science 228:190-92; During et al., 1989, Ann. Neurol., 25:351-56; Howard et al., 1989, J. Neurosurg. 71:105-12; U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463 and 5,128,326; PCT Publication Nos. 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-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters.In one embodiment, the polymer used in the sustained-release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable.

[0342] In yet another embodiment, a controlled-release or sustained-release system can be placed near a specific target tissue (e.g., the nasal passages 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, Science, 1990, Vol. 249: pp. 1527-1533. Any technique known to those skilled in the art can be used to produce sustained-release formulations comprising one or more antibodies or antigen-binding fragments thereof as described herein (see, e.g., U.S. Pat. No. 4,526,938; PCT Publication Nos. WO 91 / 05548 and WO 96 / 20698; Ning et al., 1996, Radiotherapy & Oncology 39: 179-89; Song et al., 1995, PDA J. of Pharma. Sci. & Tech. 50: 372-97; Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24: 853-54; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24: 759-60).

[0343] 7.12 Usage

[0344] In another aspect, provided herein are methods for using the antigen binding molecules provided herein, and the purposes of the antigen binding molecules provided herein. Such methods and purposes include methods of treatment and therapeutic uses, for example, relating to administering the antigen binding molecules to a subject suffering from a disease or illness, or a composition comprising the antigen binding molecules. In a specific embodiment, the subject needs treatment. In a specific embodiment, the composition is administered in an effective amount to achieve treatment of the disease or illness in the subject. Purposes include the purposes of the composition in such methods and treatments, and in preparing medicines to perform such methods of treatment. In a specific embodiment, these methods are performed by administering the composition to a subject suffering from or suspected of having a disease or illness. In a specific embodiment, these methods thus treat the disease or illness in the subject.

[0345] In specific embodiments, the treatments provided herein result in a complete or partial improvement or alleviation of a disease or condition, or a symptom, adverse effect or outcome, or a phenotype associated therewith. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. These terms include, but are not limited to, completely curing the disease or completely eliminating any symptom or the effect on all symptoms or outcomes.

[0346] As used herein, in specific embodiments, the treatment provided herein delays the development of a disease or illness, for example, postpones, hinders, slows down, postpones, stabilizes, suppresses and / or delays the development of a disease (such as cancer). This delay can be of different lengths of time, depending on the medical history of the disease and / or the individual treated. As will be apparent to those skilled in the art, sufficient or significant delay can actually encompass prevention, because the individual does not develop the disease or obstacle. For example, advanced cancer, such as the development of metastasis, can be delayed. In other embodiments, the method or purposes provided herein prevents a disease or obstacle.

[0347] In yet another aspect, there is provided herein a method for enriching, separating, isolating, purifying, sorting, selecting, capturing, detecting or depleting cells expressing one or more target antigens, comprising: providing a sample comprising cells expressing the one or more target antigens; contacting the sample with an antigen binding molecule; then enriching, isolating, separating, purifying, sorting, selecting, capturing, detecting or removing cells expressing the target antigen and binding to the antigen binding molecule, wherein the antigen binding molecule comprises a first paratope capable of binding to a first target antigen and a second paratope optionally capable of binding to a second target antigen, further optionally a third paratope capable of binding to a third target antigen, further optionally a fourth paratope capable of binding to a fourth target antigen. In a specific embodiment, the sample is a blood sample. In other embodiments, the sample is a tissue sample.

[0348] In another aspect, provided herein is a method for inhibiting or eliminating cancer cells or T cells, comprising contacting the cancer cells or T cells with an effective amount of an antigen binding molecule that binds to at least one tumor-associated antigen or tumor-specific antigen.

[0349] In another aspect, provided herein is a method of inhibiting or eliminating cancer cells or T cells in a subject having cancer, comprising administering to the subject an effective amount of an antigen binding molecule that binds to at least one tumor-associated antigen or tumor-specific antigen.

[0350] In another aspect, provided herein is a method of 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.

[0351] In another aspect, provided herein is a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antigen binding molecule that binds to at least one tumor-associated antigen or tumor-specific antigen.

[0352] In another aspect, provided herein is a method of treating a disease or disorder, wherein one or more therapeutic agents are administered to a subject in combination with an effective amount of an antigen binding molecule that binds to at least one target antigen.

[0353] In another aspect, provided herein is a use of an antigen binding molecule provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0354] 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 condition in a subject.

[0355] In a specific embodiment, a composition comprising an antigen binding molecule provided herein for preventing and / or treating a disease or condition is provided herein. In a specific embodiment, the subject is a subject in need thereof. In a specific embodiment, the subject suffers from the disease or condition. In other embodiments, the subject has the risk of suffering from the disease or condition. In a specific embodiment, administration causes the prevention, management, treatment or improvement of the disease or condition.

[0356] In another embodiment, a method for preventing and / or treating the symptoms of a disease or illness in a subject is provided herein, comprising administering an effective amount of the antigen binding molecules provided herein. In one embodiment, a method for preventing the symptoms of a disease or illness in a subject is provided herein, comprising administering an effective amount of the antigen binding molecules provided herein. In a specific embodiment, the subject is a subject in need thereof. In a specific embodiment, the subject suffers from the disease or illness. In other embodiments, the subject has the risk of suffering from a disease or illness. In a specific embodiment, administration causes the prevention or treatment of the symptoms of the disease or illness.

[0357] In a specific embodiment, the disease is cancer, and the antigen binding molecules are multiparatopes or multispecific antigen binding molecules. In a specific embodiment, the disease is cancer, and the antigen binding molecules are biparatopes and monospecific for a target antigen, and the target antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA) (for example, two paratopes are bound to a single target antigen). In a specific embodiment, the disease is cancer, and the antigen binding molecules are biparatopes and bispecific for two target antigens, and the two target antigens are selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA) or a combination thereof (for example, each paratope is bound to different target antigens). In a specific embodiment, the disease is cancer, and the antigen binding molecules are triparatopes and monospecific for a target antigen, and the target antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA) (for example, each paratope is bound to a single target antigen). In specific embodiments, the disease is cancer and the antigen binding molecule is triparatopic and bispecific for two target antigens selected from tumor associated antigens (TAAs), tumor specific antigens (TSAs), or a combination thereof (e.g., two paratopes bind to a single target antigen and one paratope binds to a different target antigen). In specific embodiments, the disease is cancer and the antigen binding molecule is triparatopic and trispecific for three target antigens selected from tumor associated antigens (TAAs), tumor specific antigens (TSAs), or a combination thereof (e.g., each paratope binds to a different target antigen). In specific embodiments, the disease is cancer and the antigen binding molecule is tetraparatopic and bispecific for two target antigens selected from tumor associated antigens (TAAs), tumor specific antigens (TSAs), or a combination thereof (e.g., two paratopes bind to one target antigen and two paratopes bind to different target antigens). In a specific embodiment, the disease is cancer and the antigen binding molecules are tetraparatopic and tetraspecific for four 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 a specific embodiment, the disease is cancer and the antigen binding molecules are as shown in Table 4 below. In a specific embodiment, the disease is cancer and the antigen binding molecules are as shown in Table 4 below, but wherein each of the target antigens TAA1, TAA2, TAA3, or TAA4 is independently and optionally replaced by a tumor specific antigen (e.g., TSA1, TSA2, TSA3, or TSA4). In a specific embodiment, the paratope or at least the first paratope of the antigen binding molecules as described herein binds to: i) a target antigen associated with cancer, or ii) a target antigen specific for cancer.

[0358] Table 4: Design of multiparatopic and multispecific antibodies

[0359]

[0360]

[0361] In specific embodiments, the tumor-associated antigen (TAA) or tumor-specific antigen (TSA) is present on the surface of a cancer cell.

[0362] In specific 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 carcinoma, head and neck cancer, Hodgkin lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, neuroendocrine tumor, non-Hodgkin lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma, spinal cancer, stomach cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer.

[0363] In specific embodiments, the tumor-associated antigen (TAA) or tumor-specific antigen (TSA) is an angiogenin, 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, fibronectin-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, surviving, BAGE family antigen, CAGE family antigen, GAGE ​​family antigen, MAGE family antigen, SAGE family antigen, XAGE family antigen, 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.

[0364] In specific embodiments, the tumor-associated antigen (TAA) or tumor-specific antigen (TSA) is HER2 or MET.

[0365] Provided herein is a method for preventing and / or treating a disease or illness by administering to a subject an effective amount of the antigen binding molecules provided herein or a pharmaceutical composition comprising the antigen binding molecules provided herein. In one aspect, the antigen binding molecules are substantially purified (i.e., substantially free of the material limiting its effect or producing undesirable side effects). In a specific embodiment, the subject is any animal, preferably a mammal (e.g., any mammal), most preferably the mankind. In a specific embodiment, the mammal is a non-primate or primate. In a specific embodiment, the mammal is selected from the group consisting of cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys and the mankind. In a preferred embodiment, the subject is the mankind. In a specific embodiment, the subject is the mankind suffering from a disease or illness. In a specific embodiment, the subject is the mankind suffering from cancer.

[0366] Various delivery systems are known and can be used to administer preventive or therapeutic agents (e.g., antigen binding molecules as provided herein), including but not limited to encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies or their antigen binding fragments / receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987)), constructing nucleic acids as part of retroviruses or other vectors, etc. Methods of administering preventive or therapeutic agents (e.g., antigen binding molecules as provided herein) or pharmaceutical compositions include but are not limited to parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, and mucosal administration (e.g., intranasal and oral routes). In a specific embodiment, preventive or therapeutic agents (e.g., antigen binding molecules as provided herein) or pharmaceutical compositions are administered intranasally, intramuscularly, intravenously, or subcutaneously. The prophylactic or therapeutic agent or composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucosal layers (e.g., oral mucosa, intranasal mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other bioactive agents. Administration can be systemic or local. In addition, pulmonary administration can also be adopted, for example, by using an inhaler or nebulizer and formulating with an aerosol. See, e.g., U.S. Patents 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 PCT Publications 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.

[0367] In specific embodiments, it may be desirable to locally administer preventive or therapeutic agents or pharmaceutical compositions provided herein to the region in need of treatment. This can be achieved by, for example, but not limited to, local infusion, topical administration (e.g., by intranasal spray), injection, or by means of an implant, wherein the implant has a porous, non-porous, or gel-like material, including a film (such as a silicone rubber membrane) or fiber. In specific embodiments, when administering the antigen binding molecules provided herein, it is necessary to be careful to use the material that the antigen binding molecules will not absorb.

[0368] In another embodiment, the prophylactic or therapeutic agents or compositions provided herein can be delivered in vesicles, particularly liposomes (see Langer, 1990, Science 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.).

[0369] In another embodiment, the prophylactic or therapeutic agent or the 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, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 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 a prophylactic or therapeutic agent (e.g., an antibody provided herein) or a composition provided herein (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press., 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; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol., 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. 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-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In embodiments, the polymer used in the sustained-release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable.In another embodiment, a controlled-release or sustained-release system can be placed near the therapeutic target (i.e., nasal passages or lungs), thus requiring only a portion of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)). Controlled-release systems are discussed in a review by Langer (1990, Science 249: 1527-1533). Any technology known to those skilled in the art can be used to produce a sustained-release formulation comprising one or more antigen binding molecules provided herein. See, e.g., U.S. Pat. No. 4,526,938; PCT Publication No. WO 91 / 05548; PCT Publication No. 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,” Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al., 1997, “Microencapsulation of Recombinant 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.

[0370] In a specific embodiment, where the compositions disclosed herein are polynucleotides encoding prophylactic or therapeutic agents (e.g., antigen-binding molecules disclosed herein), the polynucleotides can be administered in vivo to promote expression of the prophylactic or therapeutic agents encoded thereby: for example, by using retroviral vectors (see U.S. Pat. No. 4,980,286) or by direct injection or by using microparticle bombardment (e.g., a gene gun; Biolistic, Dupont) to construct the polynucleotide as part of an appropriate polynucleotide expression vector and then administer it so that it becomes an intracellular substance, or by coating it with lipids or cell surface receptors or transfection agents, or by administering it in conjunction with a homeobox-like peptide known to enter the cell nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868), etc. Alternatively, the polynucleotides can be introduced into cells by homologous recombination and incorporated into host cell DNA for expression.

[0371] In a specific embodiment, the compositions provided herein include one, two or more antigen binding molecules provided herein. In another embodiment, the compositions provided herein include one, two or more antigen binding molecules provided herein, and are not preventive or therapeutic agents of the antigen binding molecules provided herein. In one embodiment, known preventive or therapeutic agents can be used for or have been used for or are currently used to prevent, manage, treat and / or improve disease or illness. In addition to preventive or therapeutic agents, the compositions provided herein may also include excipients.

[0372] The compositions provided herein include raw material pharmaceutical compositions that can be used for preparing unit dosage forms (e.g., compositions suitable for administering to a subject or patient). In embodiments, the compositions provided herein are pharmaceutical compositions. Such compositions comprise one or more preventive or therapeutic agents (e.g., antigen binding molecules provided herein, or other preventive or therapeutic agents) for prevention or treatment of an effective amount, and pharmaceutically acceptable excipients. Pharmaceutical compositions can be formulated into a route suitable for administering to a subject.

[0373] In a specific embodiment, 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 derived from oil, animal, plant oils or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is an exemplary excipient. Saline solutions and aqueous dextrose solutions and glycerol solutions can also be used as liquid excipients, especially for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier or pH buffer. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations can include standard excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate etc. The example description of suitable pharmaceutical excipients is in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain the antigen binding molecules provided herein of prevention or treatment effective amount, such as its purified form, together with an appropriate amount of excipient, so as to be provided for the form suitably administered to the patient. The preparation should be adapted to the mode of administration.

[0374] In an embodiment, the composition is formulated as a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Typically, the composition for intravenous administration is a sterile isotonic buffered aqueous solution. Where necessary, the composition may also contain a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. However, such compositions may be administered by routes other than intravenous administration.

[0375] Typically, the ingredients of the compositions provided herein are provided separately or mixed together in unit dosage form in hermetically sealed containers such as ampoules or pouches indicating the amount of active agent, for example, as dry lyophilized powders or anhydrous concentrates. Where the composition is administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline can be used to dispense the composition. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0376] The antigen binding molecules provided herein can be packaged in an airtight sealed container (such as an ampoule or a pouch) indicating the amount of the antigen binding molecules. In one embodiment, the antigen binding molecules are provided in an airtight sealed container as a dry, sterilized lyophilized powder or anhydrous concentrate, and can be redissolved to an appropriate concentration, such as with water or saline, to be administered to a subject. The lyophilized antigen binding molecules can be stored between 2° C. and 8° C. in their original containers, and the antibody or its Fab can be administered within 12 hours after redissolution, such as within 6 hours, within 5 hours, within 3 hours, or within 1 hour. In an alternative embodiment, the antigen binding molecules provided herein are provided in a liquid form in an airtight sealed container indicating the amount and concentration of the antigen binding molecules.

[0377] The compositions provided herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed from anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like; and those formed from cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0378] The amount of the preventive or therapeutic agent (for example, antigen binding molecules provided herein) or the compositions provided herein that will effectively prevent and / or treat a disease or condition can be determined by standard clinical techniques. In addition, in vitro assays can optionally be used to help determine the optimal dose range. The precise dosage to be adopted in the preparation will also depend on the severity of the route of administration and the disease or condition, and should be determined according to the doctor's judgment and the situation of each patient.

[0379] Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0380] In specific embodiments, the approach of the antigen binding molecules provided herein used for a certain dose is intranasal, intramuscular, intravenous, subcutaneous or a combination of these approaches, but other approaches as described herein are also acceptable. Each dosage may or may not be used by the same route of administration. In specific embodiments, the antigen binding molecules provided herein can be administered to other dosages of the same or different antigen binding molecules provided herein simultaneously or subsequently via a variety of routes of administration.

[0381] In specific 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, alleviate or ameliorate a disease or its symptoms.

[0382] 7.13 Gene Therapy

[0383] In a specific embodiment, the polynucleotide comprising the sequence encoding antigen binding molecules or its functional derivatives is applied to the subject for use in the method provided herein, for example, to prevent, manage, treat and / or improve disease, disease or condition by gene therapy. This therapy encompasses the therapy performed by administering expressed or expressible polynucleotides to the subject. In one embodiment, the polynucleotide produces the antigen binding molecules of its encoding, and the antigen binding molecules mediate prevention or therapeutic effect. Any method available in the art for recombinant gene expression (or gene therapy) can be used.

[0384] For a general review of gene therapy approaches, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol., Vol. 32: pp. 573-596; Mulligan, 1993, Science, Vol. 260: pp. 926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem., Vol. 62: pp. 191-217; May 1993, TIBTECH, Vol. 11, No. 5: pp. 155-215. Useful methods generally known in the art of recombinant DNA technology 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).

[0385] In a specific embodiment, compositions comprises the polynucleotide of the antigen binding molecules that coding this paper provides, and these polynucleotides are the part of the expression vector expressing this antigen binding molecules or its heavy chain or light chain polypeptide in suitable host.Particularly, this type of polynucleotide has the promoter that is operably connected to antigen binding molecules coding region, such as heterologous promoter, and this promoter is inducible or constitutive, and is optionally tissue-specific.In another specific embodiment, in the polynucleotide molecule used, the flank of antigen binding molecules coding sequence and any other desired sequence is the region that promotes homologous recombination at desired site in genome, thus provides the chromosome of antibody encoding polynucleotide and expresses (Koller and Smithies, 1989, Proc.Natl.Acad.Sci.USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

[0386] Delivery of a polynucleotide (e.g., nucleic acid) into a subject can be direct, in which case the subject is directly exposed to the polynucleotide or a vector carrying the polynucleotide, or indirect, in which case cells are first transformed with the polynucleotide in vitro and then transplanted into the subject. These two approaches are known as in vivo gene therapy or ex vivo gene therapy, respectively.

[0387] In a specific embodiment, the polynucleotide sequences are administered directly in vivo, where they are expressed to produce the encoded product. This can be accomplished by any of a variety 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 with a defective or attenuated retroviral or other viral vector (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by using microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell surface receptors or transfection agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in conjunction with peptides known to enter the cell nucleus, by administering them in conjunction with ligands that undergo receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. Vol. 262: pp. 4429-4432) (which can be used to target cell types that specifically express the receptor), etc. In another embodiment, nucleic acid-ligand complex can be formed, wherein the part comprises fusion virus peptide to destroy endosome, thereby allowing nucleic acid to avoid lysosomal degradation. In another embodiment, by targeting specific receptors, nucleic acid can be targeted cell-specific uptake and expression in vivo (see, for example, PCT Publication WO 92 / 06180; WO 92 / 22635; WO 92 / 20316; WO 93 / 14188, WO 93 / 20221). Alternatively, nucleic acid can be introduced into cells by homologous recombination and incorporated 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).

[0388] In a specific embodiment, a viral vector containing a polynucleotide sequence encoding an antigen binding molecule is used. For example, a retroviral vector (see Miller et al., 1993, Meth.Enzymol.217:581-599) can be used. These retroviral vectors contain the correct packaging of viral genomes and the necessary components for integration into host cell DNA. The polynucleotide sequence encoding the antigen binding molecules for gene therapy can be cloned into one or more vectors, which is conducive to gene delivery into the subject. More 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 so that stem cells are more resistant to chemotherapy. Other references describing the use of retroviral vectors in gene therapy are: 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.

[0389] Adenovirus is another viral vector that can be used for recombinant production of antigen-binding molecules. Adenovirus is a particularly attractive vector for delivering genes to respiratory epithelial cells. Adenovirus naturally infects respiratory epithelial cells and causes mild disease at the epithelial cells. Other targets of adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenovirus has the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development, Vol. 3: pp. 499-503 provide a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5: 3-10 demonstrated that genes can be transferred to the respiratory epithelial cells of rhesus monkeys using adenovirus vectors. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; Mastrangeli et al., 1993, J. Clin. Invest., 91:225-234; PCT Publication WO 94 / 12649; and Wang et al., 1995, Gene Therapy 2:775-783. In a specific embodiment, an adenoviral vector is used.

[0390] Adeno-associated virus (AAV) (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med., Vol. 204: pp. 289-300; and U.S. Pat. No. 5,436,146) can also be used. In a specific embodiment, AAV vectors are used to express antigen binding molecules as provided herein. In a specific embodiment, AAV comprises polynucleotides encoding one or more polypeptides of antigen binding molecules. In a specific embodiment, AAV comprises polynucleotides encoding one or more light chain polypeptides of antigen binding molecules. In a specific embodiment, AAV comprises polynucleotides encoding one or more heavy chain polypeptides of antigen binding molecules.

[0391] Another approach to gene therapy involves transferring genes into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Typically, the transfer method involves transferring a selectable marker to the cells. The cells are then placed under selection to isolate those that have taken up and expressed the transferred gene. These cells are then delivered to the subject.

[0392] In a specific embodiment, the polynucleotide (e.g., nucleic acid) is introduced into the cell before the resulting recombinant cell is administered in vivo. This introduction can be performed by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral vector or phage vector containing the polynucleotide sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. A variety of techniques for introducing exogenous genes into cells are known in the art (see, for example, Loeffler and Behr, 1993, Meth. Enzymol. 217: 599-618; Cohen et al., 1993, Meth. Enzymol. Vol. 217: 618-644; Clin. Pharma. Ther., Vol. 29: 69-92, 1985) and can be used according to the methods provided herein, provided that the necessary development and physiological functions of the recipient cell are not destroyed. The technology should provide stable transfer of nucleic acids to cells so that the polynucleotides can be expressed by the cell, such as being heritable and expressible by its cell progeny.

[0393] The resulting recombinant cells can be delivered to a subject by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem cells or progenitor cells) can be administered intravenously. The amount of cells intended to be used depends on the desired effect, patient status, etc., and can be determined by those skilled in the art.

[0394] For the purpose of gene therapy, cells into which polynucleotides can be introduced encompass 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; various stem cells or progenitor cells, in particular hematopoietic stem cells or progenitor cells, such as hematopoietic stem cells or progenitor cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

[0395] In a specific embodiment, the cells used for gene therapy are autologous to the subject.

[0396] In one embodiment of gene therapy using recombinant cells, polynucleotide sequences encoding antigen binding molecules are introduced into cells so that they can be expressed by cells or the offspring of cells, and then the recombinant cells are administered in vivo to obtain therapeutic effects. In a specific embodiment, stem cells or progenitor cells are used. According to the embodiment of the method provided herein, any stem cells and / or progenitor cells that can be separated and maintained in vitro can potentially be used (see, for example, PCT Publication WO94 / 08598; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio., 21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).

[0397] In a specific embodiment, a polynucleotide introduced for gene therapy purposes 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 an appropriate transcription inducer.

[0398] 7.14 Diagnostic Assays and Methods

[0399] 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 condition.

[0400] The antigen binding molecules provided herein can be used to measure the antigen level in biological samples using classical immunohistological methods as described herein or known to those skilled in the art (e.g., see Jalkanen et al., 1985, J. Cell. Biol. 101: 976-985; and Jalkanen et al., 1987, J. Cell. Biol. 105: 3087-3096). Other methods based on antigen binding molecules that can be used to detect protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA). Suitable antibody assay markers are known in the art and include enzyme markers, such as glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In) and technetium (99Tc); luminescent markers, such as luminol; and fluorescent markers, such as fluorescein and rhodamine, and biotin. One aspect provided herein is the detection and diagnosis of a disease or disorder in a person.

[0401] In the art, it should be understood that the size of the experimenter and the imaging system used will determine the amount of the imaging part required for the diagnostic image. In the case of a radioisotope moiety, for human subjects, the amount of the radioactivity injected is generally in the range of about 5 millicuries to 20 millicuries of 99Tc. Then, the antigen binding molecules of the labeling will accumulate at the position of the cell containing the specific protein. In vivo tumor imaging is described in SW Burchiel et al., " Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments ", (Tumor Imaging: The Radiochemical Detection of Cancer Chapter 13, SW Burchiel and BARhodes edited, Masson Publishing Inc. (1982).

[0402] 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 in the subject's body and to allow unbound labeled antibody to be cleared to background levels is 6 hours to 48 hours, or 6 hours to 24 hours, or 6 hours to 12 hours. In another embodiment, the time interval after administration is 5 days to 20 days, or 5 days to 10 days.

[0403] In one embodiment, monitoring of the disease or condition is performed by repeating the method used to diagnose the disease or condition, for example, one month after the initial diagnosis, six months after the initial diagnosis, one year after the initial diagnosis, etc.

[0404] 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 marker used. Technicians will be able to determine the appropriate method for detecting a specific marker. The methods and devices that can be used in the diagnostic methods provided herein include, but are not limited to, computed tomography (CT), whole-body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasonography.

[0405] In a specific embodiment, the antigen binding molecules are labeled with radioisotopes and detected in patients using radiation response surgical instruments (Thurston et al., U.S. Patent number 5,441,050). In another embodiment, the antigen binding molecules are labeled with fluorescent compounds and detected in patients using fluorescence response scanning instruments. In another embodiment, the antigen binding molecules are labeled with positron emitting metals and detected in patients using positron emission tomography. In another embodiment, the antigen binding molecules are labeled with paramagnetic labels and detected in patients using magnetic resonance imaging (MRI).

[0406] 7.15 kit

[0407] Also provided herein are kits comprising the antigen binding molecules provided herein packaged into suitable packaging materials, or compositions (e.g., pharmaceutical compositions) of the antigen binding molecules. Kits optionally include labels or package inserts that include descriptions of components or instructions for use in vitro, in vivo, or in vitro of the components.

[0408] The term "packaging material" refers to the physical structure that houses the components of the kit. The packaging material can maintain the components sterilely and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0409] Kits provided herein may include labels or instructions for use. Labels or instructions for use include "printed material," such as paper or cardboard, either alone or attached to a component, kit, or packaging material (e.g., box), or attached to, for example, an ampoule, tube, or bottle containing kit components. Labels or instructions for use may additionally include computer-readable media, such as disks (e.g., hard disks, cards, storage disks), optical disks (e.g., CDs or DVD-ROM / RAMs, DVDs, MP3s, magnetic tapes), or electrical storage media (e.g., RAM and ROM) or a mixture of these (e.g., magnetic / optical storage media, FLASH media, or memory cards). Labels or instructions for use may include information identifying manufacturer information, batch number, manufacturer location, and date.

[0410] The test kit provided herein may additionally include other components. Each component of the test kit may be encapsulated in a separate container, and all the various containers may be in a single package. The test kit may also be designed for refrigeration. The test kit may also be designed to accommodate the antibody provided herein, or cells containing the polynucleotides encoding the antibodies provided herein. The cells in the test kit may be maintained under suitable storage conditions until ready for use.

[0411] Also provided herein are sets of antigen binding molecules that immunospecifically bind to a target antigen. In specific embodiments, provided herein are sets of antigen binding molecules having different association rate constants, different dissociation rate constants, different affinities for the antigen, and / or different specificities for the target antigen. In specific embodiments, provided herein are sets containing 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, about 1000 or more antibodies. Panels of antigen binding molecules can be used in, for example, 96-well or 384-well plates, such as for use in assays such as ELISA.

[0412] As used herein, numerical values ​​are generally given in the form of ranges throughout this document. The use of ranges is merely for convenience and brevity, and should not be construed as an absolute limitation to the scope of the present invention, unless the context clearly indicates otherwise. Therefore, the scope used explicitly includes all possible subranges, all single numerical values ​​within the range, and all numerical values ​​or numerical ranges (including fractions or integers of the numerical values ​​within such ranges and within the range) unless the context clearly indicates otherwise. Regardless of the width of the scope, in all contexts throughout this patent document, this construction is applicable. Therefore, for example, mentioning 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 of 90% to 100% also include 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., as well as 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc.

[0413] For the sake of brevity, certain abbreviations are used herein. An example is the single-letter abbreviations representing amino acid residues. The amino acids and their corresponding three-letter and single-letter abbreviations are shown in Table 5 below:

[0414] Table 5: Amino acid abbreviations

[0415]

[0416] As used herein, the term "percent identity" in the context of two or more polypeptides or nucleotide sequences (e.g., antigen binding molecules as described herein or fragments thereof, and polynucleotides encoding them) refers to two or more sequences or subsequences of identical amino acid residues or nucleotides with a specified percentage. Percent identity can be determined by comparing two related sequences using a sequence comparison algorithm known in the art to obtain maximum correspondence or by visual inspection.

[0417] Optimal alignment of sequences for comparison can be performed by a sequence comparison algorithm, which may be the local homology algorithm, Smith and Waterman, Adv. Appl. Math. 2:482 (1981); the homology alignment algorithm, Needleman and Wunsch, J. Mol. Biol. 48:443 (1970)); the search for similarity method, Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0418] Comparison algorithms that are also suitable for determining percentages of sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are 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 analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased.

[0419] 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 mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hit in each direction stops when: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative-scoring residue alignments; or 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 a word length (W) of 11, an expectation (E) of 25, M=5, N=-4, and a comparison of both chains by default. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0420] 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 and 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 or amino acid sequences would 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 a test polynucleotide to a reference polynucleotide is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0421] Affirmative language is used herein to describe various embodiments and generally disclose the present invention. The present invention also specifically includes embodiments in which specific subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays, or analyses, is excluded in whole or in part. Thus, even though the present invention is not generally expressed in terms of what is excluded from the present invention, aspects that are not explicitly included in the present invention are disclosed herein.

[0422] A number of embodiments of the present invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, the following examples are intended to illustrate, but not to limit, the scope of the invention described in the claims.

[0423] 8. Implementation Plan

[0424] The present invention provides the following non-limiting embodiments. As contemplated herein, any of embodiments A1 to A39, B1 to B33, C1 to C39, D1 to D31, and E1 to E16 may be combined and / or applied to the claims set forth herein.

[0425] In one set of embodiments (Group A embodiments), there is provided:

[0426] A1. An antigen-binding molecule comprising

[0427] a first light chain polypeptide comprising, in amino-terminal to carboxy-terminal order: VL1-LD1, wherein VL1 is a first light chain variable domain, and LD1 is a first light chain dimerization domain; and

[0428] a first heavy chain polypeptide comprising, in amino-terminal to carboxyl-terminal order: VH1-HD1, wherein VH1 is a first heavy chain variable domain, and HD1 is a first heavy chain dimerization domain,

[0429] in

[0430] a)i) LD1 includes the beta-2 microglobulin (B2M) domain and HD1 includes

[0431] HLA-E A3 (EA3) domain, or LD1 includes an EA3 domain and HD1 includes a B2M domain, wherein the B2M domain and the EA3 domain associate with each other to form a dimer; or

[0432] ii) LD1 comprises a first ICAM-1D1 domain and HD1 comprises a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer,

[0433] 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

[0434] ii) the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain, and

[0435] c) VL1 and VH1 form the first paratope.

[0436] A2. The antigen binding molecule according to embodiment A1,

[0437] wherein LD1 includes the B2M domain and HD1 includes the EA3 domain, or LD1 includes the EA3 domain and HD1 includes the B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer, and

[0438] The B2M domain comprises

[0439] a) Amino acid sequence

[0440] RTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKN

[0441] GERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID NO:2);

[0442] b) an amino acid sequence that has at least 91% sequence identity to SEQ ID NO: 2;

[0443] c) at amino acid positions 4, 8, 10, 54, 58, 60, 96 of SEQ ID NO: 2

[0444] an amino acid sequence having one, two, three, four, five, six, seven or eight single amino acid substitutions to the sequence of SEQ ID NO: 2 at one or more of SEQ ID NO: 97;

[0445] d) at amino acid positions 4, 8, 10, 54, 58, 60, 96 of SEQ ID NO: 2

[0446] an amino acid sequence having one, two, three, four, five, six, seven or eight single amino acid substitutions to the sequence of SEQ ID NO: 2 at one or more of 97, wherein each of the single amino acid substitutions is independently selected from the group consisting of F, W, C, S and T;

[0447] e) the amino acid sequence of the human B2M sequence shown in SEQ ID NO: 2, but having a set of substitutions of F56S, W60S and F62T, wherein positions are numbered according to the B2M amino acid numbering in Table 1, and optionally further having one, two, three, four or five single amino acid substitutions at positions K6, Y10, R12, D98 or M99, wherein positions are numbered according to the B2M amino acid numbering in Table 1;

[0448] f) an amino acid sequence having the human B2M sequence shown in SEQ ID NO: 2 but having a set of substitutions selected from the group consisting of:

[0449] i) F56S, W60S, F62T and K6C;

[0450] ii) F56S, W60S, F62T, and any one of Y10C, Y10F, and Y10W;

[0451] iii) F56S, W60S, F62T and R12C;

[0452] iv) F56S, W60S, F62T, and any one of D98C, D98F, and D98W;

[0453] v) F56S, W60S, F62T, and any of M99C, M99F, and M99W; or

[0454] The amino acid sequence of any one of SEQ ID NOs: 4-30.

[0455] A3. The antigen binding molecule according to embodiment A1 or A2, wherein LD1 comprises the B2M domain and HD1 comprises the EA3 domain, or LD1 comprises the EA3 domain and HD1 comprises the B2M domain, wherein the B2M

[0456] domain and the EA3 domain bind to each other to form a dimer, and

[0457] wherein the EA3 domain comprises

[0458] a) Amino acid sequence

[0459] LHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQ

[0460] QDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW(SEQ ID NO:33);

[0461] b) an amino acid sequence that has at least 93% sequence identity to SEQ ID NO: 33;

[0462] c) at amino acid positions 13, 23, 53, 55, 59 and 63 of SEQ ID NO: 33

[0463] an amino acid sequence having one, two, three, four, five or six single amino acid substitutions to the sequence of SEQ ID NO: 33 at one or more of the residues;

[0464] d) at amino acid positions 13, 23, 53, 55, 59 and 63 of SEQ ID NO: 33

[0465] an amino acid sequence having one, two, three, four, five or six single amino acid substitutions to the sequence of SEQ ID NO: 33 at one or more of the residues, wherein each of the single amino acid substitutions is independently selected from the group consisting of A, C, L; or

[0466] e) an amino acid sequence having the human EA3 sequence shown in SEQ ID NO: 33 but having one, two, three, four, five or six single amino acid substitutions at positions H192, R202, E232, R234, D238 or Q242, wherein positions are numbered according to the EA3 amino acid numbering in Table 2;

[0467] f) an amino acid sequence having the human EA3 sequence shown in SEQ ID NO: 33 but having a substitution or a group of substitutions selected from the following, wherein positions are numbered according to the EA3 amino acid numbering in Table 2:

[0468] i)H192C;

[0469] ii) R202A or R202C;

[0470] iii) E232C;

[0471] iv) R234A, R234L or R234C;

[0472] v)D238C;

[0473] vi) Q242A or Q242L;

[0474] vii) R234A and Q242A;

[0475] viii) R234A and Q242L;

[0476] ix) R234A and Q242A; or

[0477] x) R234L and Q242L; or

[0478] g) is the amino acid sequence of any one of SEQ ID NOs: 35-46.

[0479] A4. The antigen binding molecule according to embodiment A1, wherein LD1 comprises the first ICAM-1D1 domain and HD1 comprises the second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain associate with each other to form a dimer,

[0480] wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain each comprise a different amino acid sequence selected from the group consisting of:

[0481] a) Amino acid sequence

[0482] QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKE

[0483] LLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVY(SEQ ID NO:49);

[0484] b) an amino acid sequence that has at least 81% sequence identity to SEQ ID NO: 49;

[0485] c) at amino acid positions 2, 10, 13, 18, 20, 23,

[0486] 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 of SEQ ID NO: 49 at one or more of 34, 38, 42, 49, 51, 53, 63, 67 and 78, optionally further adding a cysteine ​​amino acid at the C-terminus;

[0487] 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 to the sequence of SEQ ID NO: 49 at one or more of amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67 and 78 of SEQ ID NO: 49, optionally further adding a cysteine ​​amino acid at the C-terminus, wherein each of the single amino acid substitutions is independently selected from the group consisting of V, T, F, W, A, K, E, C and R;

[0488] e) the amino acid sequence of human ICAM-1 D1 as shown in SEQ ID NO: 49, but having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirty-three, fourteen, or fifteen single amino acid substitutions at positions T2, I10, R13, L18, T20, T23, E34, P38, L42, R49, V51, E53, P63, S67, or T78, wherein positions are numbered according to the ICAM-1 D1 amino acid numbering in Table 3, optionally further comprising the addition of a cysteine ​​amino acid at the C-terminus (84C), wherein positions are numbered according to the ICAM-1 D1 amino acid numbering in Table 3;

[0489] f) an amino acid sequence having the human ICAM-1D1 sequence shown in SEQ ID NO: 49 but having a set of substitutions selected from the group consisting of:

[0490] i)E34K;

[0491] ii) T2V, I10T, T23A, E34K, P38T, P63V, S67A and

[0492] T78A;

[0493] iii) T2V, I10T, R13C, T23A, E34K, P38T, R49E, P63V, S67A, T78A, and 84C;

[0494] iv) T2V, I10T, R13C, T23A, E34K, P38T, E53R, P63V, S67A, T78A, and 84C;

[0495] v) T2V, I10T, R13C, L18F, T23A, E34K, P38T, P63V, S67A, T78A, and 84C;

[0496] vi)T2V, I10T, R13C, L18A, T20A, T23A, E34K,

[0497] P38T, L42A, V51A, P63V, S67A, T78A, and 84C; or

[0498] vii)T2V, I10T, R13C, L18W, T23A, E34K, P38T,

[0499] P63V, S67A, T78A, and 84C; or

[0500] g) is the amino acid sequence of any one of SEQ ID NOs: 51-58.

[0501] A5. The antigen binding molecule according to any one of embodiments A1 to A4, comprising the first light chain elbow region between VL1 and LD1.

[0502] A6. The antigen binding molecule according to any one of embodiments A1 to A5, comprising a first heavy chain elbow region between VH1 and HD1.

[0503] A7. An antigen binding molecule according to any one of embodiments A1 to A6, comprising

[0504] A first light chain polypeptide comprising, in amino-terminal to carboxyl-terminal order: VL1-LE1-LD1, wherein LE1 is the first light chain elbow region; and

[0505] A first heavy chain polypeptide, comprising, in amino-terminal to carboxyl-terminal order: VH1-HE1-HD1, wherein HE1 is the first heavy chain elbow region,

[0506] LE1 connects the carboxyl terminus of VL1 to the amino terminus of LD1, while HE1 connects the carboxyl terminus of VH1 to the amino terminus of HD1.

[0507] A8. The antigen binding molecule according to embodiment A7, wherein LE1 or HE1 is an amino acid sequence of 3 to 25 amino acids in length.

[0508] A9. The antigen binding molecule according to embodiment A8, wherein LE1 or HE1 is an amino acid sequence selected from the group consisting of: RTV; GGS; RTVGGS (SEQ ID NO: 59); RTVGGSRTV (SEQ ID NO: 60); AST; ASTK

[0509] (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

[0510] (SEQ ID NO:67); ASTKGGGGSGG (SEQ ID NO:68);

[0511] ASTKGGGGSGGS (SEQ ID NO: 69); ASTKGGGGSGGGS

[0512] (SEQ ID NO:70); ASTKGGGGSGGGGS (SEQ ID NO:71);

[0513] RTVA(SEQ ID NO:72);RTVAG(SEQ ID NO:73);RTVAGG

[0514] (SEQ ID NO:74);RTVAGGS (SEQ ID NO:75);RTVAGGGS

[0515] (SEQ ID NO:76); RTVAGGGGS (SEQ ID NO:77);

[0516] RTVAGGGGSG(SEQ ID NO:78); RTVAGGGGSGG(SEQ ID

[0517] NO:79); RTVAGGGGSGGS (SEQ ID NO:80);

[0518] RTVAGGGGGSGGGS (SEQ ID NO:81); RTVAGGGGSGGGGS

[0519] (SEQ ID NO:82); GGGGSGGGGS (SEQ ID NO:83);

[0520] GGGGSGGGGSGGGGS (SEQ ID NO:84);

[0521] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:85); and

[0522] GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 86). A10. The antigen binding molecule according to any one of embodiments A7 to A9, wherein

[0523] 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); RTVAGGGS

[0524] (SEQ ID NO:76); RTVAGGGGS (SEQ ID NO:77);

[0525] RTVAGGGGSG (SEQ ID NO:78); RTVAGGGGSGG

[0526] (SEQ ID NO:79); RTVAGGGGSGGS (SEQ ID NO:80); RTVAGGGGSGGGS (SEQ ID NO:81); and

[0527] RTVAGGGGSGGGGS (SEQ ID NO:82);

[0528] 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);

[0529] ASTKG (SEQ ID NO:62); ASTKGG (SEQ ID NO:63);

[0530] ASTKGGS (SEQ ID NO: 64); ASTKGGGS (SEQ ID NO: 65); ASTKGGGGS (SEQ ID NO: 66);

[0531] ASTKGGGGSG (SEQ ID NO: 67); ASTKGGGGSGG

[0532] (SEQ ID NO:68); ASTKGGGGSGGS (SEQ ID NO:69)

[0533] 1; ASTKGGGGSGGGS (SEQ ID NO: 70); or

[0534] ASTKGGGGSGGGGS(SEQ ID NO:71); or

[0535] c) LD1 or HD1 is the first or second ICAM-1 D1 domain, and LE1 or HE1 is an amino acid sequence selected from the group consisting of:

[0536] GGGGSGGGGS(SEQ ID NO:83);GGGGSGGGGSGGGGS

[0537] (SEQ ID NO:84); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:85); and GGGGSGGGGSGGGGSGGGGSGGGGS

[0538] (SEQ ID NO:86).

[0539] A11. The antigen binding molecule according to any one of embodiments A1 to A10, comprising the first light chain spacer fused to the C-terminus of LD1.

[0540] A12. The antigen binding molecule according to any one of embodiments A1 to A11, comprising the first heavy chain spacer fused to the C-terminus of HD1.

[0541] A13. The antigen binding molecule according to any one of embodiments A1 to A12, comprising

[0542] a first light chain polypeptide comprising, in amino-terminal to carboxyl-terminal order: VL1-LD1-LS1, wherein LS1 is a first light chain spacer, and

[0543] The first heavy chain polypeptide comprises, in order from amino terminus to carboxyl terminus, VH1-HD1-HS1, wherein HS1 is a first heavy chain spacer region.

[0544] A14. The antigen binding molecule of embodiment A13, wherein HS1 or LS1 is an amino acid sequence of 2 to 9 amino acids in length.

[0545] A15. The antigen binding molecule of embodiment A13 or A14, wherein 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).

[0546] A16. The antigen binding molecule of any one of embodiments A12 to A15, wherein the heavy chain spacer further comprises a hinge region.

[0547] A17. An antigen binding molecule according to any one of embodiments A1 to A16, wherein the first light chain polypeptide or the first heavy chain polypeptide further comprises a C-terminal tag, optionally wherein the C-terminal tag is a 6x His tag (SEQ ID NO: 93), a Strep-tag II tag, or a human influenza hemagglutinin tag.

[0548] A18. The antigen binding molecule of any one of embodiments A1 to A17, wherein the first heavy chain polypeptide further comprises a heavy chain constant domain 2 (CH2).

[0549] A19. The antigen binding molecule of any one of embodiments A1 to A18, wherein the first heavy chain polypeptide further comprises a heavy chain constant domain 3 (CH3).

[0550] A20. The antigen binding molecule according to any one of embodiments A1 to A19, which does not comprise a dimerization sequence of the heavy chain constant domain 1 (CH1) or the light chain constant domain (CL).

[0551] A21. The antigen binding molecule of any one of embodiments A1 to A20, which is an immunoglobulin, Fab, Fab', F(ab')2, an antibody, a biparatopic antibody, a bispecific antibody, a triparatopic antibody, a trispecific antibody, a tetraparatopic antibody, a tetraspecific antibody, a multiparatopic antibody, a multispecific antibody, or any fragment thereof that binds to a target antigen.

[0552] A22. The antigen binding molecule of any one of embodiments A1 to A21, further comprising a second light chain polypeptide having a second light chain variable domain (VL2) and a second heavy chain polypeptide having a second heavy chain variable domain (VH2), wherein VL2 and VH2 form a second paratope.

[0553] A23. The antigen binding molecule of embodiment A22, wherein the first paratope and the second paratope bind to different antigens.

[0554] A24. The antigen binding molecule of embodiment A22 or A23, further comprising a third light chain polypeptide having a third...

Claims

1. An antigen-binding molecule comprising a first light chain polypeptide comprising, in amino-terminal to carboxyl-terminal order: 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, in amino-terminal to carboxyl-terminal order: VH1-HD1, wherein VH1 is a first heavy chain variable domain, and HD1 is a first heavy chain dimerization domain, in a) i) LD1 includes a beta-2 microglobulin (B2M) domain and HD1 includes an HLA-EA3 (EA3) domain, or LD1 includes an EA3 domain and HD1 includes a B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer; or ii) LD1 includes a first ICAM-1D1 domain and HD1 includes a second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 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, and ii) the first ICAM-1 D1 domain is different from the second ICAM-1 D1 domain; and c) VL1 and VH1 form the first paratope.

2. The antigen-binding molecule according to claim 1, wherein LD1 includes the B2M domain and HD1 includes the EA3 domain, or LD1 includes the EA3 domain and HD1 includes the B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer, and The B2M domain comprises a) Amino acid sequence 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 one, two, three, four, five, six, seven or eight single amino acid substitutions to the sequence SEQ ID NO: 2 at one or more of amino acid positions 4, 8, 10, 54, 58, 60, 96 and 97 of SEQ ID NO: 2; d) an amino acid sequence having one, two, three, four, five, six, seven or eight single amino acid substitutions to the sequence SEQ ID NO: 2 at one or more of 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; e) is the amino acid sequence of the human B2M sequence shown in SEQ ID NO: 2, but the amino acid sequence has a set of substitutions of F56S, W60S and F62T, wherein the positions are numbered according to the B2M amino acid numbering in the following table, and optionally further has one, two, three, four or five single amino acid substitutions at positions K6, Y10, R12, D98 or M99, wherein the positions are numbered according to the B2M amino acid numbering in the following table; f) is the amino acid sequence of the human B2M sequence shown in SEQ ID NO: 2 but having a set of substitutions selected from the following, wherein the positions are numbered according to the B2M amino acid numbering in the following table: 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 of M99C, M99F, and M99W; or g) is the amino acid sequence of any one of SEQ ID NOs: 4-30.

3. The antigen binding molecule according to claim 1 or 2, wherein LD1 includes the B2M domain and HD1 includes the EA3 domain, or LD1 includes the EA3 domain and HD1 includes the B2M domain, wherein the B2M domain and the EA3 domain bind to each other to form a dimer, and The EA3 domain comprises a) Amino acid sequence LHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEG HTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRW(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 one, two, three, four, five or six single amino acid substitutions to the sequence SEQ ID NO: 33 at one or more of amino acid positions 13, 23, 53, 55, 59 and 63 of SEQ ID NO: 33; d) an amino acid sequence having one, two, three, four, five or six single amino acid substitutions to the sequence 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) an amino acid sequence having the human EA3 sequence shown in SEQ ID NO: 33 but having one, two, three, four, five or six single amino acid substitutions at positions H192, R202, E232, R234, D238 or Q242, wherein the positions are numbered according to the EA3 amino acid numbering in the table below; f) is the amino acid sequence of human EA3 shown in SEQ ID NO: 33 but having a substitution or a group of substitutions selected from the following, wherein the positions are numbered according to the EA3 amino acid numbering in the following table: 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 g) is the amino acid sequence of any one of SEQ ID NOs: 35-46.

4. The antigen binding molecule according to claim 1, wherein LD1 includes the first ICAM-1D1 domain and HD1 includes the second ICAM-1D1 domain, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain bind to each other to form a dimer, wherein the first ICAM-1D1 domain and the second ICAM-1D1 domain each comprise a different amino acid sequence selected from the group consisting of: a) Amino acid sequence QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGN NRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVY(SEQ ID NO:49); b) an amino acid sequence that has 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 to the sequence SEQ ID NO: 49 at one or more of amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67 and 78 of SEQ ID NO: 49, optionally also with the addition of a cysteine ​​amino acid at the C-terminus; 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 to the sequence SEQ ID NO: 49 at one or more of amino acid positions 2, 10, 13, 18, 20, 23, 34, 38, 42, 49, 51, 53, 63, 67 and 78 of SEQ ID NO: 49, optionally also adding a cysteine ​​amino acid at the C-terminus, wherein each of the single amino acid substitutions is independently selected from the group consisting of V, T, F, W, A, K, E, C and R; e) an amino acid sequence of the human ICAM-1 D1 sequence as shown in SEQ ID NO: 49, but having 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 ICAM-1 D1 amino acid numbering in the following table, optionally further adding a cysteine ​​amino acid (84C) at the C-terminus, wherein the positions are numbered according to the ICAM-1 D1 amino acid numbering in the following table; f) is the amino acid sequence of the human ICAM-1D1 sequence shown in SEQ ID NO: 49 but having a set of substitutions selected from the following, wherein the positions are numbered according to the ICAM-1D1 amino acid numbering in the following table: 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 g) is the amino acid sequence of any one of SEQ ID NOs: 51-58. 5 . The antigen binding molecule according to any one of claims 1 to 4 , comprising a first light chain elbow region between VL1 and LD1. The antigen binding molecule according to any one of claims 1 to 5, comprising a first heavy chain elbow region between VH1 and HD1.

7. The antigen-binding molecule according to any one of claims 1 to 6, comprising A first light chain polypeptide, comprising, in order from amino terminus to carboxyl terminus: VL1-LE1-LD1, wherein LE1 is the first light chain elbow region; and A first light chain polypeptide, the first light chain polypeptide comprises, in order from amino terminus to carboxyl terminus: VH1-HE1-HD1, wherein HE1 is the first heavy chain elbow region, LE1 connects the carboxyl terminus of VL1 to the amino terminus of LD1, while HE1 connects the carboxyl terminus of VH1 to the amino terminus of HD1. The antigen-binding molecule according to claim 7 , wherein LE1 or HE1 is an amino acid sequence of 3 to 25 amino acids in length.

9. The antigen binding molecule according to claim 8, wherein LE1 or HE1 is 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)1; 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); RTVAGGS (SEQ ID NO: 75); RTVAGGGS (SEQ ID NO: 76); RTVAGGGGS (SEQ ID NO: 77); RTVAGGGGSG (SEQ ID NO: 78); RTVAGGGGGSGG (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 GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:86).

10. The antigen-binding molecule according to any one of claims 7 to 9, wherein 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); 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); 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); or ASTKGGGGSGGGGS (SEQ ID NO:71); or c) LD1 or HD1 is the first or 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); GGGGSGGGGSGGGGS (SEQ ID NO: 84); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:85); and GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:86). The antigen binding molecule according to any one of claims 1 to 10, comprising a first light chain spacer fused to the C-terminus of LD1. 12 . The antigen-binding molecule according to claim 1 , comprising a first heavy chain spacer fused to the C-terminus of HD1.

13. The antigen-binding molecule according to any one of claims 1 to 12, comprising a first light chain polypeptide, comprising, in amino-terminal to carboxyl-terminal order: VL1-LD1-LS1, wherein LS1 is a first light chain spacer, and The first heavy chain polypeptide comprises, from amino terminal to carboxyl terminal, VH1-HD1-HS1, wherein HS1 is a first heavy chain spacer region. The antigen binding molecule according to claim 13 , wherein HS1 or LS1 is an amino acid sequence of 2 to 9 amino acids in length.

15. The antigen binding molecule according to claim 13 or 14, wherein 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).

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

17. An antigen binding molecule according to 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, optionally wherein the C-terminal tag is a 6x His tag (SEQ ID NO: 93), a Strep-tag II tag, or a human influenza hemagglutinin tag.

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

19. The antigen binding molecule according to any one of claims 1 to 18, wherein the first heavy chain polypeptide further comprises a heavy chain constant domain 3 (CH3). 20 . The antigen-binding molecule according to claim 1 , which does not comprise a dimerization sequence of the heavy chain constant domain 1 (CH1) or the light chain constant domain (CL).

21. An antigen binding molecule according to any one of claims 1 to 20, which is an immunoglobulin, Fab, Fab', F(ab')2, antibody, biparatopic antibody, bispecific antibody, triparatopic antibody, trispecific antibody, tetraparatopic antibody, tetraspecific antibody, multiparatopic antibody, multispecific antibody, or any fragment of the antigen binding molecule that binds to the target antigen.

22. according to the antigen binding molecules described in any one in claim 1 to 21, described antigen binding molecules also comprise the second light chain polypeptide with the second light chain variable domain (VL2) and the second heavy chain polypeptide with the second heavy chain variable domain (VH2), wherein VL2 and VH2 form the second paratope.

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

24. according to claim 22 or 23 described antigen binding molecules, described antigen binding molecules also comprise the third light chain polypeptide with the third light chain variable domain (VL3) and the third heavy chain polypeptide with the third heavy chain variable domain (VH3), wherein VL3 and VH3 form the 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. An antigen binding molecule according to claim 24 or 25, further comprising a fourth light chain polypeptide having a fourth light chain variable domain (VL4) and a fourth heavy chain polypeptide having 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. 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 antigen binding molecule of claim 24 or 25, wherein 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).

31. An antigen binding molecule according to claim 26 or 27, wherein 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).

32. A polynucleotide encoding the separation of an antigen binding molecule described in any one of claims 1 to 31, optionally wherein the isolated polynucleotide sequence comprises a nucleotide sequence selected from SEQ ID NO: 3 and 263-343, or a nucleotide sequence having at least 85% sequence identity with any one of SEQ ID NO: 3 and 263-343.

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, the method comprising: i) culturing the host cell of claim 34 under suitable conditions so that the antigen-binding molecule is expressed by the host cell; as well as ii) isolating the antigen-binding molecule.

Images