Antibodies for binding to cd80

Abstract

The present invention relates to antigen-binding proteins, related fragments thereof, for binding to CD80, and their use for treating various conditions such as inflammation and autoimmunity.

Description

Technical Field

[0001] This invention relates to antigen-binding proteins for binding to CD80, related fragments thereof, and their use in treating various conditions such as inflammation and autoimmunity.

[0002] Related applications

[0003] This application claims priority to Australian Provisional Application AU 2023903540, the entire contents of which are hereby incorporated by reference. Background Technology

[0004] The costimulatory ligand CD80 and the inhibitory ligand PD-L1 interact cis-with on the surface of antigen-presenting cells, including dendritic cells. In the context of the cis-CD80:PD-L1 complex, CD80 can still bind to the costimulatory receptor CD28 on interacting T cells, but PD-L1 cannot bind to the T cell inhibitor receptor PD1. Therefore, these complexes activate primordial T cells by allowing CD80 on the dendritic cell surface to trigger CD28 signaling without PD-L1 inhibition.

[0005] In addition to CD28, the costimulatory ligands CD80 and CD86 expressed by APC are also ligands of CTLA4 on the cell surface.

[0006] It regulates the constitutively abundant expression of CTLA4 in T cells (Tregs) and inhibits co-stimulation through other mechanisms, thereby depleting CD80 and CD86 from the surface of APCs via CTLA4-mediated transcellular endocytosis.

[0007] Due to the crucial role of the CD80 / CD86 co-stimulatory pathway in promoting and maintaining immune responses, therapeutics designed to antagonize this pathway are used clinically to treat autoimmune diseases and conditions. One approach is the development of abatacept (Orencia®), a CTLA4-Ig fusion protein composed of an extracellular binding domain of CTLA4 linked to the Fc domain of human IgG. Abatacept was developed to inhibit CD80-mediated co-stimulation and is approved for the treatment of rheumatoid arthritis (RA) and is in clinical trials for many other autoimmune indications.

[0008] Another approach is to develop anti-CD80 antibodies, some of which block the interaction between CD80 and CD28, and others which bind to the PD-L1 binding site on CD80. Therefore, antibodies designed to bind to CD80 can have different mechanisms of action and different effects on the CD80 co-stimulatory pathway.

[0009] There is a need for improved and / or alternative compositions and methods for modulating CD80 co-stimulation and the PD-L1 checkpoint pathway.

[0010] References to any prior art in this specification are not an admission or implication that such prior art constitutes part of common common sense in any jurisdiction, nor are they an admission or implication that such prior art can be reasonably expected or understood by a person skilled in the art, or be regarded as relevant and / or combined with other prior art. Summary of the Invention

[0011] This invention relates to antigen-binding proteins for binding to CD80, and specifically to antigen-binding proteins that block CD80:PD-L1 interaction and are therefore usable as immunosuppressants.

[0012] This invention provides an antigen-binding protein for binding to CD80, the antigen-binding protein having an antigen-binding domain comprising:

[0013] FR1 - CDR1 – FR2 – CDR2 – FR3 – CDR3 – FR4, and

[0014] FR1a – CDR1a – FR2a – CDR2a – FR3a – CDR3a – FR4a,

[0015] in:

[0016] FR1, FR2, FR3, and FR4 are each a frame region;

[0017] CDR1, CDR2 and CDR3 are each complementary determinant regions;

[0018] FR1a, FR2a, FR3a and FR4a are each a frame region;

[0019] CDR1a, CDR2a and CDR3a are each complementarity-determining regions;

[0020] The sequence of any of the frame regions or complementary decision regions mentioned herein is as described herein, preferably as described in the tables herein.

[0021] In any embodiment, CDR1, CDR2, and CDR3 refer to complementarity-determining regions (CDRs) of the variable heavy chain (VH) of the antibody, and CDR1a, CDR2a, and CDR3a are CDRs of the variable light chain (VL) of the antibody, or wherein CDR1, CDR2, and CDR3 are CDRs of the VL, and CDR1a, CDR2a, and CDR3a are CDRs of the VH. In such examples, the CDRs may be referred to as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, depending on the context.

[0022] The article mentions that proteins or antibodies that "bind to CD80 (or CD28 antigen ligand or B7-1 antigen)" provide textual support for proteins or antibodies that "bind specifically to" or "specifically binds to".

[0023] In preferred embodiments, the antigen-binding protein as described herein is capable of binding to or specifically binding to residues of the distal IgV domain of human CD80 (corresponding to residues G34 to A140 of human CD80). Optionally, the antigen-binding protein as described herein does not bind to the PD-L1-bound region or portion of CD80, but, upon binding to CD80, prevents or reduces the binding of PD-L1 to CD80. In preferred embodiments, the antigen-binding protein as described herein is capable of binding to or specifically binding to the PD-L1-bound portion of CD80. Therefore, the antigen-binding protein of the present invention preferably inhibits the binding of PD-L1 to CD80. As described elsewhere herein, preventing the binding of PD-L1 to CD80 may also be referred to as “PD-L1 release”.

[0024] In some embodiments, the antigen-binding protein does not inhibit the binding of CD28 or CD86 to CD80 after binding to CD80.

[0025] Optionally, antigen-binding proteins as defined herein inhibit the binding of CD28 to CD80 in addition to inhibiting the binding of PD-L1 to CD80.

[0026] Optionally, the antigen-binding protein as described herein can bind to or specifically bind to CD80, thereby inhibiting PD-L1 but not the binding of CD28 to CD80.

[0027] In any embodiment, the present invention provides an antigen-binding protein for binding to CD80, wherein the antigen-binding protein competitively inhibits the binding of CD80 to an antibody, wherein the antigen-binding protein:

[0028] - Includes VH, which contains a sequence as shown in SEQ ID NO: 73 or SEQ ID NO: 77; and VL, which contains a sequence as shown in SEQ ID NO: 148 or SEQ ID NO: 152;

[0029] - Includes VH, which contains a sequence as shown in SEQ ID NO: 74 or SEQ ID NO: 78; and VL, which contains a sequence as shown in SEQ ID NO: 149 or SEQ ID NO: 153;

[0030] -Comprising: VH, which comprises the sequence shown in SEQ ID NO: 75; and VL, which comprises the sequence shown in SEQ ID NO: 150; or

[0031] -Comprising: VH, which comprises the sequence shown in SEQ ID NO: 76; and VL, which comprises the sequence shown in SEQ ID NO: 151;

[0032] Preferably, the antigen-binding protein is not antibody TKMF5 (as described in WO 2020116636).

[0033] In any embodiment, the present invention provides an antigen-binding protein comprising CDRH1, CDRH2, and / or CDRH3 having an antigen-binding domain of a variable heavy chain as defined in any of SEQ ID NO: 73 to 78.

[0034] In any embodiment, the present invention provides an antigen-binding protein comprising CDRL1, CDRL2, and / or CDRL3 having an antigen-binding domain of a variable light chain as defined in any of SEQ ID NO: 148 to 153.

[0035] In any embodiment, the present invention provides an antigen-binding protein for binding to CD80, the protein comprising:

[0036] - CDR1, CDR2 and CDR3 having an antigen-binding domain of a variable heavy chain as defined in SEQ ID NO: 73 or SEQ ID NO: 77, and CDR1, CDR2 and CDR3 having an antigen-binding domain of a variable light chain as defined in SEQ ID NO: 148 or SEQ ID NO: 152;

[0037] - CDR1, CDR2 and CDR3 having an antigen-binding domain of a variable heavy chain as defined in SEQ ID NO: 74 or SEQ ID NO: 78, and CDR1, CDR2 and CDR3 having an antigen-binding domain of a variable light chain as defined in SEQ ID NO: 149 or SEQ ID NO: 153;

[0038] - CDR1, CDR2, and CDR3 having antigen-binding domains of a variable heavy chain as defined in SEQ ID NO: 75, and CDR1, CDR2, and CDR3 having antigen-binding domains of a variable light chain as defined in SEQ ID NO: 150; or

[0039] - CDR1, CDR2 and CDR3 having an antigen-binding domain of a variable heavy chain as defined in SEQ ID NO: 76, and CDR1, CDR2 and CDR3 having an antigen-binding domain of a variable light chain as defined in SEQ ID NO: 151.

[0040] In any embodiment, the antigen-binding protein described herein comprises:

[0041] FR1 – CDR1 – FR2 – CDR2 – FR3 – CDR3 – FR4 – Linker – FR1a – CDR1a –FR2a – CDR2a – FR3a – CDR3a – FR4a.

[0042] As defined herein, the linker may be chemical, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0043] In some preferred embodiments, the present invention provides an antigen-binding protein comprising, substantially comprising, or consisting of the amino acid sequences of SEQ ID NO: 73 and 148 (in N-to-C-terminus or C-to-N-terminus).

[0044] In some preferred embodiments, the present invention provides an antigen-binding protein comprising, substantially comprising, or consisting of the amino acid sequences of SEQ ID NO: 77 and 152 (in N to C-terminus or C to N-terminus).

[0045] In some preferred embodiments, the present invention provides an antigen-binding protein comprising, substantially comprising, or consisting of the amino acid sequences of SEQ ID NO: 74 and 149 (in N-to-C-terminus or C-to-N-terminus).

[0046] In some preferred embodiments, the present invention provides an antigen-binding protein comprising, substantially comprising, or consisting of the amino acid sequences of SEQ ID NO: 78 and 153 (in N-to-C-terminus or C-to-N-terminus).

[0047] In some preferred embodiments, the present invention provides an antigen-binding protein comprising, substantially comprising, or consisting of the amino acid sequences of SEQ ID NO: 75 and 150 (in N to C-terminus or C to N-terminus).

[0048] In some preferred embodiments, the present invention provides an antigen-binding protein comprising, substantially comprising, or consisting of the amino acid sequences of SEQ ID NO: 76 and 151 (in N to C-terminus or C to N-terminus).

[0049] In a particularly preferred embodiment, the antigen-binding protein comprises, is substantially composed of, or is composed of SEQ ID NO: 148 and SEQ ID NO: 73 (i.e., VL to VH) in N-to-C-terminal order. Optionally, the antigen-binding protein comprises SEQ ID NO: 148 (VL) - adapter - SEQ ID NO: 73 (VH).

[0050] In a particularly preferred embodiment, the antigen-binding protein comprises, is substantially composed of, or is composed of SEQ ID NO: 152 and SEQ ID NO: 77 (i.e., VL to VH) in N-to-C-terminal order. Optionally, the antigen-binding protein comprises SEQ ID NO: 177 (VL) - adapter - SEQ ID NO: 77 (VH).

[0051] In a particularly preferred embodiment, the antigen-binding protein comprises, is substantially composed of, or is composed of SEQ ID NO: 149 and SEQ ID NO: 74 (i.e., VL to VH) in N-to-C-terminal order. Optionally, the antigen-binding protein comprises SEQ ID NO: 149 (VL) - adapter - SEQ ID NO: 74 (VH).

[0052] In a particularly preferred embodiment, the antigen-binding protein comprises, is substantially composed of, or is composed of SEQ ID NO: 153 and SEQ ID NO: 78 (i.e., VL to VH) in N-to-C-terminal order. Optionally, the antigen-binding protein comprises SEQ ID NO: 153 (VL) - adapter - SEQ ID NO: 78 (VH).

[0053] In a particularly preferred embodiment, the antigen-binding protein comprises, in order from N to C ends, SEQ ID NO: 150 and SEQ ID NO: 75 (i.e., VL to VH), optionally SEQ ID NO: 150 (VL) - adapter - SEQ ID NO: 75 (VH), substantially composed of or composed of therein.

[0054] In a particularly preferred embodiment, the antigen-binding protein comprises, in order from N to C ends, SEQ ID NO: 151 and SEQ ID NO: 76 (i.e., VL to VH), optionally SEQ ID NO: 151 (VL) - adapter - SEQ ID NO: 76 (VH), substantially composed of or composed of.

[0055] In any embodiment, the antigen-binding domain comprises:

[0056] (i) VH, wherein the VH comprises: a complementarity determination region (CDR) 1, wherein the CDR 1 comprises a sequence as shown in SEQ ID NO: 1 (IMGT) or 13 (Kabat) or a sequence that is 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% identical to the sequence shown in SEQ ID NO: 1 (IMGT) or 13 (Kabat); and a CDR 2, wherein the CDR 2 comprises a sequence as shown in SEQ ID NO: 1 (IMGT) or 13 (Kabat). The sequence shown in NO:2 (IMGT) or 14 or 165 (Kabat) or the sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 3 (IMGT) or 15 (Kabat) or the sequence that is 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% identical to it;

[0057] (ii) VH, wherein the VH comprises a sequence as shown in SEQ ID NO: 73 or SEQ ID NO: 77 or a sequence that is 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% identical to the sequence shown in SEQ ID NO: 77;

[0058] (iii) VL, the VL comprising: a complementarity determination region (CDR) 1, the CDR 1 comprising a sequence as shown in SEQ ID NO: 79 (IMGT) or 91 (Kabat) or a sequence that is 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% identical to it; and a CDR 2, the CDR 2 comprising a sequence as shown in SEQ ID NO: 79 (IMGT) or 91 (Kabat). The sequence shown in NO:80 (IMGT) or 92 (Kabat) or a sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 81 (IMGT or Kabat) or a sequence that is 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% identical to it;

[0059] (iv) VL, wherein the VL comprises a sequence as shown in SEQ ID NO: 148 or SEQ ID NO: 152 or a sequence that is 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% identical to the sequence shown in SEQ ID NO: 152.

[0060] (v) VH, wherein VH comprises: CDR1, wherein CDR1 comprises the sequence shown in SEQ ID NO: 1; CDR2, wherein CDR2 comprises the sequence shown in SEQ ID NO: 2; and CDR3, wherein CDR3 comprises the sequence shown in SEQ ID NO: 3; or comprises: CDR1, wherein CDR1 comprises the sequence shown in SEQ ID NO: 13; CDR2, wherein CDR2 comprises the sequence shown in SEQ ID NO: 14 or 165; and CDR3, wherein CDR3 comprises the sequence shown in SEQ ID NO: 15;

[0061] (vi) VL, the VL comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 79; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 80; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 81; or comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 91; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 92; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 93;

[0062] (vii) VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 1; CDR2, which comprises the sequence shown in SEQ ID NO: 2; and CDR3, which comprises the sequence shown in SEQ ID NO: 3; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 79; CDR2, which comprises the sequence shown in SEQ ID NO: 80; and CDR3, which comprises the sequence shown in SEQ ID NO: 81; or VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 13; CDR2, which comprises the sequence shown in SEQ ID NO: 14 or 165; and CDR3, which comprises the sequence shown in SEQ ID NO: 15; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 1; CDR2, which comprises the sequence shown in SEQ ID NO: 14 or 165; and CDR3, which comprises the sequence shown in SEQ ID NO: 15; and VL, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 1; CDR2, which comprises the sequence shown in SEQ ID NO: 1 ... The sequence shown in NO:91; CDR2, which comprises the sequence shown in SEQ ID NO:92; and CDR3, which comprises the sequence shown in SEQ ID NO:93; or

[0063] (viii) VH, which comprises the sequence shown in SEQ ID NO: 73 or SEQ ID NO: 77; and VL, which comprises the sequence shown in SEQ ID NO: 148 or SEQ ID NO: 152.

[0064] When the CDR is determined according to the IMGT system, the antigen-binding domain may further include at least one of the following:

[0065] (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 25 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 26 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 27 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises SEQ ID NO: The amino acid sequence of 28 or thereof, or at least about 80%, at least 85%, 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% thereof; and

[0066] ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 101 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 102 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 103 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 104 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0067] When the CDR is determined according to the Kabat system, the antigen-binding domain may further include at least one of the following:

[0068] (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 49 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 50 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 51 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 52 or thereof, or at least about 80%, at least 85%, 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% thereof; and

[0069] ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 124 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 125 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 126 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 127 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0070] When the CDR is determined according to the IMGT system, the antigen-binding domain may further include at least one of the following:

[0071] (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 41 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 42 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 43 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: 44 amino acid sequences or sequences thereof comprising at least about 80%, at least 85%, 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% of the total amino acid sequence; and

[0072] (ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 105 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 106 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 107 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 108 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0073] When the CDR is determined according to the Kabat system, the antigen-binding domain may further include at least one of the following:

[0074] (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 53 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 54 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 55 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 56 or thereof, or at least about 80%, at least 85%, 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% thereof; and

[0075] ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 128 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 129 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 130 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 131 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0076] In any embodiment, the antigen-binding protein comprises: a variable heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 73 or SEQ ID NO: 77, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a variable light chain comprising an amino acid sequence as shown in SEQ ID NO: 148 or SEQ ID NO: 148. The amino acid sequence shown in 152 or the sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to it; wherein the variable heavy chain and / or light chain contains, in addition to the indicated CDR sequence, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added, or combinations thereof, and wherein the antigen-binding protein retains the ability to bind to CD80.

[0077] In any embodiment, the antigen-binding domain comprises:

[0078] (i) VH, wherein the VH comprises: a complementarity determination region (CDR) 1, wherein the CDR 1 comprises a sequence as shown in SEQ ID NO: 4 or 16 (Kabat) or a sequence that is 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% identical to the sequence shown in SEQ ID NO: 4 or 16 (Kabat); and a CDR 2, wherein the CDR 2 comprises a sequence as shown in SEQ ID NO: 4 or 16 (Kabat). The sequence shown in SEQ ID NO: 5 or 17 or 166 (Kabat) or the sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 6 or 18 (Kabat) or the sequence that is 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% identical to it;

[0079] (ii) VH, wherein the VH comprises a sequence as shown in SEQ ID NO: 74 or SEQ ID NO: 78 or a sequence that is 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% identical to it;

[0080] (iii) VL, the VL comprising: a complementarity determination region (CDR) 1, the CDR 1 comprising a sequence as shown in SEQ ID NO: 82 (IMGT) or 93 (Kabat) or a sequence that is 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% identical to it; and a CDR 2, the CDR 2 comprising a sequence as shown in SEQ ID NO: 82 (IMGT) or 93 (Kabat). The sequence shown in NO:83 (IMGT) or 94 (Kabat) or the sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 84 (IMGT or Kabat) or the sequence that is 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% identical to it;

[0081] (iv) VL, wherein the VL comprises a sequence as shown in SEQ ID NO: 149 or SEQ ID NO: 153 or a sequence that is 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% identical to the sequence shown in SEQ ID NO: 153.

[0082] (v) VH, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 4; CDR2, which comprises the sequence shown in SEQ ID NO: 5; and CDR3, which comprises the sequence shown in SEQ ID NO: 6; or comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 16; CDR2, which comprises the sequence shown in SEQ ID NO: 17 or 166; and CDR3, which comprises the sequence shown in SEQ ID NO: 18;

[0083] (vi) VL, the VL comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 82; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 83; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 84; or comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 93; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 94; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 84;

[0084] (vii) VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 4; CDR2, which comprises the sequence shown in SEQ ID NO: 5; and CDR3, which comprises the sequence shown in SEQ ID NO: 6; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 82; CDR2, which comprises the sequence shown in SEQ ID NO: 83; and CDR3, which comprises the sequence shown in SEQ ID NO: 84; or VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 16; CDR2, which comprises the sequence shown in SEQ ID NO: 17 or 166; and CDR3, which comprises the sequence shown in SEQ ID NO: 18; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 4; CDR2, which comprises the sequence shown in SEQ ID NO: 5; and CDR3, which comprises the sequence shown in SEQ ID NO: 6; and VL, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 6; and CDR3, which comprises the sequence shown in SEQ ID NO: 7; and CDR3, which comprises the sequence shown in SEQ ID NO: 84; and CDR3, which comprises the sequence shown in SEQ ID NO: 85; and CDR3, which comprises the sequence shown in SEQ ID NO: 86 ...6; and CDR3, which comprises the sequence The sequence shown in NO:93; CDR2, which comprises the sequence shown in SEQ ID NO:94; and CDR3, which comprises the sequence shown in SEQ ID NO:84; or

[0085] (viii) VH, which comprises the sequence shown in SEQ ID NO: 74 or SEQ ID NO: 78; and VL, which comprises the sequence shown in SEQ ID NO: 149 or SEQ ID NO: 153.

[0086] When the CDR is defined according to the IMGT system, the antigen-binding domain may further include at least one of the following:

[0087] (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 29 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 30 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 31 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 32 or thereof, or at least about 80%, at least 85%, 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% thereof; and

[0088] ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 109 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 110 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 111 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 112 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0089] When the CDR is defined according to the Kabat system, the antigen-binding domain may further include at least one of the following:

[0090] (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 57 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 58 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 59 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 60 or thereof, or the sequence thereof, comprising at least about 80%, at least 85%, 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%; and

[0091] ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 132 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 133 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 134 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises SEQ ID NO: The amino acid sequence of 135 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0092] When the CDR is defined according to the IMGT system, the antigen-binding domain may further include at least one of the following:

[0093] i) VH, wherein the VH comprises: a frame region (FR) 1, wherein the FR1 comprises the amino acid sequence of SEQ ID NO: 45 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein the FR2 comprises the amino acid sequence of SEQ ID NO: 46 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein the FR3 comprises the amino acid sequence of SEQ ID NO: 47 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein the FR4 comprises the amino acid sequence of SEQ ID NO: 48 amino acid sequences or sequences thereof comprising at least about 80%, at least 85%, 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% of the total amino acid sequence; and

[0094] ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 113 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 114 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 115 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 116 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0095] When the CDR is defined according to the Kabat system, the first antigen-binding domain may further include at least one of the following:

[0096] (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 61 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 62 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 63 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 64 or thereof, or at least about 80%, at least 85%, 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% thereof; and

[0097] ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 136 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 137 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 138 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 139 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0098] In any embodiment, the antigen-binding protein comprises: a variable heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 74 or SEQ ID NO: 78, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a variable light chain comprising an amino acid sequence as shown in SEQ ID NO: 149 or SEQ ID NO: 149. The amino acid sequence shown in 153 or the sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to it; wherein the variable heavy chain and / or light chain contains, in addition to the indicated CDR sequence, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added, or combinations thereof, and wherein the antigen-binding protein retains the ability to bind to CD80.

[0099] In any embodiment, the antigen-binding domain comprises:

[0100] (i) VH, wherein the VH comprises: a complementarity determination region (CDR) 1, wherein the CDR 1 comprises a sequence as shown in SEQ ID NO: 7 (IMGT) or 19 (Kabat) or a sequence that is 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% identical to the sequence shown in SEQ ID NO: 7 (IMGT) or 19 (Kabat); and a CDR 2, wherein the CDR 2 comprises a sequence as shown in SEQ ID NO: 7 (IMGT) or 19 (Kabat). The sequence shown in NO:8 (IMGT) or 20 (Kabat) or a sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 9 (IMGT) or 21 (Kabat) or a sequence that is 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% identical to it;

[0101] (ii) VH, wherein the VH comprises a sequence as shown in SEQ ID NO: 75 or a sequence that is 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% identical to it;

[0102] (iii) VL, the VL comprising: a complementarity determination region (CDR) 1, the CDR 1 comprising a sequence as shown in SEQ ID NO: 85 (IMGT) or 95 (Kabat) or a sequence that is 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% identical to it; and a CDR 2, the CDR 2 comprising a sequence as shown in SEQ ID NO: 85 (IMGT) or 95 (Kabat). The sequence shown in NO:86 (IMGT) or 96 (Kabat) or a sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 87 (IMGT) or 97 (Kabat) or a sequence that is 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% identical to it;

[0103] (iv) VL, wherein the VL comprises a sequence as shown in SEQ ID NO: 150 or a sequence that is 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% identical to it;

[0104] (v) VH, wherein VH comprises: CDR1, wherein CDR1 comprises the sequence shown in SEQ ID NO: 7; CDR2, wherein CDR2 comprises the sequence shown in SEQ ID NO: 8; and CDR3, wherein CDR3 comprises the sequence shown in SEQ ID NO: 9; or comprises: CDR1, wherein CDR1 comprises the sequence shown in SEQ ID NO: 19; CDR2, wherein CDR2 comprises the sequence shown in SEQ ID NO: 20; and CDR3, wherein CDR3 comprises the sequence shown in SEQ ID NO: 21; or

[0105] (vi) VL, the VL comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 85; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 86; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 87; or comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 95; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 96; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 97; or

[0106] (vii) VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 7; CDR2, which comprises the sequence shown in SEQ ID NO: 8; and CDR3, which comprises the sequence shown in SEQ ID NO: 9; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 85; CDR2, which comprises the sequence shown in SEQ ID NO: 86; and CDR3, which comprises the sequence shown in SEQ ID NO: 87; or VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 19; CDR2, which comprises the sequence shown in SEQ ID NO: 20; and CDR3, which comprises the sequence shown in SEQ ID NO: 21; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 7; CDR2, which comprises the sequence shown in SEQ ID NO: 86; and CDR3, which comprises the sequence shown in SEQ ID NO: 9; and VL, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 87; CDR2, which comprises the sequence shown in SEQ ID NO: 88; and CDR3, which comprises the sequence shown in SEQ ID NO: 89; and VL, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 89; CDR2, which comprises the sequence shown in SEQ ID NO: 89 ... and CDR3, which comprises the sequence shown in SEQ ID NO: 89; and CDR3, which comprises the sequence shown in SEQ ID NO: The sequence shown in SEQ ID NO: 95; CDR2, which comprises the sequence shown in SEQ ID NO: 96; and CDR3, which comprises the sequence shown in SEQ ID NO: 97; or

[0107] (viii) VH, which comprises the sequence shown in SEQ ID NO: 75; and VL, which comprises the sequence shown in SEQ ID NO: 150.

[0108] When the CDR is defined according to the IMGT, the antigen-binding protein may further include at least one of the following:

[0109] (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 33 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 34 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 35 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 36 or thereof, or at least about 80%, at least 85%, 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% thereof; and

[0110] ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 117 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 118 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 119 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 104 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0111] When the CDR is defined according to Kabat, the antigen-binding protein may further include at least one of the following:

[0112] (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 65 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 66 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 67 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 68 or thereof, or at least about 80%, at least 85%, 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% thereof; and

[0113] ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 140 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 141 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 142 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 143 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0114] In any embodiment, the antigen-binding protein comprises: a variable heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 75 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a variable light chain comprising an amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in 150 or the sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to it; wherein the variable heavy chain and / or light chain contains, in addition to the indicated CDR sequence, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added, or combinations thereof, and wherein the antigen-binding protein retains the ability to bind to CD80.

[0115] In any embodiment, the antigen-binding domain comprises:

[0116] (i) VH, wherein the VH comprises: a complementarity determination region (CDR) 1, wherein the CDR 1 comprises a sequence as shown in SEQ ID NO: 10 (IMGT) or 22 (Kabat) or a sequence that is 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% identical to the sequence shown in SEQ ID NO: 10 (IMGT) or 22 (Kabat); and a CDR 2, wherein the CDR 2 comprises a sequence as shown in SEQ ID NO: 10 (IMGT) or 22 (Kabat). The sequence shown in NO:11 (IMGT) or 23 (Kabat) or the sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 12 (IMGT) or 24 (Kabat) or the sequence that is 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% identical to it;

[0117] (ii) VH, wherein the VH comprises a sequence as shown in SEQ ID NO: 76 or a sequence that is 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% identical to it;

[0118] (iii) VL, the VL comprising: a complementarity determination region (CDR) 1, the CDR 1 comprising a sequence as shown in SEQ ID NO: 88 (IMGT) or 98 (Kabat) or a sequence that is 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% identical to it; and a CDR 2, the CDR 2 comprising a sequence as shown in SEQ ID NO: 88 (IMGT) or 98 (Kabat). The sequence shown in NO:89 (IMGT) or 99 (Kabat) or the sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 90 (IMGT) or 100 (Kabat) or the sequence that is 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% identical to it;

[0119] (iv) VL, wherein the VL comprises a sequence as shown in SEQ ID NO: 151 or a sequence that is 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% identical to it;

[0120] (v) VH, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 10; CDR2, which comprises the sequence shown in SEQ ID NO: 11; and CDR3, which comprises the sequence shown in SEQ ID NO: 12; or comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 22; CDR2, which comprises the sequence shown in SEQ ID NO: 23; and CDR3, which comprises the sequence shown in SEQ ID NO: 24;

[0121] (vi) VL, the VL comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 88; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 89; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 90; or comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 98; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 99; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 100;

[0122] (vii) VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 10; CDR2, which comprises the sequence shown in SEQ ID NO: 11; and CDR3, which comprises the sequence shown in SEQ ID NO: 12; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 88; CDR2, which comprises the sequence shown in SEQ ID NO: 89; and CDR3, which comprises the sequence shown in SEQ ID NO: 90; or VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 22; CDR2, which comprises the sequence shown in SEQ ID NO: 23; and CDR3, which comprises the sequence shown in SEQ ID NO: 24; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 10; CDR2, which comprises the sequence shown in SEQ ID NO: 11; and CDR3, which comprises the sequence shown in SEQ ID NO: 12; and VL, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 12; CDR3, which comprises the sequence shown in SEQ ID NO: 13; and CDR4, which comprises the sequence shown in SEQ ID NO: 12; and VL, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 13; CDR4, which comprises the sequence shown in SEQ ID NO: 14; and CDR5, which comprises the sequence shown in SEQ ID NO: 12; and CDR6, which comprises the sequence shown in SEQ ID NO: 13; and CDR7, which comprises the sequence shown in SEQ ID NO: 14; and CDR8, which comprises the sequence shown in SEQ ID NO: 12; and CDR3, which comprises the sequence shown in SEQ ID NO: 13; and CDR4, which comprises the sequence shown in SEQ ID NO: 14; and CDR5, which comprises the sequence shown in SEQ ID NO: 12; and C The sequence shown in SEQ ID NO: 98; CDR2, which comprises the sequence shown in SEQ ID NO: 99; and CDR3, which comprises the sequence shown in SEQ ID NO: 100; or

[0123] (viii) VH, which comprises the sequence shown in SEQ ID NO: 76; and VL, which comprises the sequence shown in SEQ ID NO: 151.

[0124] When the CDR is defined according to the IMGT, the antigen-binding protein may further include at least one of the following:

[0125] (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 37 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 38 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 39 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 40 or thereof, or the sequence thereof, comprising at least about 80%, at least 85%, 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%; and

[0126] ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 120 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 121 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 122 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 123 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0127] When the CDR is defined according to Kabat, the antigen-binding protein may further include at least one of the following:

[0128] (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 69 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 70 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 71 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 72 or thereof, or at least about 80%, at least 85%, 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% thereof; and

[0129] ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 144 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 145 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 146 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 147 or the sequence thereof of at least about 80%, at least 85%, 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%.

[0130] In any embodiment, the antigen-binding protein comprises: a variable heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 76 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a variable light chain comprising an amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in 151 or the sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to it; wherein the variable heavy chain and / or light chain contains, in addition to the indicated CDR sequence, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added, or combinations thereof, and wherein the antigen-binding protein retains the ability to bind to CD80.

[0131] As described herein, the antigen-binding protein may be in the following forms:

[0132] (i) Single-domain antibodies (sdAbs);

[0133] (ii) Single-stranded Fv fragments (scFv);

[0134] (iii) Dimer scFv (di-scFv); or

[0135] (iv) One of (i) or (iii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of the antibody.

[0136] Furthermore, as described herein, the antigen-binding protein may be in the following form:

[0137] (i) Bifunctional antibody;

[0138] (ii) Trifunctional antibodies;

[0139] (iii) Four-functional antibodies;

[0140] (iv) Fab;

[0141] (v)F(ab')2;

[0142] (vi)Fv;

[0143] (vii) Bispecific antibodies or other forms of multispecific antibodies (including BiTE); or

[0144] (viii) One of (i) to (vii) connected to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of the antibody.

[0145] The aforementioned antigen-binding protein can also be referred to as the antigen-binding domain of an antibody.

[0146] Preferably, the antigen-binding protein as described herein is an antibody or an antigen-binding fragment thereof. Typically, the antigen-binding protein is an antibody, such as a monoclonal antibody. The antigen-binding protein may be in the form of a recombinant or modified antibody (e.g., chimeric antibody, humanized antibody, human antibody, CDR-transplanted antibody, primate-derived antibody, deimmunized antibody, synthetic humanized antibody, hapten, bispecific antibody, trispecific antibody, or multispecific antibody). The antibody may further contain chemical modifications, such as conjugation to an active agent or radiolabeling agent, or an agent for improving solubility, or other modifications described herein.

[0147] In a preferred embodiment, the antigen-binding protein of the present invention may be monospecific or multispecific; in other words, it may bind to one or more molecular targets (i.e., to CD80 and optionally another protein target). Preferably, the antigen-binding protein of the present invention is monocomplementary, meaning that the antigen-binding protein binds to a single epitope on a given molecular target (i.e., CD80). For completeness, in a preferred embodiment, the antigen-binding protein of the present invention is not multicomplementary (in other words, it does not bind to different non-overlapping epitopes on the antigen).

[0148] As used in this article, antigen-binding proteins can be variable domains.

[0149] As used herein, the complementarity-determining region (CDR) sequence of the antigen-binding protein of the present invention may be defined according to the IMGT, Chothia, or Kabat numbering system or any other CDR numbering system known to those skilled in the art or described herein.

[0150] The present invention provides an antigen-binding protein as described herein, wherein the amino acid sequence forming one or more of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 is a human sequence.

[0151] This invention provides anti-CD80 binding proteins, immunoglobulin variable domains, antibodies, dab, scFv, Fab, Fab', F(ab')2, Fv fragments, bifunctional antibodies, trifunctional antibodies, linear antibodies, single-chain antibody molecules, or multispecific antibodies comprising antigen-binding proteins having sequences as described herein or including CDR and / or FR sequences as described herein.

[0152] As described herein, antigen-binding proteins may contain human constant regions, such as IgG constant regions, such as IgG1, IgG2, IgG3, or IgG4 constant regions, or mixtures thereof. In the presence of V... H and V L In the case of antibodies or proteins, the V H It can be connected to the heavy chain constant region, and the V L It can be connected to the constant region of light chains.

[0153] In one instance, the antigen-binding protein as described herein comprises a constant region of an IgG4 antibody or a stable constant region of an IgG4 antibody. In one instance, the protein or antibody comprises an IgG4 constant region having a proline residue at position 241 (according to Kabat's numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 1987 and / or 1991)).

[0154] In one instance, an antigen-binding protein as described herein, or a composition of an antigen-binding protein as described herein, comprises a heavy chain constant region comprising a stable heavy chain constant region comprising a mixture of sequences having, or not having, a C-terminal lysine residue, either wholly or partially.

[0155] In one instance, the antigen-binding protein comprises the V disclosed herein linked to or fused to an IgG4 constant region or a stable IgG4 constant region (e.g., as discussed above). H And V L Connected to or fused with the constant region of the κ light chain.

[0156] In any aspect of this invention, the antibody is a naked antibody. Specifically, the antibody is in a non-conjugated form and is not suitable for forming conjugates.

[0157] This invention provides a fusion protein comprising, as described herein, an antigen-binding protein, an immunoglobulin variable domain, an antibody, dab, scFv, Fab, Fab', F(ab')2, an Fv fragment, a bifunctional antibody, a trifunctional antibody, a linear antibody, a single-chain antibody molecule, or a multispecific antibody.

[0158] This invention also provides a conjugate in the form of an antigen-binding protein, an immunoglobulin variable domain, an antibody, dab, scFv, Fab, Fab', F(ab')2, an Fv fragment, a bifunctional antibody, a trifunctional antibody, a linear antibody, a single-chain antibody molecule, or a multispecific antibody or fusion protein, as described herein, conjugated to a label or cytotoxic agent. The cytotoxic agent may be a chemotherapeutic agent or other agent used to treat a disease.

[0159] In another embodiment, the antigen-binding protein includes an Fc region, which is engineered to:

[0160] -Increases the in vitro or in vivo half-life;

[0161] - It has the ability to increase the induction of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity.

[0162] - Reduce effector function; or

[0163] - Increase the co-conjugation of the antigen-binding protein.

[0164] Mutations, deletions, or modifications of amino acids in the Fc region that affect half-life, ADCC, ADCP, or complement-dependent cytotoxicity, effector function are known to those skilled in the art and are further described herein.

[0165] The functional properties of the antigen-binding protein of the present invention will be used to make necessary modifications to the antibodies of the present invention.

[0166] In any aspect of the invention and in any antigen-binding protein described herein, an Fc region is further included, said Fc region being engineered to have a reduced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, said reduced ability to induce ADCC is conferred by mutation, deletion, or modification of amino acids in the Fc region that interact with the Fc receptor.

[0167] In various aspects of the present invention relating to the formation of multiple polypeptide chains of antigen-binding proteins, the expression construct comprises a nucleic acid encoding a polypeptide comprising, for example, a VH operably linked to a promoter and a nucleic acid encoding a polypeptide comprising, for example, a VL operably linked to a promoter.

[0168] In another instance, the expression construct is a bicistronic expression construct, for example, comprising the following operatively linked components in a 5' to 3' sequence:

[0169] (i) Promoter;

[0170] (ii) The nucleic acid encoding the first polypeptide;

[0171] (iii) Internal ribosome entry sites; and

[0172] (iv) The nucleic acid encoding the second polypeptide,

[0173] The first polypeptide contains VH and the second polypeptide contains VL, or vice versa.

[0174] The present invention also contemplates separate expression constructs, one of which encodes a first polypeptide comprising VH, and another of which encodes a second polypeptide comprising VL. For example, the present invention also provides a composition comprising:

[0175] (i) a first expression construct comprising a nucleic acid encoding a polypeptide, the polypeptide comprising a VH operatively linked to a promoter; and

[0176] (ii) A second expression construct comprising a nucleic acid encoding a polypeptide, the polypeptide comprising a VL operatively linked to a promoter.

[0177] This invention provides a cell comprising the vector or nucleic acid described herein. Preferably, the cell is isolated, substantially purified, or recombinant. In one example, the cell comprises the expression construct of this invention or:

[0178] (i) a first expression construct comprising a nucleic acid encoding a polypeptide, the polypeptide comprising a VH operatively linked to a promoter; and

[0179] (ii) A second expression construct comprising a nucleic acid encoding a polypeptide, the polypeptide comprising a VL operatively linked to a promoter.

[0180] The first polypeptide associates with the second polypeptide to form the antigen-binding protein of the present invention.

[0181] Examples of cells used in this invention include bacterial cells, yeast cells, insect cells, or mammalian cells.

[0182] This invention provides a nucleic acid that encodes an antigen-binding protein, an immunoglobulin variable domain, an antibody, dab, scFv, Fab, Fab', F(ab')2, an Fv fragment, a bifunctional antibody, a trifunctional antibody, a linear antibody, a single-chain antibody molecule or a multispecific antibody, a fusion protein or a conjugate, as described herein.

[0183] The present invention provides a vector comprising the nucleic acid described herein.

[0184] The present invention provides a cell comprising the vector or nucleic acid described herein.

[0185] The present invention provides a pharmaceutical composition comprising an antigen-binding protein, or comprising CDR and / or FR sequences as described herein, or immunoglobulin variable domains, antibodies, dab, scFv, Fab, Fab', F(ab')2, Fv fragments, bifunctional antibodies, trifunctional antibodies, linear antibodies, single-chain antibody molecules or multispecific antibodies, fusion proteins or conjugates, and pharmaceutically acceptable carriers, diluents or excipients as described herein.

[0186] The present invention provides a diagnostic composition comprising an antigen-binding protein, or comprising CDR and / or FR sequences as described herein, or an antigen-binding protein, immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, bifunctional antibody, trifunctional antibody, linear antibody, single-chain antibody molecule or multispecific antibody, fusion protein or conjugate, diluent, and optionally labeling.

[0187] The present invention provides a kit or product comprising an antigen-binding protein, or comprising CDR and / or FR sequences as described herein, or immunoglobulin variable domains, antibodies, dab, scFv, Fab, Fab', F(ab')2, Fv fragments, bifunctional antibodies, trifunctional antibodies, linear antibodies, single-chain antibody molecules or multispecific antibodies, fusion proteins or conjugates as described herein.

[0188] The present invention provides the use of one or more sequences of CDR1, CDR2, FR1, FR2, FR3 and FR4 as described herein for generating an antigen-binding protein for binding to CD80.

[0189] This invention provides the use of antigen-binding proteins or CDR and / or FR sequences as described herein for generating anti-CD80 antigen-binding proteins with increased affinity for CD80.

[0190] The present invention provides a library of nucleic acid molecules generated from mutations in antigen-binding proteins or CDR and / or FR sequences as described herein, wherein at least one nucleic acid molecule in the library encodes an antigen-binding protein for binding to CD80.

[0191] The present invention provides a method for generating an antigen-binding protein as described herein for binding to CD80, the method comprising expressing a nucleic acid as described herein in a cell or animal.

[0192] The antigen-binding proteins described herein may be purified, substantially purified, isolated, and / or recombinant.

[0193] The antigen-binding protein of the present invention may be a portion of the supernatant of a culture medium in which a hybridoma expressing the antigen-binding protein of the present invention has been grown.

[0194] Furthermore, the present invention provides a pharmaceutical composition comprising the antigen-binding protein of the present invention and a physiologically or pharmaceutically acceptable carrier or diluent.

[0195] The present invention further considers the therapeutic and diagnostic uses of the antigen-binding protein of the present invention. The antigen-binding protein of the present invention is preferably used to treat immune-related conditions. In the most preferred embodiment of the present invention, the antigen-binding protein described herein is used to treat Crohn's disease, systemic lupus erythematosus (SLE), lupus nephritis, psoriatic arthritis, psoriasis, rheumatoid arthritis, ulcerative colitis, and / or transplant rejection.

[0196] Therefore, the present invention provides a method for treating inflammatory conditions in a subject in need, the method comprising administering the antigen-binding protein or pharmaceutical composition of the present invention to the subject, thereby treating the inflammatory condition in the subject.

[0197] Furthermore, the present invention provides the use of the antigen-binding protein as described herein for the preparation of a medicament for treating inflammatory conditions or symptoms or conditions requiring immunosuppression in a subject.

[0198] The present invention also provides antigen-binding proteins or pharmaceutical compositions as described herein for the treatment of inflammatory conditions or symptoms or conditions requiring immunosuppression.

[0199] As used herein, unless the context otherwise requires, the term "comprise" and variations thereof, such as "comprising," "comprises," and "comprised," are not intended to exclude additional additives, components, wholes, or steps. The terms "comprise" and "including" are used interchangeably.

[0200] Further aspects of the invention and further embodiments of the aspects described in the foregoing paragraphs will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings.

[0201] As used herein, unless the context otherwise requires, the term “comprise” and variations thereof, such as “comprising,” “comprises,” and “comprised,” are not intended to exclude additional additives, components, wholes, or steps.

[0202] Further aspects of the invention and further embodiments of the aspects described in the foregoing paragraphs will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings. Attached Figure Description

[0203] Figure 1 Schematic diagram of the CD80 / CD28 axis and the binding of PD-L1 and CD80 to therapeutic agents. (A) Homeostasis (T cell activation), where CD80 and PD-L1 bind cis to the surface of APCs, and CD80 simultaneously interacts with CD28. (B) CTLA-Ig (e.g., abatacept) binds to CD80, thereby indirectly releasing PD-L1 and blocking CD80:CD28 interaction, leading to T cell suppression. (C) Anti-CD80 mAb TKMF5 binds to CD80, thereby competitively releasing PD-L1, but does not block CD80:CD28 interaction.

[0204] Figure 2 (A) Flow cytometry of CHO cells expressing the cis-CD80:PD-L1 complex, incubated with a novel CD80 antibody from unpurified supernatant of transfected 293T cells compared to 10 µg / mL abatacept. CD80 antibody binding (hIgG1 staining), CD28-Ig (CD28mIgG2a with anti-mIgG2a secondary antibody), and free PD-L1 (MIH1 staining). Note the partial CD28-Ig blocking of 19B10 and B5 relative to TKMF5. (B) Novel CD80 mAb binding (hIgG1 staining) and free PD-L1 (MIH1 staining) relative to MFI of untreated cells.

[0205] Figure 3 Abatacept, TKMF5, and 19B10 were used to dose / response cisCD80:PD-L1 CHO cells, and PD-L1 release was measured (MIH1 staining).

[0206] Figure 4(A) CHO cells co-transduced with CD80mCherry and PD-L1mGFP at approximately equivalent levels of fluorescence, with individual MFI values ​​of MIH1 outputting a CD80mCherry+PD-L1mGFP+ population. MFI data were used to generate histograms in which cells were binned into equidistant CD80mCherry:PDL1mGFP MFI ratio ranges centered at approximately 1:1. The relative fold change in MIH1 MFI of abatacept and TKMF5 across the CD80mCherry:PDL1mGFP MFI ratio relative to untreated cells was plotted. (B) Models of cell surface CD80:PD-L1 duplexes at 1:1 (left) and 2:1 (right) ratios, with PD-L1 released via abatacept (CTLA4-Ig) and the PD-L1 competitor TKMF5. Theoretical PD-L1 release ratios for 'partial' and 'complete' PD-L1 releasers across the CD80:PD-L1 ratio range.

[0207] Figure 5 like Figure 4 As shown in Figure A, the novel CD80 antibodies 19B10 and B5 are compared with abatacept and TKMF5.

[0208] Figure 6 The novel CD80 antibody in scFv-Fc form bound to human CD80, human CD80 with the mutation L104D at the PD-L1 binding site, human CD80-ALPN202 with seven mutations (H52Y, A60E, E69D, M81L, V102M, A105G, D124G) in the IgV domain, chimeras of mouse CD80 and human IgV domain, and CHO cells containing cynomolgus monkey CD80 and mouse CD80 (hIgG1 staining).

[0209] Figure 7 (A) Primary T cell activation assay from healthy donors, measuring co-stimulatory blockade of T cell proliferation (CFSE dilution) and (B) PD-1 signaling by downregulating T cell cytokines IFN-γ and IL-2.

[0210] Figure 8 (A) Dose / response free PD-L1 (MIH1 MFI) across PD-L1 release of antibodies B5, 19B10, and TKMF5 in cisCD80:PD-L1 cells compared to abatacept. (B) Fold change of 100 µg / mL free PD-L1 relative to untreated cells across a series of CD80mCherry:PD-L1mGFP fluorescence ratios from A for B5, 19B10, TKMF5, and abatacept.

[0211] Figure 9 (A) In vitro primary human T cell activation, assessed as the percentage of IL-2+ T cells after 4 days of co-culture with artificial antigen-presenting cells (aAPCs) expressing CD80:PD-L1 in a 1:1 ratio and OKT3scFv on the cell surface. 50 µg / mL abatacept or B5 was added during the last 8 hours of co-culture. (B) Dose / response of the experimental conditions described in A, showing similar (CD4) or reduced (CD8) T cell activity of B5 relative to abatacept. (C) Activity in CD4 T cells treated as described in A, indicating the relative contributions of the anti-PD-1 antibody (nivolumab), abatacept (partial), and B5 (full) to PD-1 signaling.

[0212] Figure 10 (A) Schematic diagram of the humanization strategy for mouse CD80 and PD-L1 genes. Homologous human sequences replace exon 2 of mouse CD80 (encoding the IgV domain bound by human CD80 antibody) and exon 3 of mouse PD-L1. Double knock-in (DKI) mice maintain endogenous regulation and expression of CD80 and PD-L1, and maintain cis binding to each other and trans binding to CD28, CTLA4, or PD-1. (B) Schematic diagram of a mouse model of renal autoimmune anti-GBM glomerulonephritis. (C) Renal injury as measured by the urine albumin:creatinine ratio (uACR), showing the improved protective effect of B5 relative to abatacept.

[0213] Sequence information

[0214] A table containing sequence information:

[0215] Table 1: V H CDR sequence

[0216]

[0217] Table 2: V H Frame area

[0218]

[0219] Table 3: V H full sequence

[0220]

[0221] Table 4: V L CDR sequence

[0222]

[0223] Table 5: V L Frame area

[0224]

[0225] Table 6: VL full sequence

[0226] Detailed Implementation

[0227] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or obviously present in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0228] Further aspects of the invention and further embodiments of the aspects described in the foregoing paragraphs will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings.

[0229] Reference will now be made to certain embodiments of the invention. Although the invention will be described in conjunction with embodiments, it should be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined in the claims.

[0230] Those skilled in the art will recognize that many methods and materials can be similar to or equivalent to those described herein, and can be used to practice this invention. This invention is by no means limited to the methods and materials described. It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0231] All patents and publications mentioned in this article are incorporated herein by reference in their entirety.

[0232] For the purposes of interpreting this specification, terms used in the singular will also include the plural form, and vice versa.

[0233] The general chemical terms used in the chemical formulas in this article have their usual meanings.

[0234] The costimulatory ligand CD80 and the inhibitory ligand PD-L1 interact cis-with on the surface of antigen-presenting cells, including dendritic cells (DCs). In the context of the cis-CD80:PD-L1 complex, CD80 can still bind to the costimulatory receptor CD28 on interacting T cells, but PD-L1 cannot bind to the T cell inhibitory receptor PD-1. Therefore, these complexes activate primordial T cells by allowing CD80 on the DC surface to trigger CD28 signaling without PD-L1 inhibition.

[0235] The novel antibody of the present invention is believed to disrupt the cisCD80:PD-L1 complex by binding to CD80 and thereby releasing PD-L1 from CD80. Not wishing to be bound by theory, the inventors believe this is because the antibody binds to an epitope on CD80 near the PD-L1 interaction region. Furthermore, the antigen-binding protein of the present invention is believed to release PD-L1 from the cisCD80:PD-L1 complex more effectively than existing standards of care such as CTLA4-Ig (e.g., abatacept), and this is associated with improved T-cell suppression.

[0236] An additional advantage of certain antibodies of the present invention (e.g., B5 and 19B10) is that, in addition to effectively blocking CD80:PD-L1 interaction, they are also capable of partially disrupting the interaction between CD80 and CD28-Ig. To the knowledge of the inventors, this property of other CD80-binding antibodies (including those that block CD80:PD-L1 interaction) has not been reported, and given that simultaneous blocking of CD80:PD-L1 and partial blocking of CD80:CD28 interaction are expected to synergistically inhibit T cell activation, this property offers potential therapeutic benefits.

[0237] Therefore, the novel antibody of the present invention is considered a useful immunosuppressant in vivo and can be used to treat a range of autoimmune diseases and / or symptoms that require reduction or prevention of inflammation.

[0238] Overview

[0239] Throughout this specification, unless expressly stated otherwise or the context otherwise requires, references to a single step, a composition of substances, a group of steps, or a group of compositions of substances shall be deemed to cover one or more (i.e., one or more) of such steps, compositions of substances, groups of steps, or groups of compositions of substances. Therefore, as used herein, unless the context expressly indicates otherwise, the singular forms “a,” “an,” and “the” include the plural aspect, and vice versa. For example, reference to “a” includes one and two or more; reference to “an” includes one and two or more; reference to “the” includes one and two or more, and so on.

[0240] Those skilled in the art will understand that variations and modifications are readily possible beyond those specifically described. It should be understood that the invention encompasses all such changes and modifications. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, and any and all combinations of any two or more of said steps or features.

[0241] Those skilled in the art will recognize that many methods and materials can be similar to or equivalent to those described herein, and can be used to practice this invention. This invention is by no means limited to the methods and materials described.

[0242] All patents and publications mentioned in this article are incorporated herein by reference in their entirety.

[0243] This invention is not limited to the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are obviously within the scope of this invention.

[0244] Unless otherwise expressly stated, any instance or embodiment of the invention described herein, with necessary modifications, should be considered applicable to any other instance or embodiment of the invention.

[0245] Unless otherwise specified, all technical and scientific terms used herein should be regarded as having the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0246] Unless otherwise stated, the recombinant proteins, cell culture, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained in the following sources: J. Perbal, *A Practical Guide to Molecular Cloning*, John Wiley and Sons (1984); J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Press (1989); TA Brown (ed.), *Essential Molecular Biology: A Practical Approach*, Volumes 1 & 2, IRL Press (1991); DM Glover and BD Hames (ed.), *DNA Cloning: A Practical Approach*, Volumes 1–4, IRL Press (1995 and 1996); and FMAusubel et al. (ed.), *Current Protocols in Molecular Biology*. Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Antibodies: A Laboratory Manual (edited by Ed Harlow and David Lane), Cold Spring Harbour Laboratory (1988); and Current Protocols in Immunology (edited by JE Coligan et al.), John Wiley & Son Publishing (including all updates to date).

[0247] The descriptions and definitions of variable regions and their portions, immunoglobulins, antibodies and their fragments in this article can be further clarified by the definitions provided in this article.

[0248] The term “and / or” (e.g., “X and / or Y”) should be understood to mean “X and Y” or “X or Y”, and should be regarded as providing explicit support for both or either of these meanings.

[0249] As used herein, the term “originating from” should indicate that the specified integer can be obtained from a particular source, although not necessarily directly from said source.

[0250] A series of residues mentioned herein will be understood to be included. For example, the reference to "region containing amino acids 56 to 65" will be understood in an inclusive manner, that is, the region contains the amino acid sequence numbered 56, 57, 58, 59, 60, 61, 62, 63, 64 and 65 in the specified sequence.

[0251] Selected Definitions

[0252] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, due to its origin or derived source, does not associate with the naturally associated components that accompany it in its native state; and is substantially free of other proteins from the same source. Protein purification techniques known in the art can be used to render a protein substantially free of naturally associated components or substantially purified by isolation. "Substantially purified" means that the protein is substantially free of contaminants, for example, at least about 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98% or 99% free of contaminants.

[0253] The term "recombinant" should be understood to refer to the product of artificial genetic recombination. Therefore, in the context of recombinant proteins containing antibody-antigen-binding domains, this term does not cover antibodies naturally present in the subject's body, which are products of natural recombination occurring during B cell maturation. However, if such antibodies are isolated, they are considered isolated proteins containing antibody-antigen-binding domains. Similarly, if nucleic acids encoding proteins are isolated and expressed using recombinant methods, the resulting proteins are recombinant proteins containing antibody-antigen-binding domains. Recombinant proteins also encompass proteins expressed through artificial recombinant methods when they are present in cells, tissues, or within the subject, such as proteins expressed therein.

[0254] The term "protein" should be understood to include a single polypeptide chain, that is, a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or nonvalently linked to each other (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using suitable chemicals or disulfide bonds. Examples of nonvalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0255] The term “polypeptide” or “polypeptide chain” will be understood from the preceding paragraphs to refer to a series of consecutive amino acids linked by peptide bonds.

[0256] As used herein, the term “antigen-binding protein” is used interchangeably with “antigen-binding domain” and should be considered to refer to a region of an antibody capable of specifically binding to an antigen, i.e., VH or VL, or an Fv containing both VH and VL. The antigen-binding domain does not need to be in the context of a complete antibody; for example, it may be in a dissociative form (e.g., a domain antibody) or in another form, such as scFv as described herein.

[0257] For the purposes of this disclosure, the term "antibody" includes a protein capable of specifically binding to one or more closely related antigens via an antigen-binding domain contained within an Fv. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR transplanted antibodies, primate-derived antibodies, deimmunized antibodies, synthetic humanized antibodies, haptens, bispecific antibodies). Antibodies typically contain constant domains that may be arranged as constant regions or constant fragments or crystallizable fragments (Fc). Exemplary forms of antibodies contain a four-chain structure as their basic unit. Full-length antibodies comprise two covalently linked heavy chains (about 50 kDa to 70 kDa) and two light chains (each about 23 kDa). The light chains typically contain a variable region (if present) and a constant domain, and in mammals are κ or λ light chains. The heavy chains typically contain a variable region and one or two constant domains connected to additional constant domains via hinge regions. In mammals, the heavy chain is one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently linked to one of the heavy chains. For example, two heavy chains, as well as heavy and light chains, are held together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary in different types of antibodies. Each chain has an N-terminal variable region (V... H or V L The light chain has one or more constant domains at the C-terminus (each approximately 110 amino acids in length) and approximately 110 amino acids in length. L ) and the first constant structural domain of the heavy chain (C10, which is 330 to 440 amino acids in length) H1 Alignment and disulfide bonding. Alignment of the variable region of the light chain with the variable region of the heavy chain. The antibody heavy chain may contain two or more additional C... H Structural domains (such as C) H2 C H3 (etc.), and can contain C H1 With C H2Hinge regions between constant structural domains. Antibodies can belong to any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one instance, the antibody is a mouse (mouse or rat) antibody or a primate (e.g., human) antibody. In one instance, the antibody heavy chain has lost a C-terminal lysine residue. In one instance, the antibody is humanized, synthetically humanized, chimeric, CDR-transplanted, or deimmunized.

[0258] The terms “full-length antibody,” “intact antibody,” or “whole antibody” are used interchangeably to refer to an antibody in its substantially complete form, rather than an antigen-binding fragment of the antibody. Specifically, a whole antibody includes antibodies having a heavy chain and a light chain that includes an Fc region. The constant domain can be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or a variant of its amino acid sequence.

[0259] As used herein, a “variable region” refers to the portion of the light and / or heavy chain of an antibody that is capable of specifically binding to an antigen, as defined herein, and includes complementarity-determining regions (CDRs); namely CDR1, CDR2, and CDR3, and the amino acid sequence of a frame region (FR). For example, a variable region may contain three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) and three CDRs. H This refers to the variable region of the heavy chain. V L It refers to the variable region of a light chain.

[0260] As used herein, the term "subject" should be understood to mean any animal, including humans, such as mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, a subject is a human.

[0261] Antibodies, or immunoglobulins, or Ig are gamma globulins found in the blood or other bodily fluids of vertebrates. They function in the immune system to bind to antigens, thus identifying and neutralizing foreign substances.

[0262] Antibodies are typically heterotetraglycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by a covalent disulfide bond. Depending on the H chain isotype, the two H chains are interconnected by one or more disulfide bonds. Each H and L chain also has regularly spaced intrachain disulfide bridges.

[0263] The H and L chains define specific Ig domains. More specifically, each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isoforms. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. VL is aligned with VH, and CL is aligned with the first constant domain (CH1) of the heavy chain.

[0264] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes are further subdivided into subclasses; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The L chain from any vertebrate species can be designated as one of two distinct types, known as κ and λ, based on the amino acid sequence of its constant domain.

[0265] The constant domain includes an Fc region containing the carboxyl-terminal portions of two H chains held together by disulfide bonds. The effector function of antibodies (such as ADCC) is determined by the sequence in the Fc region, which is also the portion recognized by Fc receptors (FcRs) present on certain types of cells.

[0266] The pairing of VH and VL together forms a "variable region" or "variable domain," comprising the N-terminal domain of either the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." The V domain contains an antigen-binding protein that influences antigen binding and defines the specificity of a particular antibody for its specific antigen. The V region spans approximately 110 amino acid residues and consists of relatively invariant extensions of framework regions (FRs) (typically about four) of 15–30 amino acids each, separated by highly variable shorter regions (typically about three) of 9–12 amino acids each. The FRs largely adopt a β-sheet configuration, and the hypervariable regions form loops connecting the β-sheet structures and, in some cases, form part of the β-sheet structure.

[0267] "Hypervariant region," "HVR," or "HV" refers to a region of an antibody's variable domain that is sequence-hypervariant and / or forms a structurally defined loop. Typically, an antibody contains six hypervariant regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Many hypervariant region descriptions are used and covered herein.

[0268] As used herein, the term "complementarity-determining region" (synonymous CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the antibody variable region, the presence of which is a major factor in specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1, CDR2, and CDR3. The CDRs of VH are also referred to herein as CDR H1, CDR H2, and CDR H3, respectively, where CDR H1 corresponds to CDR 1 of VH, CDR H2 corresponds to CDR 2 of VH, and CDR H3 corresponds to CDR 3 of VH. (Alternatively, the CDRs may be named HCDR1, HCDR2, and HCDR3). Similarly, the CDRs of VL are referred to herein as CDR L1, CDR L2, and CDR L3, respectively, where CDR L1 corresponds to CDR 1 of VL, CDR L2 corresponds to CDR 2 of VL, and CDR L3 corresponds to CDR 3 of VL. (Alternatively, the CDRs in the variable region of the light chain can be named LCDR1, LCDR2, and LCDR3.)

[0269] “Frame” or “FR” residues are those variable domain residues other than the hypervariable regions or CDR residues defined herein. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively, where FR H1 corresponds to FR 1 of VH, FR H2 corresponds to FR 2 of VH, FR H3 corresponds to FR 3 of VH, and FR H4 corresponds to CDR 4 of VH. Similarly, the FRs of VL are referred to herein as FR L1, FR L2, FR L3, and FR L4, respectively, where FR L1 corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FR L3 corresponds to FR 3 of VL, and FR L4 corresponds to CDR 4 of VL.

[0270] In any embodiment, the amino acid positions assigned to CDR and FR can be defined according to Kabat, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). It should be understood that, according to the present invention, the system used to define FR and CDR is not limited to the Kabat numbering system, but includes all numbering systems, including the standardized numbering systems in the following literature: the numbering systems of Chothia and Lesk, *Journal of Molecular Biology* 196: 901-917, 1987; Chothia et al., *Nature* 342: 877-883, 1989; and / or Al-Lazikani et al., *Journal of Molecular Biology* 273: 927-948, 1997; Honnegher and Plükthun, *Journal of Molecular Biology* 309: 657-670, 2001; or the IMGT system discussed in Giudicelli et al., *Nucleic Acids Res.* 25: 206-211, 1997. In another instance, the amino acid positions assigned to CDRs and FRs can be defined according to the Martin (Enhanced Chothia) numbering scheme (http: / / www.bioinfo.org.uk / abs / info.html). In some instances, CDRs can be defined according to the AbM numbering system. The AbM system represents a compromise between the Kabat and Chothia structural rings and is used through Oxford Molecular's AbM antibody modeling software. In some instances, a CDR can be a "contact" CDR. A "contact" CDR is based on the analysis of available complex crystal structures.

[0271] In one instance, the CDR is defined according to the Kabat numbering system. Optionally, the heavy chain CDR2 according to the Kabat numbering system does not contain the five C-terminal amino acids listed herein, or any one or more of these amino acids are substituted by another naturally occurring amino acid. In this regard, Padlan et al., Journal of the Federation of American Societies of Experimental Biology (FASEB J), 9: 133-139, 1995, demonstrated that the five C-terminal amino acids of the heavy chain CDR2 are generally not involved in antigen binding.

[0272] The different systems used to specify the CDR and FR for antigen-binding domains are well known to those skilled in the art and are summarized in the table below:

[0273]

[0274] 1 Some of these definitions (particularly for the Chothia ring) vary depending on the individual publications examined.

[0275] 2 When using the Kabat numbering convention, the end of the Chothia CDR H1 ring varies between H32 and H34 depending on the ring length. This is because the Kabat numbering scheme places insertions at H35A and H35B: if neither H35A nor H35B exists, the ring terminates at H32; if only H35A exists, the ring ends at H33; and if both H35A and H35B exist, the ring ends at H34.

[0276] "Peptides used to form antigen-binding proteins" generally refer to peptides that can form conformations that confer specificity to antigens on antibodies. Examples include the entire antibody or the entire antibody-associated structure, the entire antibody fragment including variable domains, variable domains and fragments thereof (including light and heavy chains), or fragments including some, but not all, of the light and heavy chains in hypervariable or constant regions.

[0277] A “complete” or “whole” antibody is an antibody that contains an antigen-binding protein as well as CL and at least heavy chain constant domains CH1, CH2, and CH3. The constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0278] "The entire antibody-associated structure" includes the entire antibody in its polymerized form.

[0279] "The entire antibody fragment including variable domains" includes Fab, Fab', F(ab')2 and Fv fragments; bifunctional antibodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0280] The Fab fragment consists of the complete L chain, the variable region domain (VH) of the H chain, and the first constant domain (CHI) of the heavy chain. Each Fab fragment is monovalent in terms of antigen binding, meaning it has a single antigen-binding protein.

[0281] The Fab' fragment differs from the Fab fragment in that it has a small number of additional residues at the carboxyl terminus of the CHI domain, which includes one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the name for Fab' in this paper, where the cysteine ​​residues of the constant domain carry free thiol groups.

[0282] The F(ab')2 fragment roughly corresponds to two Fab fragments linked by disulfide bonds that have divalent antigen-binding activity, and it can still crosslink antigens.

[0283] "Fv" is an antibody fragment containing complete antigen recognition and antigen binding sites. This fragment consists of a dimer of a tightly non-covalently associated heavy chain variable region domain and a light chain variable region domain.

[0284] In single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate into a "dimer" structure similar to the dimer structure in double-chain Fv species. The folding of these two domains yields six hypervariable rings (three rings each in the H and L chains), which provide amino acid residues for antigen binding and impart antigen-binding specificity to the antibody.

[0285] "Single-chain Fv" is also abbreviated as "sFv" or "scFv" and is an antibody fragment containing VH and VL antibody domains that connect to form a single polypeptide chain. Preferably, the scFv polypeptide further includes a polypeptide linker located between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0286] The “single variable domain” is half of the Fv that has the ability to recognize and bind antigens (containing only three CDRs that are specific to the antigen), but with a lower affinity than the complete binding site.

[0287] A "bifunctional antibody" is an antibody fragment with two antigen-binding sites, the fragment containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). Small antibody fragments are prepared by constructing an sFv fragment with a short linker (approximately 5-10 residues) between the VH and VL domains (see previous paragraph), enabling interchain rather than intrachain pairing of the V domains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites.

[0288] Bifunctional antibodies can be divalent or bispecific. A bispecific bifunctional antibody is a heterodimer of two “cross-linked” sFv fragments, wherein the VH and VL domains of the two antibodies are located on different polypeptide chains. Trifunctional and tetrafunctional antibodies are also commonly known in the art.

[0289] "Isolated antibodies" are antibodies that have been identified, isolated, and / or recovered from components of their prior environment. Contaminant components are materials that can interfere with the therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes.

[0290] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies produced by humans and / or having been prepared using any techniques disclosed herein for preparing human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering antigens to transgenic animals that have been modified to produce such antibodies in response to antigen stimulation, but whose endogenous loci have been disabled.

[0291] A “humanized” form of a nonhuman (e.g., rodent) antibody is a chimeric antibody containing a minimal sequence derived from a nonhuman antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a nonhuman species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate, possessing the desired antibody specificity, affinity, and capability. In some cases, frame region (FR) residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies may contain residues not present in either the recipient or donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody will contain all or substantially all of its hypervariable loops corresponding to regions of the nonhuman immunoglobulin and all or substantially all of its FRs being at least one FR of the human immunoglobulin sequence and, typically, substantially all of the two variable domains. Optionally, a humanized antibody will also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region.

[0292] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that individual antibodies comprising this group are identical, except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies exhibit high specificity against a single antigenic site or determinant on an antigen. In addition to their specificity, a key advantage of monoclonal antibodies is that they can be synthesized without contamination from other antibodies. Monoclonal antibodies can be prepared using hybridoma methods or by using recombinant DNA methods in bacterial, eukaryotic, or plant cells. Monoclonal antibodies can also be isolated from phage antibody libraries.

[0293] The monoclonal antibodies discussed in this article include “chimeric” antibodies, in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity. The chimeric antibodies of interest discussed in this article include “primatized” antibodies comprising a variable domain antigen-binding sequence derived from non-human primates (e.g., Old World monkeys, apes, etc.) and a human constant region sequence.

[0294] The terms "anti-CD80 antibody," "CD80-binding antibody," or "CD80-binding protein or antibody" refer to proteins or antibodies capable of binding to CD80 with sufficient affinity, such that the antibody can be used as a diagnostic and / or therapeutic agent targeting proteins or cells that express or present CD80. Preferably, the anti-CD80 antibody binds to unrelated tags or proteins to less than about 10% of the antibody's binding to CD80, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the dissociation constant (Kd) of the CD80-binding antibody is < 1 μM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM.

[0295] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Typically, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between the members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies typically bind antigens slowly and tend to dissociate readily, while high-affinity antibodies typically bind antigens more quickly and tend to remain bound for longer periods. Various methods for measuring binding affinity are known in the art, and any of these methods may be used for the purposes of this invention.

[0296] As used herein, when referring to the interaction between an antigen-binding protein or its antigen-binding domain and an antigen, the term "binding" means that the interaction depends on the presence of a specific structure on the antigen (e.g., an antigenic determinant or epitope). For example, an antibody recognizes and binds to a specific protein structure rather than a protein in general. If an antibody binds to epitope "A", in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the antibody.

[0297] As used herein, the terms “specifically binds” or “binds specifically” should be considered to mean that the antigen-binding proteins of the present invention react or associate with a specific antigen or a cell expressing said antigen more frequently, more rapidly, for a longer duration, and / or with a greater affinity than reacting or associating with alternative antigens or cells.

[0298] As used herein, the term "undetectable binding" should be understood to mean that an antigen-binding protein (e.g., an antibody) binds to a candidate antigen at a level less than 10%, 8%, 6%, or 5% above background. Background can be the level of binding signal detected in the absence of the protein and / or in the presence of a negative control protein (e.g., an allotype control antibody) and / or in the presence of a negative control antigen. Binding levels are detected using biosensor analysis (e.g., Biacore), in which the antigen-binding protein is immobilized and contacted with the antigen.

[0299] As used herein, the term "insignificant binding" should be understood to mean that the binding level of the antigen-binding protein of the present invention to the peptide is not statistically significantly higher than the background level, for example, the binding signal level detected in the absence of the antigen-binding protein and / or in the presence of a negative control protein (e.g., an allotype control antibody) and / or in the presence of a negative control peptide. Binding levels are detected using biosensor analysis (e.g., Biacore), in which the antigen-binding protein is immobilized and contacted with the antigen.

[0300] Affinity-matured antibodies are antibodies with one or more alterations in one or more of their HVRs, which improve the antibody's affinity for the antigen compared to the parent antibody without those alterations. Preferred affinity-matured antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced using procedures known in the art.

[0301] “ADCC” refers to a process known as antibody-dependent cytotoxicity, an immune response in humans primarily mediated by natural killer (NK) cells. In ADCC, FcyRIII on the surface of NK cells recognizes the Fe region of an antibody that binds to an antigen displayed on the surface of a target cell. This activates the NK cells, which release perforin and granzymes, thereby causing lysis and apoptosis of the target cells.

[0302] "CDC" refers to a complex process known as complement-dependent cytotoxicity, which leads to cell killing through a series of protein actions that can function through either of two main pathways.

[0303] "ADCP" refers to a process known as antibody-dependent cell-mediated phagocytosis. In this Fe receptor-mediated process, the target cells to which antibodies bind are phagocytosed by phagocytes (such as macrophages, monocytes, neutrophils, and dendritic cells). Multiple Fc receptors are involved in this process.

[0304] "Blocking" antibodies or "antagonist" antibodies are antibodies that inhibit or reduce the biological activity of the antigens they bind to. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0305] As used in this article, an "agonist antibody" is an antibody that mimics at least one functional activity of the peptide of interest.

[0306] As referred to herein, an "Fc region" is a dimer consisting of two polypeptide chains linked by one or more disulfide bonds, each chain containing a hinge domain plus part or all of the CH2 and CH3 domains. Each of the polypeptide chains is referred to as an "Fc polypeptide chain". To distinguish the two Fc polypeptide chains, one is referred to herein as "chain A" and the other as "chain B". More specifically, the Fc region considered for use in this invention is the IgG Fc region, which may be a mammalian or human IgG1, IgG2, IgG3, or IgG4 Fc region. In the human IgG1 Fc region, at least two allelic types are known.

[0307] As used herein, "Fc-containing protein" refers to a protein that includes an Fc region as described herein and a binding region that binds to a target molecule. The term "Fc-containing protein" also encompasses antibodies or Fc fusion proteins containing an Fc region.

[0308] The phrase “therapeutic effective amount” generally refers to the amount of antigen-binding protein of the present invention, which (i) treats a particular disease, symptom or condition, (ii) reduces, improves or eliminates one or more symptoms of a particular disease, symptom or condition, or (iii) delays the onset of one or more symptoms of a particular disease, symptom or condition described herein.

[0309] The term "treat" or "treatment" refers to therapeutic treatment aimed at alleviating (reducing) undesirable physiological changes or symptoms. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, reduction of disease severity, stable (i.e., non-deterioration) state of disease, delay or slowing of disease progression, improvement or alleviation of disease status, and mitigation (whether partial or overall), whether detectable or undetectable. Treatment can also mean prolonged survival compared to the expected survival without treatment. Treatment does not necessarily have to result in the complete eradication of the disease or symptom, but can reduce or minimize complications and side effects of infection and the progression of the disease or symptom. The success of treatment can be monitored through individual physical examination, cytopathology, serological DNA or mRNA detection techniques, etc.

[0310] The terms “prevent” and “prevention” generally refer to preventive or preventative measures taken to protect or prevent an individual who does not have a given disease or condition from developing that disease or condition.

[0311] The phrase “pharmaceutically acceptable” means that a substance or composition must be chemically and / or toxicologically compatible with other ingredients containing the compound and / or with the mammals treated with the compound.

[0312] Protein mutation

[0313] The present invention also provides an antigen-binding protein or a nucleic acid encoding the antigen-binding protein that has at least 80% identity with the sequence disclosed herein. In one example, the antigen-binding protein or nucleic acid of the present invention comprises at least about 85%, or 90%, or 95%, or 97%, or 98% or 99% sequence identity with the sequence disclosed herein.

[0314] Alternatively or additionally, the antigen-binding protein contains at least about 80%, or 85%, or 90%, or 95%, or 97%, or 98% or 99% of the same CDR as VH or VL as described herein according to any example (e.g., three CDRs).

[0315] In another example, the nucleic acid of the present invention comprises at least about 80%, or 85%, or 90%, or 95%, or 97%, or 98% or 99% identical to the sequence encoding an antigen-binding protein having the function described herein according to any example. The present invention also covers nucleic acids encoding the antigen-binding protein of the present invention that differ from the sequences exemplified herein due to the degeneracy of the genetic code.

[0316] The identity percentage of nucleic acids or peptides was determined by GAP (Needleman and Wunsch. Molecular Biology, 48, 443-453, 1970) analysis (GCG procedure), where a vacancy generation penalty of 5 and a vacancy extension penalty of 0.3 were applied. The query sequence was at least 50 residues long, and the GAP analysis aligned the two sequences over a region of at least 50 residues. For example, the query sequence was at least 100 residues long, and the GAP analysis aligned the two sequences over a region of at least 100 residues. Alternatively, the two sequences were aligned over their entire length.

[0317] This invention also considers nucleic acids that hybridize with nucleic acids encoding antigen-binding proteins described herein under stringent hybridization conditions. “Medium stringency” is defined herein as hybridization and / or washing performed in 2 x SSC buffer, 0.1% (w / v) SDS at temperatures ranging from 45°C to 65°C or equivalent conditions. “High stringency” is defined herein as hybridization and / or washing performed in 0.1 x SSC buffer, 0.1% (w / v) SDS, or lower salt concentrations at temperatures of at least 65°C or equivalent conditions. References to specific levels of stringency herein cover equivalent conditions using wash / hybridization solutions other than SSC known to those skilled in the art. For example, methods for calculating the temperature at which the strands of a double-stranded nucleic acid will dissociate (also referred to as the melting temperature or Tm) are known in the art. Temperatures similar to (e.g., within 5°C or 10°C) or equal to the Tm of the nucleic acid are considered high stringency. Medium stringency is considered to be within 10°C to 20°C or 10°C to 15°C of the calculated Tm of the nucleic acid.

[0318] The present invention also contemplates mutant forms of the antigen-binding protein of the present invention, which contain one or more conserved amino acid substitutions compared to the sequence shown herein. In some instances, the antigen-binding protein contains 10 or fewer, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 conserved amino acid substitutions. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain and / or hydropathicity and / or hydrophilicity.

[0319] Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The hydrophilic index is described, for example, in Kyte and Doolittle, *Journal of Molecular Biology*, 157: 105-132, 1982, and the hydrophylic index is described, for example, in US4554101.

[0320] The present invention also considers non-conservative amino acid alterations. For example, particular interest is found in replacing charged amino acids with another charged amino acid and neutral or positively charged amino acids. In some instances, antigen-binding proteins contain 10 or fewer, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-conservative amino acid substitutions.

[0321] In one instance, the mutation occurs within the FR of the antigen-binding domain of the antigen-binding protein of the present invention. In another instance, the mutation occurs within the CDR of the antigen-binding protein of the present invention.

[0322] Exemplary methods for generating mutant forms of antigen-binding proteins include:

[0323] - DNA mutagenesis (Thie et al., Methods in Molecular Biology 525: 309-322, 2009) or RNA (Kopsidas et al., Immunology Letters 107:163-168, 2006; Kopsidas et al., BMC Biotechnology, 7: 18, 2007; and WO1999 / 058661).

[0324] - Introducing nucleic acids encoding polypeptides into mutant cells, such as XL-1Red, XL-mutS, and XL-mutS-Kanr bacterial cells (Stratagene);

[0325] -DNA shuffling, for example, as disclosed in Stemmer, Nature 370: 389-91, 1994; and

[0326] - Site-directed mutagenesis, for example, as described in Dieffenbach (ed.) and Dveksler (ed.) (PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).

[0327] Exemplary methods for determining the biological activity (e.g., antigen binding) of the mutant antigen-binding proteins of the present invention will be obvious to those skilled in the art and / or described herein. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation, and therapeutic efficacy are described herein.

[0328] As used herein, the properties of amino acids are defined in the following table:

[0329]

[0330] constant region

[0331] This invention covers antigen-binding proteins and / or antibodies comprising constant regions of antibodies as described herein. This includes antigen-binding fragments of antibodies fused to Fc.

[0332] The sequences that can be used to generate the constant regions of the proteins of the present invention can be obtained from many different sources. In some instances, the constant region of the protein, or a portion thereof, is derived from a human antibody. The constant region, or a portion thereof, can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one instance, the constant region is human isotype IgG1 or a stable IgG1 constant region.

[0333] In various embodiments of the invention, the Fc region of the antibody may contain one or more substitutions for altering effector function (including increasing or decreasing effector function) and circulating half-life. Examples of such substitutions and modifications are described in Chapter 1296 of Saunders (2019) Frontiers in Immunology, which is incorporated herein by reference in its entirety.

[0334] In one instance, for example, the Fc region of a constant region has a reduced ability to induce effector function compared to the Fc region of natural or wild-type human IgG1 or IgG3. In one instance, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the effector function levels of proteins containing Fc regions are known in the art and / or described herein.

[0335] In one instance, the Fc region is the IgG4 Fc region (i.e., derived from the IgG4 constant region), such as the human IgG4 Fc region. The sequence of a suitable IgG4 Fc region is obvious to a person skilled in the art and / or available from publicly available databases (e.g., from the National Center for Biotechnology Information).

[0336] In one instance, the constant region is the stable IgG4 constant region. The term "stable IgG4 constant region" will be understood as referring to an IgG4 constant region that has been modified to reduce or undergo Fab arm exchange, or to have a tendency to form a hapten. "Fab arm exchange" refers to a type of protein modification against human IgG4 in which the IgG4 heavy chain and its attached light chain (half a molecule) are exchanged for a heavy-light chain pair from another IgG4 molecule. Thus, the IgG4 molecule can acquire two different Fab arms that recognize two different antigens (producing a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro using purified blood cells or reducing agents such as reduced glutathione. A "hapten" is formed when an IgG4 antibody dissociates to form two molecules, each containing a heavy chain and a light chain.

[0337] In one instance, according to Kabat's system (Kabat et al., Sequences of Immunologically Significant Proteins, Washington DC, United States Department of Health and Human Services, 1987 and / or 1991), the stable IgG4 constant region contains proline at position 241 of the hinge region. This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Immunologically Significant Proteins, Washington DC, United States Department of Health and Human Services, 2001 and Edelman et al., Proceedings of the National Academy of Sciences (Proc. Natl. Science USA), 63, 78-85, 1969). In human IgG4, this residue is typically serine. Following the substitution of proline with serine, the IgG4 hinge region contains the sequence CPPC (SEQ ID NO: 164). In this regard, those skilled in the art will recognize that the "hinge region" is the proline-rich portion of the antibody heavy chain constant region connecting the Fc and Fab regions, which imparts fluidity to the two Fab arms of the antibody. The hinge region comprises cysteine ​​residues involved in the inter-heavy chain disulfide bond. According to the Kabat numbering system, it is generally defined as extending from Glu226 to Pro243 of human IgG1. Hinge regions of other IgG isotypes can be compared with the IgG1 sequence by placing the first and last cysteine ​​residues forming the inter-heavy chain disulfide bond (SS) in the same position (see, for example, WO2010 / 080538).

[0338] Another example of a stable IgG4 antibody is an antibody in which arginine at position 409 in the heavy chain constant region of human IgG4 (according to the EU numbering system) is replaced by lysine, threonine, methionine, or leucine (e.g., as described in WO2006 / 033386). The Fc region of the constant region may additionally or alternatively contain residues selected from the group consisting of alanine, valine, glycine, isoleucine, and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region contains proline at position 241 (i.e., the CPPC sequence, SEQ ID NO: 164) (as described above).

[0339] In another instance, the Fc region is a modified region with reduced effector function, i.e., a “non-immunostimulatory Fc region.” For example, the Fc region is an IgG1 Fc region containing substitutions at one or more sites selected from the group consisting of: 268, 309, 330, and 331. In another instance, the Fc region is an IgG1 Fc region containing the deletion of one or more of the following variations: E233P, L234V, L235A, and G236, and / or one or more of the following variations: A327G, A330S, and P331S (Armour et al., *Eur J Immunol.* 29:2613-2624, 1999; Shields et al., *Journal of Biol Chem.* 276(9):6591-604, 2001). Other examples of non-immunostimulatory Fc regions are described in, for example, the following literature: Dall'Acqua et al., *Journal of Immunology* 177: 1129-1138 2006; and / or Hezareh, *Journal of Virology* 75: 12161-12168, 2001.

[0340] Antibodies with reduced effector function include antibodies having substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (as described in U.S. Patent No. 6,737,056, which is incorporated herein by reference). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant (U.S. Patent No. 7,332,581) which substitutes residues 265 and 297 for alanine. For example, antibody variants may comprise an Fc region with one or more amino acid substitutions that weaken FcγR binding (e.g., substitutions at positions 234 and 235 of the Fc region (EU numbers of the residues)). For example, the substitutions are L234A and L235A (LALA) (see, for example, WO 2012 / 130831). These substitutions may additionally include substitution of the proline residue at position 329, such as the P329G mutation, to prevent it from binding to FcR. Further, alterations may be made in the Fc region resulting in altered (i.e., weakened) C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and in Idusogie et al., *Journal of Immunology* 164: 4178-4184 (2000).

[0341] In some respects, the Fc region includes mutations at complement (C1q) and / or Fcγ receptor (FcγR) binding sites. In some respects, such mutations can render the antibody incapable of antibody-directed cytotoxicity (ADCC) and complement-directed cytotoxicity (CDC). An example of a CDC-deficient antibody is one containing substitutions at one or more of Glu318, Lys320, Pro 329, Pro331, and Lys322 (e.g., K322A), wherein the residues in the Fc region are numbered according to the EU index as described in Kabat et al.

[0342] In another example, the Fc region is a chimeric Fc region, for example, comprising at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgG1 antibody, wherein the Fc region comprises substitutions at one or more amino acid positions selected from the group consisting of (e.g., as described in WO2010 / 085682): 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409, and 427 (EU numbers). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0343] Other modifications

[0344] The present invention also considers further modifications to antibodies or antigen-binding proteins containing Fc regions or constant regions.

[0345] Neonatal Fc receptors (FcRn) are important for the metabolic fate of IgG antibodies in vivo. The function of FcRn is to rescue IgG from lysosomal degradation pathways, resulting in reduced clearance and prolonged half-life. FcRn binds with high affinity to the CH2-CH3 portion of the Fc region of IgG antibodies. The interaction between IgG antibodies and FcRn is pH-dependent and occurs at a stoichiometric ratio of 1:2, meaning that one IgG antibody molecule can interact with two FcRn molecules via its two heavy chain Fc region peptides (see, for example, Huber, AH et al., Journal of Molecular Biology 230 (1993) 1077-1083).

[0346] In some embodiments of the invention, the antibody may contain one or more amino acid substitutions that increase the half-life of the protein. For example, the antibody contains an Fc region containing one or more amino acid substitutions that increase the affinity of the Fc region for neonatal Fc regions (FcRn). For example, the Fc region has increased affinity for FcRn at lower pH (e.g., about pH 6.0) to promote Fc / FcRn binding in endosomes. In one instance, the Fc region has increased affinity for FcRn at about pH 6 compared to affinity at about pH 7.4, which promotes the re-release of Fc into the bloodstream after cell recirculation. These amino acid substitutions can be used to prolong the half-life of the protein by reducing clearance from the blood.

[0347] According to the EU numbering system, exemplary amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US7083784.

[0348] In another embodiment, the antibody comprises one or more amino acid substitutions that reduce the half-life of the protein. For example, the antibody comprises an Fc region containing one or more amino acid substitutions that reduce or decrease the affinity of the Fc region for the neonatal Fc region (FcRn).

[0349] Therefore, the present invention provides an antibody having substitutions in the CH2 and / or CH3 domains of a constant region and including substitutions at one or more of residues His310, His435, His436 and Ile253 (Kabat number), thereby altering the FcRn binding affinity and / or serum half-life of the antibody relative to naturally occurring antibodies.

[0350] In some instances, the amino acid at positions 310 and / or 435 of the antibody may be alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine.

[0351] Preferably, the residue at position 310 is selected from alanine, glutamic acid, or glutamine; or the amino acid residue 435 from the heavy chain constant region is selected from arginine, glutamine, or alanine. In other preferred embodiments, the antibody has an alanine residue at position 310 and a glutamine residue at position 435.

[0352] In a preferred embodiment of the invention, the binding affinity of the modified antibody to FcRn and / or the serum half-life of the modified antibody is reduced by at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times. In a preferred embodiment of the invention, the binding affinity of the modified antibody to FcRn and / or the serum half-life of the modified antibody is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

[0353] Antibodies may also contain amino acid substitutions at residues corresponding to Ser228 and Leu235 in the constant heavy chain region, such as Ser228Pro and / or Leu235Glu.

[0354] Proteins containing antibody-binding domains

[0355] In another embodiment, an antigen-binding protein as described above is provided, wherein the amino acid sequence forming one or more of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 is derived from or in the form of a human sequence.

[0356] Antigen-binding proteins can exist in humanized forms, including non-human (e.g., mouse) and human immunoglobulin sequences. Typically, all sequences of an antigen-binding protein, except for the CDR sequence, are derived from a non-human species, such as mouse, rat, or rabbit. In some cases, the framework residues of an antigen-binding protein can also be non-human. When antigen-binding proteins are provided as complete antibodies, at least a portion of the immunoglobulin constant region (Fc) is typically human, thereby allowing for a variety of human effector functions.

[0357] Methods for humanizing non-human antigen-binding proteins are well known in the art, and examples of suitable processes include Jones et al., (1986) Nature, 321:522; Riechmann et al., (1988) Nature, 332:323; Verhoeyen et al., (1988) Science, 239:1534.

[0358] The variable domains, including the CDR and FR of this invention, can be made less immunogenic by substituting surface-exposed residues, thereby enabling the antibody to act as itself to the immune system. An exemplary method is provided in Padlan, EA, 1991, *Molecular Immunology*, 28, 489. Typically, affinity is preserved because the internal stacking of amino acid residues near the antigen-binding protein remains unchanged, and CDR residues or adjacent residues that typically affect binding properties are not substituted during these processes.

[0359] In another embodiment, an anti-CD80 binding protein, an immunoglobulin variable domain, an antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, a bifunctional antibody, a trifunctional antibody, a linear antibody, a single-chain antibody molecule, or a multispecific antibody as described herein is provided, preferably having a sequence as shown in the table herein.

[0360] In some embodiments, the antigen-binding protein is provided as a single-chain Fv fragment (scFv). Fv and scFv are suitable for reducing nonspecific binding during in vivo use because they have complete binding sites lacking constant regions. Fusion proteins including scFv can be constructed to produce fusion of effector proteins at the amino or carboxyl termini of the scFv.

[0361] In another embodiment, a bifunctional or trifunctional antibody, or other multispecific antibody, comprising the antigen-binding protein described above, is provided. Multispecific antibodies can be assembled using polypeptide domains that allow for polymerization. Examples include the CH2 and CH3 regions of the Fc domain, and the CH1 and Cκ / λ regions. Other naturally occurring protein polymerization domains can be used, including leucine zipper domains (bZIP), helical-loop-helical motifs, Src homology domains (SH2, SH3), EF hands, phosphotyrosine-binding (PTB) domains, or other domains known in the art.

[0362] In another embodiment, a fusion domain or heterologous protein is provided, comprising an antigen-binding protein, an immunoglobulin variable domain, an antibody, dab, scFv, Fab, Fab', F(ab')2, an Fv fragment, a bifunctional antibody, a trifunctional antibody, a linear antibody, a single-chain antibody molecule, or a multispecific antibody as described herein.

[0363] Heterogeneous polypeptides can be recombinantly fused or chemically conjugated to the N or C terminus of the antigen-binding protein of the present invention or a molecule containing said antigen-binding protein.

[0364] Furthermore, the antigen-binding proteins, immunoglobulin variable domains, antibodies, dab, scFv, Fab, Fab', F(ab')2, Fv fragments, bifunctional antibodies, trifunctional antibodies, linear antibodies, single-chain antibody molecules, or multispecific antibodies of the present invention can be modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protective / blocking groups, protein hydrolysis, or linkage with cell ligands or other proteins.

[0365] Further details of the antigen-binding protein of the present invention

[0366] The antigen-binding protein of the present invention can be composed of amino acids linked together by peptide bonds or modified peptide bonds (i.e., peptide isosteres), and can contain amino acids in addition to the 20 amino acids encoded by genes. The antigen-binding protein of the present invention can be modified by natural processes (e.g., post-translational processing) or by chemical modification techniques well known in the art. Such modifications are well described in the basic text and research literature. Modifications can occur anywhere in the antigen-binding protein, including the peptide backbone, amino acid side chains, and amino or carboxyl terminals, or on moieties such as carbohydrates. It should be understood that the same type of modification can be present in the same or different amounts at several sites in a given antigen-binding protein. Furthermore, a given antigen-binding protein can contain many types of modifications. The antigen-binding protein can be branched, for example, as a result of ubiquitination, and it can be cyclic, with or without branching. Circular, branched, and branched-circular antigen-binding proteins can be produced by post-translational natural processes or can be prepared by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamic acid, formylation, γ-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristylation, oxidation, polyethylene glycolation, proteolytic processing, phosphorylation, isopentenylation, racemization, selenylation, sulfation, and transfer-RNA-mediated addition of amino acids to proteins, such as argininoylation and ubiquitination.

[0367] In another embodiment, conjugates are provided in the form of antigen-binding proteins, immunoglobulin variable domains, antibodies, Fab, dab, scFv, bifunctional antibodies, trifunctional antibodies, or fusion proteins as described above, said conjugates being conjugated to cytotoxic agents such as chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., bacterial, fungal, plant, or animal-derived enzyme-active toxins or fragments thereof) or labels such as radioisotopes (i.e., radioactive conjugates). On the other hand, the invention further provides methods of using immunoconjugates. In one aspect, the immunoconjugate comprises any of the aforementioned variable domains covalently attached to a cytotoxic agent or a detectable agent.

[0368] In another embodiment, an antibody is provided for binding to antigen-binding proteins, immunoglobulin variable domains, antibodies, dab, scFv, Fab, Fab', F(ab')2, Fv fragments, bifunctional antibodies, trifunctional antibodies, linear antibodies, single-chain antibody molecules or multispecific antibodies, fusion proteins or conjugates as described above.

[0369] In another embodiment, a nucleic acid is provided that encodes an antigen-binding protein, an immunoglobulin variable domain, an antibody, dab, scFv, Fab, Fab', F(ab')2, an Fv fragment, a bifunctional antibody, a trifunctional antibody, a linear antibody, a single-chain antibody molecule or a multispecific antibody, a fusion protein or a conjugate as described above.

[0370] The polynucleotide encoding a CDR or FR according to any of the general formulas described above, or an antigen-binding protein containing said polynucleotide, can be generated from nucleic acids from any source, such as by chemical synthesis or isolation from cDNA or genomic libraries. For example, a cDNA library can be generated from antibody-producing cells such as B cells, plasma cells, or hybridoma cells, and the relevant nucleic acids can be isolated by PCR amplification using oligonucleotides targeting the specific clone of interest. The isolated nucleic acids can then be cloned into a vector using any method known in the art. The relevant nucleotide sequence can then be mutagenized using methods known in the art, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al., ed., 1998, The Laboratory Manual of Contemporary Molecular Biology, John Wiley & Sons, NY, to produce antigen-binding proteins with different amino acid sequences, for example, by producing amino acid substitutions, deletions, and / or insertions.

[0371] connector

[0372] In any embodiment, the VH and VL regions of the antigen-binding domain as described herein can be linked together via a linker. Furthermore, antigen-binding fragments of antibodies (such as scFv) and their variants can be linked to the Fc region via linkers. The following sections describe linkers that can be used to link any peptide described herein.

[0373] The term "linker" is used to refer to a polypeptide comprising two or more amino acid residues linked by peptide bonds and used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proceedings of the National Academy of Sciences 90:6444-6448; Poljak, RJ et al. (1994) Structure 2:1121-1123). Various linkers can be used in some of the embodiments described herein to covalently link the Fc region to a fusion partner.

[0374] The term "linker" as used herein is also referred to as "linker sequence," "spacer," "tethered sequence," or their grammatical equivalents. Bifunctional or heterobifunctional linkers are well known (see the 1994 Pierce Chemical Company catalog, section on crosslinking agent technology, pp. 155-200, which is incorporated herein by reference in its entirety). Many strategies can be used to covalently link molecules together. These strategies include, but are not limited to, polypeptide bonds between the N-terminus and C-terminus of proteins or protein domains, bonds via disulfide bonds, and bonds via chemical crosslinking agents. In one aspect of this embodiment, the linker is a peptide bond generated through recombinant technology or peptide synthesis. The linker peptide may primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length sufficient to link the two molecules in a manner that allows them to present the correct conformation relative to each other, thereby retaining the desired activity. In one embodiment, the linker length is from about 1 to 50 amino acids, preferably from about 1 to 30 amino acids. In one embodiment, a linker length of 1 to 20 amino acids can be used. Useful linkers include glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers, wherein the glycine-serine polymers include, for example, (GS)n, (GSGGS)n (SEQ ID NO: 154), (GGGGS)n (SEQ ID NO: 155), and (GGGS)n (SEQ ID NO: 156), where n is an integer of at least one. Alternatively, various non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene, or copolymers of polyethylene glycol and polypropylene glycol, can be used as linkers. The fusion protein of the present invention may include a linker region (or spacer) located between the first and second portions.

[0375] Linkers are typically peptides with a length of up to 20 amino acids. The terms "linked to" or "fused to" refer to a covalent bond (e.g., a peptide bond) formed between two parts. Therefore, in the context of this invention, the length of a linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 or more amino acids.

[0376] Linkers can be flexible (e.g., linkers containing repeating sequences of glycine and serine residues), rigid (e.g., linkers containing glutamic and lysine residues flanking alanine repeating sequences), and / or cleavable (e.g., sequences susceptible to protease cleavage). Examples of such linkers are known to those skilled in the art and are described, for example, in Chen et al., (2013) Advanced Drug Delivery Reviews, 65:1357-1369.

[0377] In some aspects, peptide linkers can include glycine and serine amino acids of various lengths and combinations. In some aspects, peptide linkers can include the sequences Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS, SEQ ID NO: 156), or Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 155), as well as their variants or repeating sequences. In some aspects, peptide linkers can include the amino acid sequence GGGGGS (a linker of 6 amino acids in length, SEQ ID NO: 157) or even longer. The linker can be a series of repeating glycine and serine residues of different lengths (GS), i.e., (GS). n , where n is any number from 1 to 15 or greater. For example, the connector can be (GS)3 (i.e., GSGSGS, SEQ ID NO: 158) or longer (GS). 11 (SEQ ID NO: 159) or longer. It should be understood that n can be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or greater. Fusion proteins with linkers of this length are included within the scope of this invention. Similarly, the linker can be a series of repeating glycine residues separated by serine residues. For example, (GGGGS)3 (i.e., the linker can contain the amino acid sequence GGGGSGGGGSGGGGS, (G4S)3, SEQ ID NO: 160) and its variants.

[0378] Peptide linkers can consist of a series of Thr-Pro (TP) repeat sequences comprising one or more additional amino acid N-termini and C-termini. For example, a linker can comprise or consist of the sequence GTPTPTPTPTGEF (SEQ ID NO: 161) (also known as the TP5 linker). In another aspect, the linker can be a short and / or α-helical rigid linker (e.g., A(EAAAK)3A, SEQ ID NO: 162; PAPAP, SEQ ID NO: 163; or a dipeptide, such as LE).

[0379] In some respects, the adapter can be flexible and cleavable. Such adapters preferably contain one or more recognition sites for proteases to enable cleavage.

[0380] Preferred linkers may comprise sequences derived from the antibody hinge region. Hinge region sequences from any antibody isotype can be used, including, for example, hinge sequences from IgG1, IgG2, IgG3, and / or IgG4. The linker sequence may also comprise any sequence of any length of the CL / CH1 domain, not all residues of the CL / CH1 domain; for example, the first 5-12 amino acid residues of the CL / CH1 domain. The linker may be derived from any isotype of immunoglobulin heavy chain, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. The linker may be derived from immunoglobulin light chains, such as Cκ or Cλ. The linker sequence may also be derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), hinge region-derived sequences, and other natural sequences from other proteins.

[0381] Protein production

[0382] In another embodiment, a method for generating an anti-CD80 antigen-binding protein as described above is provided, the method comprising expressing the nucleic acid as described above in cells or non-human animals.

[0383] The generation of the antigen-binding protein of the present invention typically requires an expression vector containing a polynucleotide encoding the antigen-binding protein of the present invention. The polynucleotide encoding the antigen-binding protein of the present invention can be obtained using recombinant DNA technology and subcloned into a vector for generating the antigen-binding protein, using techniques well known in the art (including those described herein). Many different expression systems are considered, including those using mammalian cells (including human cells) to generate and secrete the antigen-binding protein. Examples of cells include the 293F, CHO, and NSO cell lines.

[0384] Expression vectors containing protein-coding sequences and appropriate transcription and translation control signals can be constructed using methods known in the art. These include in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. In some embodiments, a reproducible vector is provided having nucleic acid encoding an antigen-binding protein operatively linked to a promoter.

[0385] Cells transfected with expression vectors can be cultured using conventional techniques to produce antigen-binding proteins. Therefore, in some embodiments, host cells or cell transfectants containing polynucleotides encoding the antigen-binding proteins of the present invention operatively linked to a promoter are provided. The promoter can be heterologous. Various host expression vector systems can be utilized, and in some systems, the transcription machinery of the vector system is particularly well-matched to the host cell. For example, mammalian cells such as Chinese hamster ovary cells (CHO) can be transfected with vectors comprising promoter elements of major intermediate early genes derived from human cytomegalovirus. Additionally or alternatively, host cells that regulate the expression of the inserted sequence or modify and process gene products (including various forms of post-translational modifications) as needed can be used. Examples of mammalian host cells with specific post-translational modification processes include CHO, VERY, BHK, HeIa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NSO, CRL7O3O, and HsS78Bst cells.

[0386] Many bacterial expression vectors can be advantageously selected depending on their intended use for protein molecules. In one instance, when producing large quantities of antigen-binding proteins, vectors that result in easily purified fusion protein products leading to high levels of expression can be used, such as the E. coli expression vector pUR278. The expression product can be produced as a fusion protein with lacZ. Other bacterial vectors include pIN vectors, etc. pGEX vectors can also be used to express exogenous peptides as fusion proteins with glutathione-S-transferase (GST). These fusion proteins are generally soluble and can be easily purified from lysed cells by adsorption and binding to a glutathione-agarose affinity matrix, followed by elution in the presence of free glutathione. Thrombin and / or factor Xa protease cleavage sites can be provided in the expressed peptide so that the cloned target gene product can be released from the GST moiety.

[0387] Alfalfa silver-striped armyworm nucleopolyhedrovirus (AcNPV) can be used as a vector to express exogenous genes in insect systems including Spodoptera frugiperda cells. The specific promoter used can depend on the location where the protein code is inserted into the sequence. For example, the sequence can be cloned separately into the polyhedromic protein gene and placed under the control of the polyhedromic protein promoter.

[0388] Virus-based expression systems can be used in mammalian cells, such as adenoviruses, whereby the coding sequence of interest can be linked to the adenovirus late promoter and triple leader sequence. This chimeric gene can then be inserted into the adenovirus genome using in vitro or in vivo recombination. Insertion into region E1 or E3 will produce a live recombinant virus capable of expressing antigen-binding proteins in infected host cells. Specific initiation signals, including the ATG start codon and neighboring sequences, are likely required for efficient translation of the inserted antigen-binding protein coding sequence. Initiation and translation control signals, as well as codons, can be obtained from various sources, both natural and synthetic. Transcriptional enhancer elements and transcription terminators can be used to enhance the expression efficiency of virus-based systems.

[0389] When long-term, high-yield production of recombinant proteins is required, stable expression is preferred. Typically, selectable marker genes are used, whereby after transfection, cells are grown in enrichment medium for 1-2 days and then transferred to a selective medium in which cells containing the corresponding selectable marker, such as antibiotic resistance, can be screened. As a result, cells with the plasmid stably integrated into their chromosome grow and form lesions, which can then be cloned and amplified into cell lines. Herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes are examples of genes that can be used for tk-cells, hgprt-cells, or aprT-cells, respectively, thereby providing a suitable selection system. The following genes: dhfr, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to aminoglycoside G-418; and hygro, which confers resistance to hygromycin, are examples of genes that can be used for antimetabolite selection systems.

[0390] The antigen-binding protein of the present invention can be purified by a recombinant expression system using known methods, including ion exchange chromatography, affinity chromatography (especially affinity for specific antigen protein A or protein G), gel filtration chromatography, centrifugation, differential solubility, or any other standard technique used for protein purification. Purification can be facilitated or aided by providing the antigen-binding protein in fusion protein form.

[0391] Large quantities of the antigen-binding proteins of the present invention can be produced in a research laboratory using a scale-up process starting from a pilot-scale expression system, the scale-up being extended to analytical-scale bioreactors (typically from 5 L to about 50 L bioreactors) or production-scale bioreactors (e.g., but not limited to 75 L, 100 L, 150 L, 300 L, or 500 L). Desired scale-up processes include those where low to undetectable aggregation levels exist, typically no more than 5% by weight to no more than 0.5% by weight of protein aggregation, as measured by HPSEC or rCGE. Additionally or alternatively, in the scale-up process, fragmentation based on undetectable levels measured according to the total peak area representing the intact antigen-binding protein may be desired, such that at least 80% and up to 99.5% or higher of the total peak area represents the intact antigen-binding protein. In other embodiments, the scale-up process of the present invention produces antigen-binding proteins at a production efficiency of about 10 mg / L to about 300 mg / L or higher.

[0392] Various techniques have been developed for generating antibody fragments, including proteolytic digestion of intact antibodies and recombinant expression in host cells. Regarding the latter, as described below, Fab, Fv, and scFv antibody fragments can all be expressed and secreted in *E. coli*, antibody fragments can be isolated from antibody phage libraries, and Fab'-SH fragments can be directly recovered from *E. coli* and chemically coupled to form F(ab')2 fragments. In another approach, F(ab')2 fragments are isolated directly from recombinant host cell cultures.

[0393] In another embodiment, a vector comprising the aforementioned nucleic acid is provided. The vector may be in the form of, for example, plasmids, granules, viral particles, or bacteriophages. A suitable nucleic acid sequence can be inserted into the vector using various procedures. Typically, DNA is inserted into a suitable restriction endonuclease site using techniques known in the art. Vector components typically include, but are not limited to, one or more of the following: signal sequences, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. The construction of suitable vectors containing one or more of these components employs standard ligation techniques known to those skilled in the art.

[0394] Antigen-binding sites can be generated not only through direct recombination but also through recombination with heterologous polypeptides, which can be signal sequences or other polypeptides having specific cleavage sites at the N-terminus of mature proteins or polypeptides. Typically, the signal sequence can be a component of a vector, or it can be a portion of the DNA encoding the antigen-binding site inserted into the vector. The signal sequence can be, for example, a prokaryotic signal sequence selected from the group consisting of alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leader sequences. For yeast secretion, the signal sequence can be, for example, a yeast invertase leader sequence, an α-factor leader sequence, an acid phosphatase leader sequence, or a Candida albicans glucosylamylase leader sequence. In mammalian cell expression, mammalian signal sequences can be used to guide protein secretion, such as signal sequences of secretory polypeptides from the same or related species, and viral secretion leader sequences.

[0395] The polynucleotide sequence encoding the polypeptide component of the antigen-binding protein of the present invention can be obtained using standard recombinant techniques as described above. The polynucleotide can be synthesized using a nucleotide synthesizer or PCR. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing the heteropolynucleotide in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of this invention. The selection of a suitable vector will depend primarily on the size of the nucleic acid inserted into the vector and the specific host cell transformed with the vector. Each vector contains various components depending on its function (amplification or expression of the heteropolynucleotide or both) and its compatibility with the specific host cell in which it resides.

[0396] Typically, plasmid vectors containing replicons and control sequences derived from species compatible with the host cell are used in conjunction with these hosts. Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, as well as marker sequences that provide phenotypic selection in transformed cells. Such sequences are well known for a wide variety of bacteria, yeasts, and viruses. Origin of replication from plasmid pBR322 (which contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides a simple means of identifying transformed cells) is suitable for most Gram-negative bacteria, 2 μm plasmid origins are suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) can be used for cloning vectors in mammalian cells. pBR322, its derivatives, or other microbial plasmids or phages may also contain or be modified to contain promoters that can be used by microbial organisms to express endogenous proteins.

[0397] In addition, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors that bind to these hosts. For example, bacterial phages (such as λGEM.TM.-11) can be used to prepare recombinant vectors that can be used to transform susceptible host cells (such as Escherichia coli LE392).

[0398] The expression vector of the present invention may contain two or more promoter-cistron pairs (cistrons are DNA segments containing all the information needed to produce a single polypeptide). A promoter is an untranslated regulatory sequence located upstream (5') of a cistron that regulates cistron expression. Prokaryotic promoters are typically classified into two categories: inducible and constitutive. Inducible promoters are promoters that initiate an increased transcriptional level of cistron under their control in response to changes in culture conditions (e.g., the presence or absence of nutrients, or changes in temperature).

[0399] A large number of promoters recognized by various potential host cells are well known. By removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention, the selected promoter can be operatively linked to cistrans DNA encoding either the light or heavy chain. Both native promoter sequences and many heterologous promoters can be used to direct the amplification and / or expression of target genes. In some embodiments, heterologous promoters are used because they generally allow for greater transcription and higher yields of the expressed target gene compared to native target polypeptide promoters.

[0400] Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the PhoA promoter, β-galactanase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and hybrid promoters such as the tac or trc promoters. Promoters for bacterial systems will also contain a Shain-Dalgano (SD) sequence operatively linked to the DNA encoding the antigen-binding protein of the present invention. However, other promoters functional in bacteria (such as other known bacterial or bacteriophage promoters) are also suitable. Their nucleotide sequences have been disclosed, thereby enabling those skilled in the art to operatively link them to the cistrans molecules encoding the target light and heavy chains using adapters or integrators to supply any desired restriction sites.

[0401] In one aspect of the invention, each cistron within the recombinant vector contains a secreted signal sequence component that directs the translocation of the expressed polypeptide across the membrane. Typically, the signal sequence can be a component of the vector, or it can be a portion of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of this invention should be a signal sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and process the native signal sequence of the heterologous polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected from, for example, the group consisting of: alkaline phosphatase, penicillinase, Ipp or heat-stable enterotoxin II (STII) leader sequence, LamB, PhoE, PeIB, OmpA, and MBP. In one embodiment of the invention, the signal sequences used in the two cistrons of the expression system are STII signal sequences or variants thereof.

[0402] On the other hand, the production of immunoglobulins according to the invention can occur in the cytoplasm of host cells, and therefore does not require the presence of a secretory signal sequence in each cistron. In this regard, the immunoglobulin light and heavy chains are expressed, folded, and assembled in the cytoplasm to form functional immunoglobulins. Certain host strains (e.g., *Escherichia coli* trxB strain) provide cytoplasmic conditions favorable for disulfide bond formation, thereby allowing for the proper folding and assembly of the expressed protein subunits.

[0403] This invention provides an expression system in which the quantitative ratio of expressed polypeptide components can be adjusted to maximize the yield of secreted and correctly assembled antigen-binding proteins of the invention. Such adjustment is achieved, at least in part, by simultaneously modulating the translational intensity of the polypeptide components.

[0404] For expression in eukaryotic host cells, vector components typically include, but are not limited to, one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer element, promoter, and transcription termination sequence.

[0405] Vectors used in eukaryotic host cells may also contain a signal sequence or other polypeptides having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The selected heterologous signal sequence is preferably a heterologous signal sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In mammalian cell expression, mammalian signal sequences, as well as viral secretion leader sequences (e.g., herpes simplex virus gD signal), are available.

[0406] The DNA used for this type of precursor region is linked to the DNA encoding the antibody within the reading frame.

[0407] Typically, mammalian expression vectors do not require a replication origin component. For example, the SV40 origin can typically be used simply because it contains an early promoter.

[0408] Expression and cloning vectors typically contain selection genes, also known as selectable markers. Typical selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline); (b) supplement auxotrophic deficiencies; or (c) supply key nutrients that cannot be obtained from complex culture media, such as genes encoding D-alanine racemic enzymes in Bacilli.

[0409] One example of a selection strategy involves using drugs to inhibit the growth of host cells. Cells successfully transformed with a heterologous gene produce drugs-resistant proteins and thus withstand the selection strategy. Examples of this type of dominant selection use drugs such as neomycin, mycophenolic acid, and hygromycin.

[0410] Examples of suitable selectable markers for mammalian cells are those capable of identifying cells that can absorb nucleic acids encoding antigen-binding proteins, such as DHFR or thymidine kinase, metallothionein I and II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc. When using wild-type DHFR, suitable host cells are DHFR-deficient CHO cell lines (e.g., ATCC CRL-9096) that are prepared and propagated. For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx) (a competitive antagonist) containing DHFR. Alternatively, host cells transformed or co-transformed with a DNA sequence encoding an antibody, wild-type DHFR protein, and another selectable marker (such as aminoglycoside 3'-phosphotransferase (APH)) (especially wild-type hosts containing endogenous DHFR) can be selected by cell growth in a medium containing a selector against the selectable marker (such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418).

[0411] Expression and cloning vectors typically contain promoters that are operatively linked to a nucleic acid sequence encoding an antigen-binding protein to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well-known.

[0412] Eukaryotic genes typically have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Another sequence found 70 to 80 bases upstream of the transcription start site in many genes is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence, which can be a signal used to add a poly-A tail region to the 3' end of the coding sequence. All these sequences are appropriately inserted into eukaryotic expression vectors.

[0413] Examples of suitable promoter sequences for use with a yeast host include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, including enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0414] Other yeast promoters (which are inducible promoters with the additional advantage of transcription controlled by growth conditions) are the promoter regions of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, nitrogen metabolism-related degradative enzymes, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the utilization of maltose and galactose.

[0415] Antigen-binding proteins are transcribed from vectors in mammalian host cells under controlled conditions, for example, by promoters obtained from: viral genomes, such as polyomaviruses, fowlpox viruses, adenoviruses (e.g., adenovirus 2), bovine papillomaviruses, avian sarcoma viruses, cytomegaloviruses, retroviruses, hepatitis B viruses, and simian virus 40 (SV40); heterologous mammalian promoters, such as actin promoters or immunoglobulin promoters; and heat shock promoters, provided that such promoters are compatible with the host cell system.

[0416] The transcription of DNA encoding antigen-binding proteins can be increased in higher eukaryotes by inserting enhancer sequences into vectors. Enhancer sequences include those known from mammalian genes (globulins, elastases, albumins, alpha-fetoproteins, and insulin). However, enhancers from eukaryotic viruses are typically used. Examples include the SV40 enhancer (bp 100-270) located post-OMI, the cytomegalovirus early promoter enhancer, the polyoma enhancer located post-OMI, and the adenovirus enhancer.

[0417] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for terminating transcription and stabilizing the mRNA. These sequences are typically derived from the 5' and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed into polyadenylated fragments in the untranslated portion of mRNA encoding antigen-binding proteins.

[0418] In another embodiment, a cell comprising the vector or nucleic acid described above is provided. The nucleic acid molecule or vector may exist as an independent molecule outside the genome, preferably as a replicable molecule in a genetically modified host cell or host, or the nucleic acid molecule or vector may be stably integrated into the genome of the host cell or host.

[0419] The host cell of this invention can be any prokaryotic or eukaryotic cell.

[0420] Examples of prokaryotic cells are those commonly used for cloning, such as *Escherichia coli* or *Bacillus subtilis*. Eukaryotic cells, on the other hand, include, for example, fungal or animal cells.

[0421] Suitable examples of fungal cells are yeast cells, preferably yeast cells of the genus *Saccharomyces* and most preferably yeast cells of the species *Saccharomyces cerevisiae*.

[0422] Examples of animal cells include insect cells, vertebrate cells, and preferably mammalian cells such as HEK293, NSO, CHO, MDCK, U2-OS, HeLa, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, and C33A. These host cells (e.g., CHO cells) can provide post-translational modifications to the antibody molecules of the present invention, including leader peptide removal, folding and assembly of the H (heavy) and L (light) chains, glycosylation of the molecule on the correct side, and secretion of functional molecules.

[0423] Other suitable cell lines known in the art can be obtained from cell line collections such as the American Type Culture Collection (ATCC).

[0424] In another embodiment, an animal comprising the cells described above is provided. In some embodiments, the transgenic animal and its tissues can be used to generate the antigen-binding protein of the present invention. The introduction of nucleic acid molecules as transgenics into a non-human host and their subsequent expression can be used to generate antigen-binding proteins; for example, expressing such transgenics in the milk of a transgenic animal provides a means of obtaining a quantifiable antigen-binding protein. In this regard, useful transgenics comprise nucleic acid molecules of the present invention operatively linked to promoter and / or enhancer structures from mammary gland-specific genes such as casein or β-lactoglobulin, such as coding sequences for the antigen-binding proteins described herein. The animal can be a non-human mammal, most preferably a mouse, rat, sheep, calf, dog, monkey, or ape.

[0425] Binding to target antigen

[0426] Methods for determining the successful binding of the antigen-binding protein of the present invention to its target antigen (i.e., CD80) are well known in the art. Non-limiting examples of such methods are described in the embodiments herein. Methods for confirming the specificity and binding affinity of the antigen-binding protein include the use of Western blotting, ELISA, immunohistochemistry, and Biacore methods, all of which are within the skill of those skilled in the art.

[0427] Binding to CD80 and release of PD-L1 cis-binding to CD80

[0428] It should be understood that, in preferred embodiments, after the antigen-binding protein of the present invention binds to CD80, PD-L1 is prevented (or partially prevented) from binding to CD80 (or partially or completely released from binding to CD80), such that substantially all PD-L1 molecules do not bind cis to CD80 (and therefore bind freely to PD-1). This contrasts with the binding of prior art anti-CD80 antibodies, which may not release PD-L1, such that after binding, PD-L1 remains substantially cis-bound to CD80. This document in [the context of the previous text is missing]. Figure 1 Section B provides a schematic diagram of the graded (partial) release of PD-L1 compared to non-release of PD-L1. As shown, binding of abatacept (a prior art CD80 binder containing CTLA4-Fc) provides only partial release of PD-L1 from CD80, while antigen-binding proteins B5, 19B10 (and also 2B29, 2B30, and TKMF5) promote complete release of PD-L1 (compared to abatacept). Furthermore, the inventors believe that the extent of PD-L1 release provided by abatacept generally depends on the ratio of the number of PD-L1 to CD80 molecules on the cell surface. In contrast, the antigen-binding proteins described herein that release PD-L1 binding are generally independent of this ratio.

[0429] As used herein, the terms “liberation” and “release” in relation to the interaction between CD80 and PD-L1 are used interchangeably. The terms will be understood to refer to a reduction in the molecular interaction between CD80 and PD-L1 such that, upon “liberation” or “release,” PD-L1 no longer binds to CD80 on the cell surface (and thus makes PD-L1 available for binding to PD-1).

[0430] As used herein, "maintaining substantially cis-binding to CD80" means the minimum possible liberation / release of PD-L1 from its binding to CD80. For example, "maintaining substantially cis-binding to CD80" indicates that, after binding to the antigen-binding protein described herein, no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of PD-L1 is liberated (i.e., released) from its cis-binding to CD80. Typically, the degree of binding can be compared to abatacept.

[0431] When PD-L1 is partially released from its cis-binding with CD80, the percentage of released PD-L1 molecules is no more than 15%, no more than 20%, no more than 25%, no more than 30%, no more than 40%, no more than 50%, no more than 60%, no more than 70%, or no more than 80%, preferably no more than 50%. Typically, the degree of binding can be compared with abatacept.

[0432] As used herein, "substantially released from CD80" means the complete or near-complete release of PD-L1 from its binding to CD80. Preferably, "substantially released from CD80" indicates that after the antigen-binding protein of the present invention binds to CD80, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, or no more than 2% of PD-L1 remains cis-bound to CD80. Preferably, "substantially released from CD80" indicates that after the antigen-binding protein of the present invention binds to CD80, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, or less than 2% of PD-L1 remains cis-bound to CD80. In other words, "substantially released from CD80" means that after the antigen-binding protein described herein binds to CD80, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or all of PD-L1 is released from CD80. Typically, the degree of binding can be compared to abatacept.

[0433] Those skilled in the art will be familiar with the methods used to determine the binding of any antigen-binding protein of the present invention to CD80, including any of the methods described herein in the examples.

[0434] Furthermore, those skilled in the art will be able to determine the binding of antigen-binding proteins to CD80, including whether the protein of the present invention thereby promotes the release of PD-L1 from its binding to CD80. In other words, determining whether PD-L1 remains bound to CD80 after the protein of the present invention has bound to CD80, including compared to other known CD80-binding proteins, will be within the capabilities of those skilled in the art. Such methods are also described in the examples herein.

[0435] Further examples of methods for determining the extent of protein binding to CD80 and / or PD-L1 release when a protein binds to CD80 are described in the following references: Sugiura et al., (Nature Immunol. 2022, 23: 399-410); Tekguc et al., (Proceedings of the National Academy of Sciences (PNAS), 2021; 119(30)e2023739117); and Oxley et al., (2024) Cell Reports, 43:114834, each of which is incorporated herein by reference.

[0436] Composition

[0437] The antigen-binding protein of the present invention can be provided in pharmaceutically acceptable compositions for administration to individuals in need. For example, the antigen-binding protein prepared according to the present invention can be used to treat various conditions requiring immunosuppression, such as various inflammatory diseases or conditions.

[0438] Pharmaceutical compositions having been considered, in which the antigen-binding protein disclosed herein is formulated and one or more additional therapeutic agents, are also considered. Formulations of the antigen-binding protein disclosed herein are prepared for storage by mixing the antigen-binding protein, having a desired degree of purity, with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A., 1980, the entire reference is incorporated herein by reference) in the form of a lyophilized formulation or an aqueous solution.

[0439] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used, and include buffers such as phosphates, citrates, acetates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin or gelatin; and hydrophilic polymers. Such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrose; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; sweeteners and other flavoring agents; fillers such as microcrystalline cellulose, lactose, corn, and other starches; binding agents; additives; colorants; salt-forming counterions such as sodium; metal complexes (e.g., Zn protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0440] In one embodiment, a pharmaceutical composition comprising the antigen-binding protein disclosed herein may be in a water-soluble form, such as as a pharmaceutically acceptable salt, intended to include both acid and base addition salts. A “pharmaceutically acceptable acid addition salt” refers to a salt formed from inorganic and organic acids that retains the biological effectiveness of the free base and is undesirable non-biologically or otherwise. The inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; the organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. A “pharmaceutical acceptable base addition salt” includes base addition salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. Some embodiments include at least one of ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Formulations intended for in vivo administration can be sterile. This can be easily achieved through filtration using sterile membranes or other methods.

[0441] The antigen-binding proteins disclosed herein can also be formulated into immunoliposomes. Liposomes are small vesicles containing various types of lipids, phospholipids, and / or surfactants, which can be used to deliver therapeutic agents to mammals. Liposomes containing antigen-binding proteins are prepared using methods known in the art. The components of liposomes are typically arranged in a bilayer, similar to the lipid arrangement of biological membranes. Particularly useful liposomes can be produced by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with defined pore sizes to produce liposomes with desired diameters.

[0442] Antigen-binding proteins and other therapeutic agents can also be encapsulated in microcapsules prepared by methods including, but not limited to, coagulation techniques, interfacial polymerization (e.g., using hydroxymethyl cellulose or gelatin microcapsules, or poly-(methyl methacrylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), and macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. editor, 1980, which is incorporated herein by reference in its entirety. Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer, said matrix being in the form of a molded article, such as a membrane or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot® (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), poly-D-(-)-3-hydroxybutyric acid, and ProLease® (commercially available from Alkermes), a microsphere-based delivery system composed of desired bioactive molecules incorporated into a poly(DL-lactide-co-glycolic acid) (PLG) matrix.

[0443] Treatment

[0444] The antigen-binding proteins disclosed herein are applicable to a wide range of products. In one embodiment, the antigen-binding proteins disclosed herein are therapeutic agents, diagnostic agents, or investigational agents. The antigen-binding proteins can be used in monoclonal or polyclonal compositions. The antigen-binding proteins disclosed herein can be used for therapeutic purposes. The antigen-binding proteins can be administered to patients to treat conditions.

[0445] "Patients" for the purposes disclosed herein include humans and other animals, such as other mammals. Therefore, the antigen-binding proteins disclosed herein have both human therapeutic and veterinary applications. As disclosed herein, the terms "treatment" or "treating" refer to both therapeutic treatment and preventative measures targeting a disease or condition. Thus, for example, successful administration of an antigen-binding protein before the onset of a disease constitutes treatment of the disease. As another example, successful administration of an optimized antigen-binding protein to combat the symptoms of a disease after the clinical manifestation of the disease constitutes treatment of the disease. "Treatment" and "treating" also encompass the administration of an optimized antigen-binding protein after the onset of a disease to eradicate it. Successful administration of an agent after the onset of the disease and after the appearance of clinical symptoms, which may alleviate clinical symptoms and may improve the disease, constitutes treatment of the disease. Those "requiring treatment" include mammals that already have the said disease or condition, as well as those predisposed to having the said disease or condition, including those for which prevention of the said disease or condition is desired.

[0446] The antigen-binding proteins described herein are preferably used to treat diseases or conditions in which it may be desirable to suppress the immune response.

[0447] The antigen-binding proteins described herein are preferably used to treat immune-related conditions or illnesses. Immune-related conditions include, but are not limited to, autoimmune diseases, inflammatory conditions, and the prevention of immune responses associated with donor tissue rejection.

[0448] The antigen-binding proteins described in this article can be used to treat autoimmune diseases. "Autoimmune diseases" in this article include allogeneic islet transplant rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies (ANCA), adrenal autoimmune diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's syndrome, celiac spruce-dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, and Churg-Strauss syndrome. Syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, primary mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Grave's disease, Guillain-Barré syndrome, Goodpasture's syndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki disease, lichen planus, lupus erythema, Meniere's disease Diseases including mixed connective tissue disease, multiple sclerosis, type 1 diabetes, myasthenia gravis, pemphigus vulgaris, and pernicious anemia.Polyarteritis nodosa, polychondritis polyangiitis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Reynolds' phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, solid organ transplant rejection, stiff-person syndrome, systemic lupus erythematosus, takayasuarteritis, transient arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis, uveitis, vasculitis such as herpes-like dermatitis vasculitis, vitiligo, and Wegner's granulomatosis.

[0449] The antigen-binding proteins described in this article can be used to treat inflammatory conditions. "Inflammatory conditions" in this article include acute respiratory distress syndrome (ARDS), acute septic arthritis, adjuvant arthritis, juvenile idiopathic arthritis, allergic encephalomyelitis, allergic rhinitis, allergic vasculitis, allergy, asthma, atherosclerosis, chronic inflammation due to chronic bacterial or viral infection, chronic obstructive pulmonary disease (COPD), coronary artery disease, encephalitis, inflammatory bowel disease, inflammatory osteolysis, inflammation associated with acute and delayed-type hypersensitivity reactions, inflammation associated with tumors, peripheral nerve injury, or demyelinating diseases, inflammation associated with tissue trauma such as burns and ischemia, inflammation due to meningitis, multiple organ injury syndrome, pulmonary fibrosis, sepsis and septic shock, Stevens-Johnson syndrome, undifferentiated arthropathy, and undifferentiated spondyloarthropathy.

[0450] The antigen-binding proteins described herein can be used to prevent or suppress immune responses associated with rejection of donor tissue, cells, grafts, or organ transplants in recipient subjects. Graft-related diseases or conditions include graft-versus-host disease (GVDH), such as that associated with bone marrow transplantation, and immune conditions caused by or associated with rejection of organ, tissue, or cell grafts (e.g., tissue or cell allogeneic or xenografts), including grafts such as skin, muscle, neurons, islets, organs, liver parenchymal cells, etc. Regarding donor tissue, cells, grafts, or solid organ grafts in recipient subjects, it is believed that such molecules of the invention disclosed herein can effectively prevent acute rejection of such grafts in recipients and / or be used for long-term maintenance therapy, thereby preventing rejection of such grafts in recipients (e.g., suppressing rejection of insulin-producing islet cell grafts from donors in recipients with diabetes).

[0451] Preferred immune-related conditions that can be treated with the antigen-binding proteins disclosed herein include Crohn's disease, systemic lupus erythematosus (SLE), lupus nephritis, psoriatic arthritis, psoriasis, rheumatoid arthritis, ulcerative colitis, and transplant rejection, including but not limited to kidney transplant, liver transplant, and pancreas transplant.

[0452] The administration of pharmaceutical compositions containing the antigen-binding proteins disclosed herein, for example in the form of sterile aqueous solutions, can be carried out in a variety of ways, including but not limited to oral, subcutaneous, intravenous, intranasal, intraocular, transdermal, topical (e.g., gels, ointments, lotions, creams, etc.), intraperitoneal, intramuscular, intrapulmonary, vaginal, parenteral, rectal, or intraocular administration. In some cases, such as for the treatment of wounds, the antigen-binding proteins can be applied directly as solutions or sprays. As is known in the art, pharmaceutical compositions can be formulated accordingly based on the manner of introduction.

[0453] Subcutaneous administration can be used in situations where patients can self-administer the pharmaceutical composition. Many protein therapeutics are not potent enough to allow for the formulation of a therapeutically effective dose at the maximum acceptable volume for subcutaneous administration. This problem can be partially addressed by using protein formulations comprising arginine-HCl, histidine, and polysorbate. The antigen-binding proteins disclosed herein are more suitable for subcutaneous administration due to, for example, increased potency, improved serum half-life, or enhanced solubility. As is known in the art, protein therapeutics are typically delivered via IV infusion or bolus injection. The antigen-binding proteins disclosed herein can also be delivered using such methods. For example, administration can be performed via intravenous infusion of 0.9% sodium chloride as the infusion medium.

[0454] Lung delivery can be achieved using an inhaler or nebulizer and a formulation containing a nebulizing agent. For example, the AERx® inhalable technology, commercially available from Aradigm, or the inhance™ lung delivery system, commercially available from Nektar Therapeutics, can be used. Furthermore, the antigen-binding proteins disclosed herein are suitable for oral delivery.

[0455] In addition, any of the many delivery systems known in the art can be used to administer the antigen-binding proteins disclosed herein. Examples include, but are not limited to, encapsulation in liposomes, microparticles, microspheres (e.g., PLA / PGA microspheres), etc. Alternatively, implants of porous, non-porous, or gel-like materials, including membranes or fibers, can be used. Sustained-release systems may comprise polymeric materials or matrices such as polyesters, hydrogels, poly(vinyl alcohol), polylactide, copolymers of L-glutamic acid and L-glutamic acid ethyl ester, ethylene-vinyl acetate, lactic acid-glycolic acid copolymers such as Lupron Depot® and poly-D-(-)-3-hydroxybutyric acid. Nucleic acids encoding the antigen-binding proteins disclosed herein can also be administered, for example, through retroviral infection, direct injection, or coating with lipids, cell surface receptors, or other transfection agents. In all cases, controlled-release systems can be used to release the antigen-binding proteins disclosed herein at or near the desired site of action.

[0456] In one embodiment, the dosage and frequency of administration that are effective for treatment or prevention are selected. As is known in the art, adjustments may be necessary for protein degradation, systemic versus local delivery, and the rate of new protease synthesis, as well as for age, weight, general health condition, sex, diet, timing of administration, drug interactions, and severity of symptoms, and will be determined by those skilled in the art through routine experiments.

[0457] The concentration of the therapeutically active antigen-binding protein in the formulation can vary from about 0.1% by weight to 100% by weight. In one embodiment, the concentration of the antigen-binding protein is in the range of 0.003 mol to 1.0 mol. For the purpose of treating a patient, a therapeutically effective dose of the antigen-binding protein disclosed herein can be administered. "Therapeutically effective dose" as used herein means the dose that produces the effect of the administered protein. The precise dose will depend on the purpose of treatment and will be determined by those skilled in the art using known techniques. The dose range can be from 0.0001 mg / kg to 100 mg / kg body weight or greater, for example, 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or 50 mg / kg body weight. In one embodiment, the dose range is from 1 mg / kg to 10 mg / kg.

[0458] In some embodiments, only a single dose of the antigen-binding protein is used. In other embodiments, multiple doses of the antigen-binding protein are administered. The time elapsed between administrations can be less than 1 hour, about 1 hour, about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 2-4 days, about 4-6 days, about 1 week, about 2 weeks, or more than 2 weeks.

[0459] In other embodiments, the antigen-binding proteins disclosed herein are administered using metronome dosing regimens, through continuous infusion or frequent administration without prolonged rest periods. Such metronome dosing can involve administration at constant intervals without rest periods. Typically, such regimens cover prolonged periods of low-dose or continuous infusion, such as 1-2 days, 1-2 weeks, 1-2 months, or up to 6 months or longer. Using lower doses minimizes the need for side effects and rest periods.

[0460] Reagent test kit

[0461] In another embodiment, a kit or article of manufacture is provided, comprising an antigen-binding protein, an immunoglobulin variable domain, an antibody, dab, scFv, Fab, Fab', F(ab')2, an Fv fragment, a bifunctional antibody, a trifunctional antibody, a linear antibody, a single-chain antibody molecule or a multispecific antibody, a fusion protein, a conjugate or a pharmaceutical composition as described above.

[0462] In other embodiments, a kit is provided for the purposes described above, the kit comprising:

[0463] - A container for containing a therapeutic composition, said therapeutic composition being in one or more of the following forms: antigen-binding protein, immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, bifunctional antibody, trifunctional antibody, linear antibody, single-chain antibody molecule or multispecific antibody, fusion protein, conjugate or pharmaceutical composition;

[0464] - Includes a label or packaging insert with an instruction manual.

[0465] The kit or "article" may include a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. The container can be formed from a variety of materials, such as glass or plastic. The container contains a therapeutic composition for effectively treating a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). The label or packaging insert indicates that the therapeutic composition is intended to treat the selected condition. In one embodiment, the label or packaging insert includes instructions for use.

[0466] The kit may comprise (a) a therapeutic composition; and (b) a second container containing a second active ingredient. The kit in this embodiment of the invention may further include a packaging insert indicating that the active ingredient and other active ingredients can be used to treat a condition or prevent complications arising from inflammatory symptoms. Alternatively or additionally, the kit may further comprise a second (or third) container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0467] In some embodiments, the therapeutic composition may be provided in the form of a disposable or reusable device, including a receiver for containing the therapeutic composition. In one embodiment, the device is a syringe. The device may contain 1-2 mL of the therapeutic composition. The therapeutic composition may be provided in the device in a ready-to-use state or in a state where mixing or adding additional components is required.

[0468] In other embodiments, a kit is provided for the above-described diagnostic applications, the kit comprising:

[0469] - A container for containing a diagnostic composition in one or more of the following forms: antigen-binding protein, immunoglobulin variable domain, antibody, Fab, dab, scFv, bifunctional antibody, trifunctional antibody, fusion protein or conjugate;

[0470] - Includes a label or packaging insert with an instruction manual.

[0471] The kit may contain (a) a diagnostic composition; and (b) a second container therein containing a second diagnostic agent or a second label. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, etc.

[0472] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or obviously present in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0473] The following examples are intended to illustrate and are by no means limiting of the invention.

[0474] Example

[0475] The inventors have developed a series of antibodies that bind to CD80 and competitively release PD-L1 (superior to abatacept / CTLA4-Ig) from CD80.

[0476] Example 1: Materials and Methods

[0477] Cell Culture. Cells were cultured at 37°C in a 10% CO2 incubator in medium containing 100 U / mL penicillin, 100 µg / mL streptomycin (Gibco), and 10% FCS (Sigma-Aldrich). CHO cells were basally cultured in α-minimum essential medium (α-MEM; Gibraltar), 293T cells in Dulbecco's Modified Eagle Medium (DMEM; Gibraltar), and primary human T cells in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Gibraltar). Cells were isolated for passage or harvested using trypsin-EDTA (Gibco). For retroviral production, expression and packaging vectors were introduced into 293T cells via calcium phosphate transfection, and retroviral transduction of CHO cells was performed using standard protocols.

[0478] Flow cytometry of cultured cells. Harvested cells were washed with PBS. To assess protein binding, unless otherwise specified, cells were incubated with 20 µL of 10 µg / mL protein for 20 min, washed, and stained with the relevant anti-Fc secondary antibody for 20 min. After washing to remove excess protein and antibody, the cell pellet was resuspended in 100 µL FACS buffer (PBS containing 10% FCS). Flow cytometry of live cells (based on SSC / FSC) was performed using BD LSRII or LSR Tortessa (BD Biosciences). Human PD-L1 was detected using PD-L1-PE (clone MIH1, Thermo Fisher Scientific). Antibodies and proteins were detected using secondary antibodies against hIgG1-APC (clone IS11-12E4.23.20, Miltenyi) and antiIgG2a-PE-Cy7 (clone m2a-15F8, Thermo Fisher Scientific).

[0479] CD80mCherry:PD-L1mGFP population analysis. At least 250,000 mCherry / mGFP double-positive cells were recorded by flow cytometry. The mCherry, mGFP, and free PD-L1 MIH1 MFI values ​​for double-positive events were derived. Assuming a 1:1 infection ratio, the mCherry and mGFP MFI values ​​were independently normalized relative to the mean of all double-positive events, and the mCherry:mGFP ratio for each event was determined. The MIH1 MFI and log2 (mCherry:mGFP ratio) for each event were transferred to a pivot table. For events grouped at intervals of 0.1–0.2 log2 (mCherry:mGFP ratio), the mean MIH1 MFI was determined. The fold change between untreated and treated samples was determined.

[0480] Protein expression and purification. For crude supernatant analysis, 293T was expressed at 5 x 10⁻⁶ ppm. 5 Cells were plated in 10 cm plates. Cells were transfected with 5 µg of the vector and washed after 8 hours. The supernatant was collected after 3 days and concentrated directly or in a MicrosepAdvance centrifuge (PALL, MCP030C46) before use in experiments. For purified protein, Freestyle™ 293-F cells (Thermo Fisher Scientific) were grown to 1 × 10⁻⁶ cells in FreeStyle 293 expression medium at 37°C, 8% CO₂, and 130 rpm. 6 Cells were transfected at a density of [density missing] mL with plasmid DNA and polyethyleneimine (PEI) at a 3:1 PEI:DNA ratio (1 mg DNA / L). Cells were allowed to grow for 7 days post-transfection, supplemented with Glutamax (Thermo Fisher Scientific), 0.2 mM butyrate (Sigma-Aldrich), and 5 g / L lupin (Solabia) on days 1 and 4 post-transfection. The secreted recombinant protein was purified from the supernatant using Protein G resin (Cytiva). The protein was concentrated and applied to a Superdex 200 size exclusion column (Cytiva) equilibrated in DPBS (Gibco). The purest fractions, as determined by non-reducing SDS-PAGE, were combined, concentrated, filtered sterile, and stored at 4°C.

[0481] Primary human T-cell assay. Consenting blood donors were healthy controls participating in a low-risk ethical study that examined blood leukocyte subset analysis in accordance with the principles of the Declaration of Helsinki (Monash University 2020-26385 and 2022-35867) and was approved by the Monash University Human Research Ethics Committee (MUHREC). T cells (>95%) were purified from thawed erythrocyte sedimentation rate (ESR) amber layer using MACS cell isolation via human CD3 microbeads (Miltenyi Biotec). T cells were stained with 2.5 µM CFSE (Ingenieur) for 10 min for proliferation assay. Cells were seeded at 50,000 T cells / well in flat-bottomed 96-well plates pre-seeded with 10,000 CHO aAPC cells. Cells were co-cultured in 200 µL RPMI containing 10% FCS and penicillin / streptomycin. Treatment consisted of 50 µL of concentrated crude supernatant. At harvest, live T cells were gated based on SytoxBlue (Thermo Fisher Scientific) negative titers. T cell proliferation was assessed by flow cytometry using CFSE dilution. T cell activation was assessed by CD69 surface expression. For cytokine assays, 2 x 10⁻⁶ cells were used in the presence of CD80 antibody (40–50 µg / mL), with or without nivolumab (100 µg / mL). 6 1 T cell and 3 x 10 5 CHOaAPC cells were seeded together in 6-well plates for 3–4 days, then re-seeded into 96-well plates containing brevidin A (3 µg / mL) and monensin (2 µM). After 8–12 hours, cells were isolated and stained for CD4 (clone SK3, BioLegend) and CD8 (clone SK1, BioLegend), then fixed with 100 µL IC fixation buffer (Thermo Fisher Scientific). Cells were stained with anti-IL2 (clone MQ1-17H12, BioLegend) and anti-IFNγ (clone 4S.B3, BioLegend) in permeabilization buffer (Thermo Fisher Scientific), washed, and resuspended in FACS buffer for analysis.

[0482] Experimental model of basement membrane glomerulonephritis. Six- to ten-week-old transgenic C57BL / 6 mice with double knock-in humanized CD80 and PD-L1 were housed under specific pathogen-free conditions. Mice were subcutaneously sensitized with 0.5 mg of normal sheep globulin in Freund's complete adjuvant [FCA]. Four days later, 4.5 mg of sheep anti-mouse GBM globulin was administered intravenously via the tail vein, and 100 µg of abatacept and B5 were delivered intraperitoneally on days 5 and 7. On day 8, urine was collected for albuminuria and creatinine measurements.

[0483] Example 2: Development of novel anti-CD80 antibodies

[0484] Mice were immunized with the mouse pre-B cell line 300.19, which expresses human CD80 retrovirally, via four injections. The isolated B cells were analyzed using the Beacon platform (Berkeley Lights) to screen for antibody-producing B cells. These cells were selected based on their binding to CHO cells expressing human CD80 retrovirally, their binding to the same CHO cells pre-bound with MEDI5265-Ig (positive or negative), their binding to CHO cells expressing chimeric mouse CD80 with a human distal membrane domain retrovirally, and their binding to anti-IgG beads. cDNA was prepared from the selected B cells and sequenced.

[0485] Example 3: Characterization of antibodies

[0486] Figure 1 A schematic diagram is provided depicting the CTLA4 / CD80 / PD-L1 axis at the T cell and APC interface and how this axis is thought to be affected by the binding of various biological agents. For example, Figure 1 A shows the axis when there is no therapeutic modulation. Figure 1 B illustrates what the inventors understand to happen after the application of abatacept. Figure 1 C illustrates what the inventors understand to happen when the prior art anti-CD80 antibody TKMF5 binds, and it is understood that when TKMF5 binds to CD80, TKMF5 releases PD-L1 from its cis-interaction with CD80.

[0487] In the context of this understanding of the CTLA4 / CD80 / PD-L1 axis, the inventors evaluated the function of the novel anti-CD80 antibody of the present invention. When tested on CHO cells expressing the surface cis-CD80:PD-L1 complex, the novel anti-CD80 antibody clones B5, 19B10, 2B29, and 2B30 bound, as detected by anti-hIgG1 flow cytometry. Figure 2 AB).

[0488] When tested on CHO cells expressing the cis-CD80:PD-L1 complex, antibody clones 19B10, B5, 2B29, and 2B30 efficiently released PD-L1 from CD80. Figure 2 AB Figure 3 , Figure 5 Specifically, clones B5 and 19B10 competitively release PD-L1 from CD80 (superior to abatacept / CTLA4-Ig) and also partially disrupt the binding of CD80 to CD28. Figure 2 A).

[0489] Notably, 19B10 and B5 exhibited PD-L1 release activity exceeding that of standard care biologics CTLA4-Ig (e.g., abatacept). Figure 2 A). This indicates that the divalent binding of the CTLA4-Ig homodimer to two CD80 molecules releases a single PD-L1 molecule (A). Figure 1 B), while the divalent binding of 19B10 and B5 may actually release two PD-L1 molecules, similar to TKMF5 ( Figure 1 C).

[0490] To test whether TKMF5 (also a prior art antibody that blocks the cis-CD80:PD-L1 complex) and B5 release PD-L1 at different CD80:PD-L1 ratios, the inventors co-transduced CHO cells with two retroviral vectors: one encoding CD80 fused to an intracellular monomeric Cherry (mCherry), and the other encoding PD-L1 fused to an intracellular monomeric green fluorescent protein (mGFP). This produced a co-transduced cell population in which the mCherry:mGFP fluorescence ratio broadly reflected the relative surface CD80:PD-L1 expression (…). Figure 4 ).

[0491] 19B10, B5, 2B29, and 2B30 effectively block CD80:PD-L1 interaction, with 19B10 and B5 also having a partial effect on CD80:CD28 interaction. Figure 2 It is predicted that these two effects will synergistically inhibit T cell activation.

[0492] As a low-resolution method for defining the CD80 region binding to each antibody clone, the inventors evaluated binding with CHO cells expressing different forms of CD80 (anti-IgG1 flow cytometry). Figure 6 ):

[0493] -Human CD80 Wild Type

[0494] -Human CD80-L104D mutant

[0495] -Human CD80-ALPN202 compound mutants (H52Y, A60E, E69D, M81L, V102M, A105G, D124G)

[0496] - Human / mouse chimeric CD80 (mouse framework, but human G34-A140 residues)

[0497] -Monkey CD80 (the distal IgV domain of the membrane is a mutant relative to the human T135M).

[0498] - Mouse CD80

[0499] All antibody clones bound to wild-type human CD80, the human CD80-ALPN202 compound mutant, human / mouse chimeric CD80, and cynomolgus monkey CD80. Therefore, all antibodies appear to bind to the distal IgV domain (residues G34-A140) of human CD80.

[0500] Antibody clone 2B29 failed to bind to the CD80-L104D mutant. Figure 6 The binding of the clone to mouse CD80 was low / background / negative. Figure 6 ).

[0501] Example 4: Immunosuppressive effect of the antibody of the present invention

[0502] In a co-stimulation-driven human primary T cell activation / provocation assay, antibodies 19B10 and B5 were more effective than TKMF5 in inhibiting CD28-dependent co-stimulation. Figure 7 A). This is consistent with partial CD80:CD28 blocking in 19B10 and B5.

[0503] Additionally, in a human primary T-cell assay system, where PD1 activating inhibition was achieved through chronic stimulation of preactivated T cells, unquantified 19B10 and B5 cells more effectively activated PD1 than abatacept (reversed by the anti-PD1 blocking antibody nivolumab). Figure 7 This is consistent with the difference in PD-L1 release or protein production between 19B10 and B5 and abatacept.

[0504] Purified proteins allow for more accurate comparisons of biologics. When quantified, the potency of B5, 19B10, and TKMF5 can be assessed head-to-head at equivalent concentrations (e.g., ...). Figure 8 and Figure 9 As shown in the image).

[0505] Figure 8 A shows the dose / response free PD-L1 (MIH1 MFI) of releasing antibodies B5, 19B10 and TKMF5 across PD-L1 in cisCD80:PD-L1 cells compared to abatacept.

[0506] Figure 8 B shows from Figure 8 The fold change in the free PD-L1 relative to untreated A at 100 µg / mL B5, 19B10, TKMF5 and abatacept across a series of CD80mCherry:PD-L1mGFP fluorescence ratios.

[0507] Overall, these results show that the B5 antibody is superior to the prior art antibody TKMF5 in releasing PD-L1 after binding to CD80.

[0508] Figure 9 A shows the percentage of IL-2+ T cells activated in vitro, as assessed by co-culturing for 4 days with artificial antigen-presenting cells (aAPCs) expressing CD80:PD-L1 in a 1:1 ratio and OKT3scFv on the cell surface. 50 µg / mL abatacept or B5 was added during the last 8 hours of co-culture.

[0509] Figure 9 B shows Figure 9 The dose / response of the experimental conditions described in A demonstrates similar (CD4) or reduced (CD8) T cell activity of B5 relative to abatacept.

[0510] Figure 9 C shows, as Figure 9 The activity in CD4 T cells described in A indicates the relative contributions of abatacept (partial) and B5 (full) to PD-1 signaling by the addition of anti-PD-1 antibody (nivolumab).

[0511] These results indicate that, compared to abatacept, the B5 antibody can induce greater immunosuppression in vitro. Furthermore, B5 immunosuppression is primarily driven by PD-L1 / PD-1 agonism.

[0512] Example 5: In vivo immunosuppression of the antibody of the present invention

[0513] The inventors then tested the immunosuppressive activity of the antibodies of the present invention in an in vivo model. The anti-GBM mouse model of glomerulonephritis is a well-known model (see, for example, Odobasic et al., (2014), *Curr. Protoc. Immunol.* 106:15.26.1-15.26.20) and is used to evaluate treatments for reducing inflammatory nephropathy. The biologics were administered to mice to alleviate the disease. Figure 10 A provides a schematic diagram of the humanization strategy for the mouse CD80 and PD-L1 genes. Homologous human sequences replace exon 2 of mouse CD80 (which encodes an IgV domain that binds to human CD80 antibodies) and exon 3 of mouse PD-L1. Double knock-in (DKI) mice maintain endogenous regulation and expression of CD80 and PD-L1, and retain cis binding to each other and trans binding to CD28, CTLA4, or PD-1.

[0514] Figure 10 B provides a schematic diagram of a mouse model of renal autoimmunity against GBM glomerulonephritis.

[0515] The result is Figure 10As shown in Figure C. Compared to abatacept, the B5 antibody provides improved protection against kidney injury, as measured by the urine albumin:creatinine ratio (uACR). These results indicate that the antibody of the present invention is also capable of providing in vivo immunosuppression and demonstrates improved immunosuppressive capacity compared to abatacept.

[0516] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or obviously present in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

Claims

1. An antigen-binding protein for binding to CD80, wherein the protein comprises an antigen-binding domain, the antigen-binding domain comprising: CDRH1, CDRH2, and / or CDRH3 having an antigen-binding domain of a variable heavy chain as defined in any of SEQ ID NO: 73, 74, 75, 76, 77, or 78; and / or CDRL1, CDRL2 and / or CDRL3 having an antigen-binding domain of a variable heavy chain as defined in any of SEQ ID NO: 148, 149, 150, 151, 152 or 153.

2. The antigen-binding protein according to claim 1, wherein the antigen-binding protein inhibits the binding of PD-L1 to CD80.

3. The antigen-binding protein according to claim 1 or 2, wherein the antigen-binding protein inhibits the binding of CD28 to CD80.

4. The antigen-binding protein according to any one of claims 1 to 3, wherein the protein comprises: - CDR1, CDR2 and CDR3 having an antigen-binding domain of a variable heavy chain as defined in SEQ ID NO: 73, and CDR1, CDR2 and CDR3 having an antigen-binding domain of a variable light chain as defined in SEQ ID NO: 148; - CDR1, CDR2 and CDR3 having antigen-binding domains of variable heavy chains as defined in SEQ ID NO: 77, and CDR1, CDR2 and CDR3 having antigen-binding domains of variable light chains as defined in SEQ ID NO: 152; - CDR1, CDR2 and CDR3 having antigen-binding domains of variable heavy chains as defined in SEQ ID NO: 74, and CDR1, CDR2 and CDR3 having antigen-binding domains of variable light chains as defined in SEQ ID NO: 149; - CDR1, CDR2 and CDR3 having antigen-binding domains of variable heavy chains as defined in SEQ ID NO: 78, and CDR1, CDR2 and CDR3 having antigen-binding domains of variable light chains as defined in SEQ ID NO: 153; - CDR1, CDR2, and CDR3 having antigen-binding domains of a variable heavy chain as defined in SEQ ID NO: 75, and CDR1, CDR2, and CDR3 having antigen-binding domains of a variable light chain as defined in SEQ ID NO: 150; or - CDR1, CDR2 and CDR3 having an antigen-binding domain of a variable heavy chain as defined in SEQ ID NO: 76, and CDR1, CDR2 and CDR3 having an antigen-binding domain of a variable light chain as defined in SEQ ID NO:

151.

5. The antigen-binding protein according to any one of claims 1 to 4, comprising, substantially consisting of, or consisting of the amino acid sequences of SEQ ID NO: 73 and 148 (in the order of N to C-terminus or C to N-terminus).

6. The antigen-binding protein according to any one of claims 1 to 4, comprising, substantially comprising, or consisting of the amino acid sequences of SEQ ID NO: 74 and 149 (in the order of N to C-terminus or C to N-terminus).

7. The antigen-binding protein according to any one of claims 1 to 4, comprising, substantially comprising, or consisting of the amino acid sequence of SEQ ID NO: 77 and 152 (in the order of N to C-terminus or C to N-terminus).

8. The antigen-binding protein according to any one of claims 1 to 4, comprising, substantially comprising, or consisting of the amino acid sequence of SEQ ID NO: 78 and 153 (in the order of N to C-terminus or C to N-terminus).

9. The antigen-binding protein according to any one of claims 1 to 4, comprising, substantially consisting of, or consisting of the amino acid sequences of SEQ ID NO: 75 and 150 (in the order of N to C-terminus or C to N-terminus).

10. The antigen-binding protein according to any one of claims 1 to 4, comprising, substantially comprising, or consisting of the amino acid sequence of SEQ ID NO: 76 and 151 (in the order of N to C-terminus or C to N-terminus).

11. The antigen-binding protein according to any one of claims 1 to 4, wherein the protein comprises an antigen-binding domain, the antigen-binding domain comprising: (i) VH, wherein the VH comprises: a complementarity determination region (CDR) 1, wherein the CDR 1 comprises a sequence as shown in SEQ ID NO: 1 (IMGT) or 13 (Kabat) or a sequence that is 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% identical to the sequence shown in SEQ ID NO: The sequence shown in SEQ ID NO: 3 (IMGT) or 14 or 165 (Kabat) or the sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 3 (IMGT) or 15 (Kabat) or the sequence that is 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% identical to it; (ii) VH, wherein the VH comprises a sequence as shown in SEQ ID NO: 73 or SEQ ID NO: 77 or a sequence that is 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% identical to the sequence shown in SEQ ID NO: 77; (iii) VL, the VL comprising: a complementarity determination region (CDR) 1, the CDR 1 comprising a sequence as shown in SEQ ID NO: 79 (IMGT) or 91 (Kabat) or a sequence that is 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% identical to it; and a CDR 2, the CDR 2 comprising a sequence as shown in SEQ ID NO: The sequence shown in SEQ ID NO: 80 (IMGT) or 92 (Kabat) or a sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 81 (IMGT or Kabat) or a sequence that is 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% identical to it; (iv) VL, wherein the VL comprises a sequence as shown in SEQ ID NO: 148 or SEQ ID NO: 152 or a sequence that is 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% identical to the sequence shown in SEQ ID NO:

152. (v) VH, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 1; CDR2, which comprises the sequence shown in SEQ ID NO: 2; and CDR3, which comprises the sequence shown in SEQ ID NO: 3; or comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 13; CDR2, which comprises the sequence shown in SEQ ID NO: 14 or 165; and CDR3, which comprises the sequence shown in SEQ ID NO: 15; (vi) VL, the VL comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 79; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 80; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 81; comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 91; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 92; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 93; (vii) VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 1; CDR2, which comprises the sequence shown in SEQ ID NO: 2; and CDR3, which comprises the sequence shown in SEQ ID NO: 3; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 79; CDR2, which comprises the sequence shown in SEQ ID NO: 80; and CDR3, which comprises the sequence shown in SEQ ID NO: 81; or VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 13; CDR2, which comprises the sequence shown in SEQ ID NO: 14 or 165; and CDR3, which comprises the sequence shown in SEQ ID NO: 15; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 1; CDR2, which comprises the sequence shown in SEQ ID NO: 14 or 165; and CDR3, which comprises the sequence shown in SEQ ID NO: 15; and VL, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 1; CDR2, which comprises the sequence shown in SEQ ID NO: 1 ... The sequence shown in NO: 91; CDR2, which comprises the sequence shown in SEQ ID NO: 92; and CDR3, which comprises the sequence shown in SEQ ID NO: 93; or (viii) VH, which comprises the sequence shown in SEQ ID NO: 73; and VL, which comprises the sequence shown in SEQ ID NO: 148; or VH, which comprises the sequence shown in SEQ ID NO: 77; and VL, which comprises the sequence shown in SEQ ID NO:

152.

12. The antigen-binding protein of claim 11, wherein the protein further comprises at least one of the following: (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 25 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 26 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 27 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 28 or thereof, or at least about 80%, at least 85%, 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% thereof; and ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 101 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 102 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 103 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 104 or the sequence thereof, which is at least about 80%, at least 85%, 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%. or (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 49 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 50 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 51 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 52 or thereof, or at least about 80%, at least 85%, 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% thereof; and ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 124 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 125 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 126 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 127 or the sequence thereof, which is at least about 80%, at least 85%, 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%. or (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 41 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 42 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 43 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: 44 amino acid sequences or sequences thereof comprising at least about 80%, at least 85%, 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% of the total amino acid sequence; and (ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 105 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 106 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 107 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 108 or the sequence thereof, which is at least about 80%, at least 85%, 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%. or (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 53 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 54 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 55 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 56 or thereof, or at least about 80%, at least 85%, 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% thereof; and ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 128 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 129 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 130 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 131 or the sequence thereof of at least about 80%, at least 85%, 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%.

13. The antigen-binding protein of claim 11 or 12, wherein the antigen-binding protein comprises: a variable heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 73 or SEQ ID NO: 77, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a variable light chain comprising an amino acid sequence as shown in SEQ ID NO: 148 or SEQ ID NO:

77. The amino acid sequence shown in 152 or the sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to it; wherein the variable heavy chain and / or light chain contains, in addition to the indicated CDR sequence, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added, or combinations thereof, and wherein the antigen-binding protein retains the ability to bind to CD80.

14. The antigen-binding protein according to any one of claims 1 to 4, wherein the protein comprises an antigen-binding domain, the antigen-binding domain comprising: (i) VH, wherein the VH comprises: a complementarity determination region (CDR) 1, the CDR 1 comprising a sequence as shown in SEQ ID NO: 4 (IMGT) or 16 (Kabat) or a sequence that is 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% identical to it; and a CDR 2, the CDR 2 comprising a sequence as shown in SEQ ID NO: The sequence shown in SEQ ID NO: 5 (IMGT) or 17 or 166 (Kabat) or a sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 6 or 18 (Kabat) or a sequence that is 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% identical to it; (ii) VH, wherein the VH comprises a sequence as shown in SEQ ID NO: 74 or SEQ ID NO: 78 or a sequence that is 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% identical to it; (iii) VL, the VL comprising: a complementarity determination region (CDR) 1, the CDR 1 comprising a sequence as shown in SEQ ID NO: 82 (IMGT) or 93 (Kabat) or a sequence that is 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% identical to it; and a CDR 2, the CDR 2 comprising a sequence as shown in SEQ ID NO: The sequence shown in SEQ ID NO: 83 (IMGT) or 94 (Kabat) or a sequence that is 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% identical to it; and CDR3, which comprises the sequence shown in SEQ ID NO: 84 (IMGT or Kabat) or a sequence that is 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% identical to it; (iv) VL, wherein the VL comprises a sequence as shown in SEQ ID NO: 149 or SEQ ID NO: 153 or a sequence that is 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% identical to the sequence shown in SEQ ID NO:

153. (v) VH, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 4; CDR2, which comprises the sequence shown in SEQ ID NO: 5; and CDR3, which comprises the sequence shown in SEQ ID NO: 6; or comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 16; CDR2, which comprises the sequence shown in SEQ ID NO: 17 or 166; and CDR3, which comprises the sequence shown in SEQ ID NO: 18; (vi) VL, the VL comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 82; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 83; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 84; or comprising: CDR1, the CDR1 comprising the sequence shown in SEQ ID NO: 93; CDR2, the CDR2 comprising the sequence shown in SEQ ID NO: 94; and CDR3, the CDR3 comprising the sequence shown in SEQ ID NO: 84; (vii) VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 4; CDR2, which comprises the sequence shown in SEQ ID NO: 5; and CDR3, which comprises the sequence shown in SEQ ID NO: 6; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 82; CDR2, which comprises the sequence shown in SEQ ID NO: 83; and CDR3, which comprises the sequence shown in SEQ ID NO: 84; or VH, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 16; CDR2, which comprises the sequence shown in SEQ ID NO: 17 or 166; and CDR3, which comprises the sequence shown in SEQ ID NO: 18; and VL, comprising: CDR1, which comprises the sequence shown in SEQ ID NO: 4; CDR2, which comprises the sequence shown in SEQ ID NO: 5; and CDR3, which comprises the sequence shown in SEQ ID NO: 6; and VL, which comprises: CDR1, which comprises the sequence shown in SEQ ID NO: 6; and CDR3, which comprises the sequence shown in SEQ ID NO: 7; and CDR3, which comprises the sequence shown in SEQ ID NO: 84; and CDR3, which comprises the sequence shown in SEQ ID NO: 85; and CDR3, which comprises the sequence shown in SEQ ID NO: 86 ...6; and CDR3, which comprises the sequence The sequence shown in NO: 93; CDR2, which comprises the sequence shown in SEQ ID NO: 94; and CDR3, which comprises the sequence shown in SEQ ID NO: 84; or (viii) VH, which comprises the sequence shown in SEQ ID NO: 74; and VL, which comprises the sequence shown in SEQ ID NO: 149; or VH, which comprises the sequence shown in SEQ ID NO: 78; and VL, which comprises the sequence shown in SEQ ID NO:

153.

15. The antigen-binding protein of claim 14, wherein the antigen-binding domain further comprises at least one of the following: (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 29 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 30 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 31 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 32 or thereof, or at least about 80%, at least 85%, 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% thereof; and ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 109 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 110 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 111 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 112 or the sequence thereof, which is at least about 80%, at least 85%, 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%. (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 57 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 58 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 59 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 60 or thereof, or the sequence thereof, comprising at least about 80%, at least 85%, 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%; and ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 132 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 133 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 134 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises SEQ ID NO: The amino acid sequence of 135 or thereof, or at least about 80%, at least 85%, 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% thereof. or i) VH, wherein the VH comprises: a frame region (FR) 1, wherein the FR1 comprises the amino acid sequence of SEQ ID NO: 45 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein the FR2 comprises the amino acid sequence of SEQ ID NO: 46 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein the FR3 comprises the amino acid sequence of SEQ ID NO: 47 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein the FR4 comprises the amino acid sequence of SEQ ID NO: 48 amino acid sequences or sequences thereof comprising at least about 80%, at least 85%, 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% of the total amino acid sequence; and ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 113 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 114 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 115 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 116 or thereof, or at least about 80%, at least 85%, 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% thereof. or (i) VH, comprising: a frame region (FR) 1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 61 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 62 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 63 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 64 or thereof, or at least about 80%, at least 85%, 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% thereof; and ii) VL, comprising: FR1, wherein FR1 comprises the amino acid sequence of SEQ ID NO: 136 or is at least about 80%, at least 85%, 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% identical thereto; FR2, wherein FR2 comprises the amino acid sequence of SEQ ID NO: 137 or is at least about 80%, at least 85%, 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% identical thereto; FR3, wherein FR3 comprises the amino acid sequence of SEQ ID NO: 138 or is at least about 80%, at least 85%, 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% identical thereto; and FR4, wherein FR4 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of 139 or the sequence thereof of at least about 80%, at least 85%, 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%.

16. The antigen-binding protein of claim 14 or 15, wherein the antigen-binding protein comprises: a variable heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 74 or SEQ ID NO: 78, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a variable light chain comprising an amino acid sequence as shown in SEQ ID NO: 149 or SEQ ID NO:

78. The amino acid sequence shown in 153 or the sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to it; wherein the variable heavy chain and / or light chain contains, in addition to the indicated CDR sequence, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residues substituted, deleted, or added, or combinations thereof, and wherein the antigen-binding protein retains the ability to bind to CD80.

17. The antigen-binding protein according to any one of claims 1 to 16, wherein the antigen-binding protein is in the following form: (i) Single-domain antibodies (sdAbs); (ii) Single-stranded Fv fragments (scFv); (iii) Variable structural domain; (iv) Dimer scFv (di-scFv); or (v) One of (i) or (iv) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of the antibody.

18. The antigen-binding protein according to any one of claims 1 to 17, wherein the antigen-binding protein is in the following form: (i) Bifunctional antibody; (ii) Trifunctional antibodies; (iii) Four-functional antibodies; (iv) Fab; (v)F(ab')2; (vi)Fv; (vii) Bispecific, trispecific antibodies, or other forms of multispecific antibodies (including BiTE); or (viii) One of (i) to (vii) connected to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of the antibody.

19. The antigen-binding protein according to any one of claims 1 to 18, wherein the protein is a monoclonal antibody.

20. The antigen-binding protein according to any one of claims 1 to 19, wherein the protein is a naked antibody.

21. The antigen-binding protein according to any one of claims 1 to 20, wherein the protein is in the form of a fusion protein.

22. The antigen-binding protein according to any one of claims 1 to 21, wherein the protein comprises chemical modifications, such as conjugation with an active agent or a radiolabel or a pharmaceutical agent for improving solubility.

23. A nucleic acid molecule encoding an antigen-binding protein according to any one of claims 1 to 21.

24. A vector or expression construct comprising the nucleic acid according to claim 23.

25. A recombinant host cell comprising the nucleic acid of claim 23 or the vector or expression construct of claim 24.

26. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 22 and a pharmaceutically acceptable carrier, diluent, or excipient.

27. A method of treating an inflammatory condition in a subject in need, the method comprising administering to the subject an antigen-binding protein according to any one of claims 1 to 22 or a pharmaceutical composition according to claim 26, thereby treating the subject's inflammatory condition.

28. Use of an antigen-binding protein according to any one of claims 1 to 22, for the preparation of a medicament for treating inflammatory conditions in subjects of need.

29. The method or use according to claim 27 or 28, wherein the inflammatory condition is an immune-related condition or a symptom or condition requiring immunosuppression.

30. The method or use according to claim 29, wherein the immune-related condition or condition requiring immunosuppression is selected from: Crohn's disease, systemic lupus erythematosus (SLE), lupus nephritis, psoriatic arthritis, psoriasis, rheumatoid arthritis, ulcerative colitis, transplant rejection and / or graft-versus-host disease (GvHD).

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