anti-CD28 antibodies

Abstract

Antibodies and antigen-binding fragments thereof that bind to CD28, and methods of using the same, are provided.

Description

[Technical Field]

[0001] Related Applications This application claims priority to European Patent Application No. 22315222.4 (filed September 30, 2022), the disclosure of which is hereby incorporated by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing The contents of the electronically submitted Sequence Listing in XML format (Name: 746135_SA9-345PC_SL.xml; Size: 541,509 bytes; and Creation Date: September 26, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to novel antibodies and antigen-binding fragments thereof that specifically bind to CD28, and methods of using the same. [Background technology]

[0004] Cluster of differentiation 28 (CD28) is a member of the costimulatory protein family expressed on the surface of T receptor cells. Because costimulatory receptors can regulate T cell activation and thus the course of specific immune responses, they have been tested for controlling T cell responses in both oncology and inflammatory settings. Therefore, CD28 antagonists are being investigated for treating autoimmune and inflammatory diseases, and CD28 agonists are being tested for tumor diseases. However, administration of agonistic anti-CD28 antibodies has been associated with severe systemic inflammatory responses, including cytokine storms. These dangerous inflammatory side effects significantly limit the therapeutic window and therapeutic use of anti-CD28 antibodies. Therefore, there is a need for novel anti-CD28 antibodies that are not associated with severe inflammatory responses. Summary of the Invention [Means for solving the problem]

[0005] Provided herein are anti-CD28 antibodies (e.g., anti-CD28 VHH antibodies) and antigen-binding fragments thereof. Methods for inhibiting the activity of CD28 or treating CD28-associated diseases are also provided.

[0006] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to CD28, comprising an immunoglobulin single variable domain (ISVD), wherein the ISVD domain comprises a CDR-H1 amino acid sequence, a CDR-H2 amino acid sequence, and a CDR-H3 amino acid sequence selected from any one of the CDR-H3, CDR-H2, and CDR-H3 amino acid sequences in Table 1.

[0007] In certain embodiments, the ISVD domain comprises any one of the ISVD amino acid sequences in Table 2.

[0008] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to CD28, comprising an immunoglobulin single variable domain (ISVD) comprising a CDR-H1 sequence, a CDR-H2 sequence, and a CDR-H3 sequence; a) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 1, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 2, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 3; b) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:4, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:5, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:6; c) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:7, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:8, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:9; d) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 10, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 11, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 12; e) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 13, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 14, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 15; f) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 16, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 17, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 18; g) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 19, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 20, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 21; h) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 22, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 23, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 24; i) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 25, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 26, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 27; j) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 28, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 29, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 30; k) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 31, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 32, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 33; l) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 34, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 35, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 36; m) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 37, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 38, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 39; n) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 40, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 41, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 42; o) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 43, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 44, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 45; p) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 46, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 47, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 48; q) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 49, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 50, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 51; r) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 52, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 53, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 54; s) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 55, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 56, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 57; t) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 58, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 59, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 60; u) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 61, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 62, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 63; v) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 64, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 65, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 66; w) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 67, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 68, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 69; x) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 70, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 71, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 72; y) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 73, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 74, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 75; z) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 76, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 77, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 78; aa) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 79, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 80, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 81; ab) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 82, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 83, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 84; ac) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 85, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 86, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 87; ad) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 88, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 89, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 90; ae) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 91, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 92, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 93; af) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 94, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 95, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 96; ag) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 97, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 98, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 99; ah) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 100, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 101, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 102; ai) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 103, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 104, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 105; aj) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 106, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 107, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 108; a k) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 109, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 110, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 111; al) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 112, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 113, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 114; am) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 115, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 116, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 117; an) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 118, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 119, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 120; ao) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 121, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 122, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 123; a) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 124, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 125, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 126; aq) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 127, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 128, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 129; ar) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 130, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 131, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 132; as) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 133, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 134, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 135; at) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 136, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 137, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 138; au) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 139, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 140, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 141; av) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 142, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 143, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 144; aw) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 145, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 146, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 147; ax) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 148, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 149, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 150; ay) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 151, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 152, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 153; a-z) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 154, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 155, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 156; ba) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 157, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 158, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 159; bb) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 160, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 161, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 162; bc) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 163, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 164, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 165; bd) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 166, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 167, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 168; be) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 169, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 170, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 171; bf) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 172, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 173, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 174; bg) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 175, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 176, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 177; bh) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 178, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 179, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 180; bi) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 181, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 182, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 183; bj) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 184, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 185, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 186; bk) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 187, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 188, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 189; bl) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 190, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 191, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 192; bm) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 193, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 194, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 195; bn) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 196, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 197, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 198; bo) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 199, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 200, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 201; bp) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 202, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 203, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 204; bq) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 205, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 206, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 207; br) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 208, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 209, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 210; bs) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 211, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 212, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 213; bt) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 214, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 215, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 216; bu) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 217, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 218, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 219; bv) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 220, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 221, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 222; bw) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 223, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 224, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 225; bx) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 226, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 227, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 228; by) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 229, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 230, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 231; bz) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 232, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 233, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 234; ca) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 235, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 236, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 237; cb) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 238, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 239, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 240; cc) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 241, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 242, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 243; cd) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 244, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 245, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 246; ce) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 247, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 248, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 249; cf) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 250, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 251, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 252; cg) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 253, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 254, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 255; ch) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 256, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 257, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 258; ci) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 259, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 260, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 261; cj) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 262, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 263, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 264; ck) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 265, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 266, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 267; cl) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 268, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 269, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 270; cm) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 271, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 272, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 273; cn) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 274, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 275, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 276; co) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 277, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 278, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 279; cp) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 280, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 281, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 282; cq) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 283, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 284, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 285; cr) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 286, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 287, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 288; cs) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 289, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 290, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 291; ct) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 292, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 293, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 294; cu) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 295, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 296, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 297; cv) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 298, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 299, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 300; cw) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 301, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 302, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 303; cx) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 304, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 305, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 306; cy) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 307, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 308, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 309; cz) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 310, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 311, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 312; da) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 313, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 314, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 315; db) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 316, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 317, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 318; dc) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 319, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 320, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 321; dd) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 322, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 323, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 324; de) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 325, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 326, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 327; df) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 328, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 329, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 330; dg) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 331, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 332, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 333; dh) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 334, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 335, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 336; di) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 337, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 338, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 339; dj) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 340, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 341, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 342; dk) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 343, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 344, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 345; dl) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 346, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 347, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 348; dm) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 349, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 350, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 351; dn) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 352, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 353, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 354; do) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 355, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 356, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 357; dp) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 358, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 359, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 360; dq) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 361, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 362, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 363; dr) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 364, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 365, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 366; ds) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 367, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 368, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 369; dt) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 370, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 371, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 372; du) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 373, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 374, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 375; dv) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 376, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 377, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 378; dw) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 379, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 380, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 381; dx) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 382, ​​the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 383, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 384; dy) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 385, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 386, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 387; dz) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 388, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 389, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 390; ea) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 391, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 392, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 393; eb) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 394, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 395, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 396; ec) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 397, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 398, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 399; ed) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 400, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 401, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 402; ee) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 403, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 404, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 405; e) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 406, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 407, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 408; e.g., the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 409, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 410, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 411; eh) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 412, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 413, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 414; ei) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 415, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 416, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 417; ej) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 418, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 419, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 420; e) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 421, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 422, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 423; el) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 424, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 425, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 426; em) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 427, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 428, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 429; en) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 430, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 431, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 432; eo) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 433, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 434, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 435; ep) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 436, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 437, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 438; eq) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 439, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 440, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 441; er) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 442, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 443, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 444; es) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 445, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 446, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 447; et) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 448, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 449, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 450; or eu) The CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 451, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 452, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 453.

[0009] In certain embodiments, a) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 454; b) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 455; c) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 456; d) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 457; e) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 458; f) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 459; g) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 460; h) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 461; i) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 462; j) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 463; k) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 464; l) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 465; m) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 466; n) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 467; o) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 468; p) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 469; q) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 470; r) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 471; s) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 472; t) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 473; u) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 474; v) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 475; w) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 476; x) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 477; y) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 478; z) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 479; aa) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 480; ab) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 481; ac) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 482; ad) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 483; ae) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 484; af) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 485; ag) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 486; ah) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 487; ai) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 488; aj) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 489; a) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 490; al) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 491; am) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 492; an) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 493; ao) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 494; ap) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 495; aq) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 496; ar) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 497; as) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 498; at) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 499; au) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 500; av) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 501; aw) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 502; ax) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 503; ay) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 504; a-z) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 505; ba) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 506; bb) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 507; bc) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 508; bd) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 509; be) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 510; bf) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 511; bg) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 512; bh) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 513; bi) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 514; bj) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 515; bk) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 516; bl) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 517; bm) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 518; bn) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 519; bo) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 520; bp) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 521; bq) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 522; br) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 523; bs) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 524; bt) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 525; bu) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 526; bv) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 527; bw) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 528; bx) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 529; by) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 530; bz) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 531; ca) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 532; cb) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 533; cc) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 534; cd) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 535; ce) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 536; cf) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 537; cg) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 538; ch) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 539; ci) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 540; cj) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 541; ck) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 542; cl) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 543; cm) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 544; cn) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 545; co) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 546; cp) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 547; cq) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 548; cr) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 549; cs) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 550; ct) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 551; cu) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 552; cv) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 553; cw) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 554; cx) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 555; cy) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 556; cz) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 557; da) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 558; db) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 559; dc) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 560; dd) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 561; de) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 562; df) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 563; dg) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 564; dh) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 565; di) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 566; dj) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 567; dk) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 568; dl) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 569; dm) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 570; dn) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 571; do) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 572; dp) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 573; dq) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 574; dr) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 575; ds) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 576; dt) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 577; du) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 578; dv) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 579; dw) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 580; dx) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 581; dy) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 582; dz) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 583; ea) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 584; eb) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 585; ec) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 586; ed) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 587; ee) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 588; ef) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 589; e.g., the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 590; eh) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 591; ei) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 592; ej) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 593; e) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 594; el) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 595; em) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 596; en) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 597; eo) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 598; ep) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 599; eq) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 600; er) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 601; es) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 602; et) the ISVD domain comprises the amino acid sequence set forth in SEQ ID NO: 603; or The eu) ISVD domain comprises the amino acid sequence set forth in SEQ ID NO:604.

[0010] In certain embodiments, the antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.

[0011] In certain embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0012] In certain embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody.

[0013] In certain embodiments, the bispecific antibody comprises an antigen-binding domain that comprises binding affinity for a tumor-associated antigen (TAA).

[0014] In certain embodiments, the ISVD is operably linked to a CH1 domain and / or a CL domain.

[0015] In certain embodiments of the bispecific antibody, an ISVD having binding affinity for CD28 is operably linked to a CH1 domain, and an antigen binding domain having binding affinity for a TAA is operably linked to a CL domain.

[0016] In certain embodiments of the bispecific antibody, an ISVD having binding affinity for CD28 is operably linked to a CL domain, and an antigen binding domain having binding affinity for a TAA is operably linked to a CH1 domain.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof is operably linked to an Fc region.

[0018] In a specific embodiment, the Fc region is a human IgG1 Fc region.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an antagonist antibody or antigen-binding fragment thereof.

[0020] In one aspect, the disclosure provides an isolated nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof, or the above-described bispecific antibody.

[0021] In one aspect, the present disclosure provides an expression vector comprising the above-described nucleic acid molecule.

[0022] In one aspect, the present disclosure provides a host cell comprising the above-described expression vector.

[0023] In one aspect, the disclosure provides a method for inhibiting CD28 activity in a subject, the method comprising administering to the subject an antibody or antigen-binding fragment thereof described above, thereby inhibiting CD28 activity in the subject.

[0024] In one aspect, the present disclosure provides a method for treating a disease associated with CD28 activity in a subject, the method comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof as described above.

[0025] In certain embodiments, the disease is an autoimmune disease.

[0026] In certain embodiments, the disease is cancer.

[0027] The summary of the disclosure set forth above is non-limiting, and other features and advantages of the disclosed antigen binding proteins and methods will be apparent from the following brief description of the drawings, detailed description of the disclosure, and claims.

[0028] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments, taken in conjunction with the accompanying drawings. This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0029] [Figure 1]Figure 1 shows the general workflow for CD28xTAA bispecific screening. [Figure 2] The sequence diversity of all CD28 binders identified in the primary screen is shown. [Figure 3] Figure 3 shows epitope binning of CD28 bispecific Fab (bsFab). Competition between anti-CD28 VHHs was investigated by SPR. Figure 3A shows the experimental scheme of competition between anti-CD28 VHHs investigated by SPR. Figure 3B shows the collected competition data in a heat map. [Figure 4] Figure 4 shows evaluation of CD28 bsFab and bivalent Fab (bvFab) in an IL-2 luciferase reporter assay with CD3 preactivation. Figure 4A shows monovalent anti-CD28x anti-foot-and-mouth disease virus (FMDV) bsFab with and without cross-linking. Figure 4B shows bivalent CD28 bvFab with and without cross-linking. Figure 4C shows monovalent CD28xFMDV bsFab against bivalent CD28 bvFab with and without cross-linking. Figure 4D shows monovalent CD28xFMDV bsFab against bivalent CD28 bvFab with cross-linking. [Figure 5] Figure 5 shows the evaluation of CD28 bsFab and bvFab in a T cell activation assay. Figure 5A shows the experimental setup. Figure 5B shows IFNγ secretion after 6 days of treatment at 100 nM. [Figure 6] Figure 6 shows the evaluation of CD28 bvFab in an MLR assay. Figure 6A shows the experimental setup for the MLR assay. Figure 6B shows IFNγ secretion after 4 days of treatment at 10 nM. [Figure 7] FIG. 1 shows a table containing the complete epitope binning results for 48 CD28 VHHs. [Figure 8] Competition of CD28 bvFab with CD80 using flow cytometry. [Figure 9] 1 shows a CD28xTAA bsFab that simultaneously binds to two cells expressing either CD28 or TAA. [Figure 10]Figure 10 shows evaluation of CD28 bsFab and bvFab in an IL-2 luciferase reporter assay. Figure 10A shows monovalent CD28xFMDV bsFab with and without cross-linking in the absence of CD3 preactivation. Figure 10B shows bivalent CD28 bsFab with and without cross-linking in the absence of CD3 preactivation. Figure 10C shows monovalent CD28xFMDV bsFab with and without cross-linking after CD3 preactivation. Figure 10D shows bivalent CD28 bvFab with and without cross-linking after CD3 preactivation. Figure 10E shows monovalent CD28xFMDV bsFab relative to bivalent CD28 bvFab without cross-linking after CD3 preactivation. Figure 10F shows monovalent CD28xFMDV bsFab relative to bivalent CD28 bvFab with cross-linking after CD3 preactivation. [Figure 11] The complete epitope binning results of 48 CD28 VHHs are shown. DETAILED DESCRIPTION OF THE INVENTION

[0030] Before the present disclosure is described, it is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. The scope of the present disclosure will be limited only by the appended claims, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0032] Although any methods and materials similar or equivalent to those described herein can be used in the practice of this disclosure, exemplary methods and materials are now described. All publications mentioned herein are incorporated by reference as if set forth in their entirety.

[0033] The term "about" or "approximately" means within about 20%, such as within about 10%, within about 5%, or within about 1% or less of a given value or range.

[0034] As used herein, the term "antibody" or "antigen-binding protein" refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with an antigen or epitope, and includes both polyclonal and monoclonal antibodies, as well as functional antibody fragments thereof. The term "antibody" or "antigen-binding protein" includes immunoglobulin single variable domain (ISVD or ISV) antibodies (e.g., sdAb, sdFv, Nanobody®, VHH). The term "antibody" also includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, meditope-enabled antibodies, heteroconjugate antibodies (e.g., multispecific antibodies, bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv), and the like.

[0035] As used herein, the term "functional antibody fragment" refers to an antibody fragment that has at least 80%, at least 85%, at least 90%, or at least 95% of the affinity as the antibody of interest from which the fragment is derived.

[0036] The term "multispecific antibody," as used herein, refers to bispecific, trispecific, or multispecific antibodies, and antigen-binding fragments thereof. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for epitopes of two or more target polypeptides. A multispecific antibody may be a single multifunctional polypeptide, or it may be a multimeric complex of two or more polypeptides that are covalently or noncovalently bound to each other. The term "multispecific antibody" includes antibodies of the present disclosure that may be linked to or coexpressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be operatively linked (e.g., by chemical bonding, genetic fusion, noncovalent bonding, or otherwise) to one or more other molecular entities, such as proteins or fragments thereof, to generate a bispecific or multispecific antibody with a second binding specificity. In certain exemplary embodiments, an antibody of the present disclosure is operatively linked to another antibody or antigen-binding fragment thereof to generate a bispecific antibody with a second binding specificity. In a particular embodiment, the second binding specificity is for a tumor-associated antigen (TAA).

[0037] As used herein, "monovalent" with respect to an antibody refers to an antibody having a single antigen recognition site specific for a target antigen. Examples of monovalent antibodies include monovalent immunoglobulin single variable domain antibodies (e.g., VHHs) or monovalent antibody fragments. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single-chain Fv fragments (scFvs). Furthermore, multispecific antibodies can have multiple antigen-binding sites, each of which recognizes a different target antigen. Thus, each antigen-binding site is monovalent with respect to the target antigen.

[0038] As used herein, "multivalent" with respect to an antibody refers to an antibody having multiple (two or more) antigen recognition sites specific for a target antigen.

[0039] As used herein, the term "complementarity determining region" or "CDR" refers to a sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. Generally, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3). The term "framework region" or "FR" is known in the art to refer to the non-CDR portion of the heavy chain variable region. Generally, there are four FRs in each heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4).

[0040] The precise amino acid sequence boundaries of a given CDR or FR can be determined using the methods described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol, 2003 January;27(1):55-77 ("IMGT" numbering scheme), and Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 June.8;309(3):657-70, (AHo numbering scheme).

[0041] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions accommodated by an insertion letter, e.g., "30a," and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbering. The contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0042] The "CDRs" or "complementarity-determining regions" of a given antibody or region thereof, such as its variable region, or individual designated CDRs (e.g., "CDR-H1," "CDR-H2," "CDR-H3"), should be understood to encompass complementarity-determining regions defined (or specific) by any of the known schemes. Similarly, the "FRs" or "framework regions" of a given antibody or region thereof, such as its variable region, or individual designated FRs (e.g., "FR-H1," "FR-H2"), should be understood to encompass framework regions defined (or specific) by any of the known schemes. In some instances, CDRs defined by a scheme for identifying specific CDRs or FRs, such as the IMGT, Kabat, Chothia, AbM, or contact method, are designated. In other cases, the specific amino acid sequences of the CDRs or FRs are provided. Unless otherwise specified, all specific CDR amino acid sequences described in this disclosure are IMGT CDRs. However, alternative CDRs defined by other schemes, such as those determined by the abysis Key Annotation (Website: abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), are also encompassed by the present disclosure. Exemplary heavy chain CDR (HCDR) sequences of anti-CD28 VHH antibodies are listed in Table 1 below.

[0043]

Table 1

[0044]

Table 2

[0045]

Table 3

[0046]

Table 4

[0047]

Table 5

[0048]

Table 6

[0049]

Table 7

[0050]

Table 8

[0051] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are generally human antibodies (recipient antibodies) in which residues from one or more CDRs are replaced with residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, llama, or non-human primate antibody, with the desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced with corresponding framework region residues from the donor antibody. Humanized antibodies can also contain residues that are not found in either the recipient antibody or the donor antibody. Such modifications can be made to further refine antibody function. A humanized sequence can be identified by its primary sequence and does not necessarily indicate the process by which the antibody was generated.

[0052] As used herein, the terms "specifically binds," "specifically binding," "binding specificity," or "specifically recognized" refer to an antigen-binding protein or antigen-binding fragment thereof that exhibits significant affinity for an antigen (e.g., CD28 antigen) and does not exhibit significant cross-reactivity with targets that are not CD28 proteins. As used herein, the term "affinity" refers to the strength of the interaction between the antigen-binding site of an antigen-binding protein or antigen-binding fragment thereof and the epitope to which it binds. In certain exemplary embodiments, affinity is measured by surface plasmon resonance (SPR), for example, in a Biacore instrument. As will be readily understood by one of skill in the art, antigen-binding protein affinity may be reported as a dissociation constant (KD) in molar concentration (M).

[0053] Specific binding can be determined according to any art-recognized means for determining such binding. In some embodiments, specific binding is determined by competitive binding assays (e.g., ELISA) or Biacore assays. In certain embodiments, the assays are performed at about 20°C, 25°C, 30°C, or 37°C.

[0054] The term "agonist," as used herein with respect to an antibody, means that the antibody stimulates or activates signal transduction through a target protein upon binding to the target protein expressed on the surface of a cell.

[0055] The term "antagonist," as used herein with respect to an antibody, means that the antibody inhibits signal transduction through a target protein upon binding to the target protein expressed on the surface of a cell.

[0056] Immunoglobulin Single Variable Domains (ISVDs) The term "immunoglobulin single variable domain" (ISV or ISVD), used interchangeably with "single variable domain," defines an immunoglobulin molecule in which the antigen-binding site is present on and formed by a single immunoglobulin domain. This term distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, dis-scFv), in which two immunoglobulin domains, in particular two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V H ) and the light chain variable domain (V L ) interact to form the antigen-binding site. In this case, V H and V L Both complementarity-determining regions (CDRs) of the nucleotides contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0057] In view of the above definition, the antigen-binding domain of a conventional four-chain antibody or an Fab fragment, an F(ab')2 fragment, an Fv fragment such as a disulfide-linked Fv, or an scFv fragment, or a diabody derived from such a conventional four-chain antibody (all known in the art), is not usually considered to be an immunoglobulin single variable domain, because in these cases, binding to each epitope of an antigen is usually not by one (single) immunoglobulin domain, but by a pair of (related) immunoglobulin domains, such as a light and heavy chain variable domain, i.e., the V of immunoglobulin domains which jointly bind to the respective epitope of the antigen. H -V L This is because they arise in pairs.

[0058] In contrast, an immunoglobulin single variable domain can specifically bind to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain consists of a single V H , a single V HH , or a single V L Formed by domains.

[0059] Therefore, a single variable domain may be used with any light chain variable domain sequence (e.g., V), as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit). L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., V H -sequence or V HH sequence) or a suitable fragment thereof.

[0060] Immunoglobulin single variable domains (ISVs) can be, for example, heavy chain ISVs, e.g., camelized V H or humanized V HH V containing H , V HH In one embodiment, this may be a camelized V H or humanized V HHV containing HH The heavy chain ISV can be derived from a traditional four-chain antibody or from a heavy chain antibody.

[0061] For example, immunoglobulin single variable domains can be single domain antibodies (or amino acid sequences suitable for use as single domain antibodies), "dAbs" or dAbs (or amino acid sequences suitable for use as dAbs), Nanobody® ISVs (as defined herein, V HH (including but not limited to), other single variable domains, or any suitable fragment of any one of these.

[0062] In particular, the immunoglobulin single variable domains are Nanobody® ISVs (e.g., humanized V HH Or Camelization V H V containing HH ) or a suitable fragment thereof. [Note: Nanobody® and Nanobodies® are registered trademarks of Ablynx NV.]

[0063] "V HH Domain" is V HH , V HH Antigen fragments, and V HH Also known as antibodies, they were originally described as the antigen-binding immunoglobulin variable domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains"; Hamers-Casterman et al. Nature 363:446-448, 1993). HH The term "variable domain" refers to these variable domains, as compared to the heavy chain variable domains (herein referred to as "V" domains) present in conventional four-chain antibodies. H domain) present in conventional four-chain antibodies and the light chain variable domain (referred to herein as "V L The domain name was chosen to distinguish it from the domains referred to as "domains." HH For further discussion, see the review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0064] The generation of immunoglobulin sequences such as VHHs has been widely described in various published literature, including WO 94 / 04678, Hamers-Casterman et al. 1993, and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001). In these methods, camelids are immunized with a target antigen to induce an immune response against the target antigen. The repertoire of VHHs obtained from this immunization is further screened for VHHs that bind to the target antigen.

[0065] In these instances, antibody generation requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources or during recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0066] Immunoglobulin sequences of different origins, including mouse, rat, rabbit, donkey, human, and camelized immunoglobulin sequences, can be sequenced using the methods described herein. Also, fully human, humanized, or chimeric sequences can be sequenced using the methods described herein. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs as described by Ward et al. (see, e.g., WO 94 / 04678 and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531-537, 1996), can be sequenced using the methods described herein. Furthermore, ISVs can be fused to form multivalent and / or multispecific constructs (one or more V HHFor multivalent and multispecific polypeptides containing domains and their preparation, see Conrath et al., J. Biol. Chem., Vol. 276, 10, 7346-7350, 2001, as well as, for example, WO 96 / 34103 and WO 99 / 23221).

[0067] "Humanized V HH " is a naturally occurring V HH corresponding to the amino acid sequence of the naturally occurring V HH One or more amino acid residues in the amino acid sequence (and particularly in the framework sequences) of the V H The term "humanized" includes amino acid sequences that have been "humanized" by substituting one or more amino acid residues (e.g., as shown above) present at the corresponding positions in the domain. This can be carried out in a manner known per se, for example, based on the prior art (e.g., WO 2008 / 020079), as will be clear to those skilled in the art. Furthermore, such humanized V HH It should be noted that can be obtained in any suitable manner known per se and is therefore not strictly limited to polypeptides obtained using naturally occurring VHH domain-containing polypeptides as starting material.

[0068] "Camelization V H " is a naturally occurring V H corresponding to the amino acid sequence of the naturally occurring V domain from a conventional four-chain antibody. H One or more amino acid residues in the amino acid sequence of the domain are replaced by the V HHThe term "camelized" includes amino acid sequences that have been "camelized" by substituting one or more amino acid residues present at the corresponding positions in the V domain. This can be carried out in a manner known per se, as will be clear to those skilled in the art, for example as described in the prior art (e.g., Davies and Riechman (1994 and 1996), supra). Such "camelized" substitutions are, as defined herein, H -V L The amino acids are inserted at positions that form and / or are present at interfaces and / or so-called camelid hallmark residues (see, e.g., WO 94 / 04678 and Davies and Riechmann (1994 and 1996), supra). In one embodiment, camelized V H V, which is used as a starting material or starting point for generating or designing H The sequence is V from mammals H Sequences, e.g., V of human origin H Array, e.g. V H 3 sequence. However, such camelized V H can be obtained in any suitable manner known per se, and therefore can be obtained without using naturally occurring V as starting material. H It should be noted that the polypeptide obtained using the polypeptide containing the domain is not strictly limited.

[0069] The structure of an immunoglobulin single variable domain sequence can be considered to consist of four framework regions ("FRs"), which are referred to in the art and herein as "framework region 1" ("FR1"); "framework region 2" ("FR2"); "framework region 3" ("FR3"); and "framework region 4" ("FR4"), respectively, interrupted by three complementarity-determining regions ("CDRs"), which are referred to in the art and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"), respectively.

[0070] In such immunoglobulin sequences, the framework regions may be any suitable framework sequence, and examples of suitable framework sequences will be clear to the skilled person on the basis of, for example, standard handbooks and the further disclosure and prior art referred to herein.

[0071] The framework sequences are immunoglobulin framework sequences or (suitable combinations of) framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization). For example, the framework sequences may be those of a light chain variable domain (e.g., V L sequence) and / or heavy chain variable domain (e.g., V H Array or V HH In a particular embodiment, the framework sequences may be derived from V HH -framework sequences derived from conventional V sequences (in which the framework sequences may optionally be partially or fully humanized) or camelized (as defined herein) H It can be either an array.

[0072] In particular, the framework sequences present in the ISV sequences used in the methods described herein may contain one or more hallmark residues (as defined herein), such that the ISV sequence is, for example, a humanized V HH or V containing camelized VH HH and Nanobody® ISVs such as: Non-limiting examples of (suitable combinations of) such framework sequences will be clear from the disclosure herein.

[0073] V H Domain and V HH The total number of amino acid residues in a domain will usually be in the range of 110 to 120, often 112 to 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

[0074] It should be noted, however, that the ISVs contained in the multivalent ISV polypeptides sequenced by this method are not limited by the origin of the ISV sequence (or of the nucleotide sequence used to express it), nor by the manner in which the ISV sequence or nucleotide sequence is (or was) generated or obtained. Thus, the ISV sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In one specific, but non-limiting embodiment, the ISV sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including, but not limited to, a "humanized" (as defined herein) immunoglobulin sequence (e.g., a partially or fully humanized mouse or rabbit immunoglobulin sequence, and particularly a partially or fully humanized VHV sequence). HH sequences), "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized V H sequences), as well as ISVs obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, joining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for manipulating immunoglobulin sequences well known to those of skill in the art; or any suitable combination of any of the foregoing.

[0075] Likewise, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and may be, for example, a sequence isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (and in particular an expression library), a nucleotide sequence prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using techniques for DNA synthesis known per se.

[0076] Generally, Nanobody® ISVs (especially (partially) humanized V HH Sequence and camelized V H V containing arrays HH A Nanobody® ISV may be characterized by the presence of one or more "hallmark residues" (also as further described herein) in one or more of the framework sequences (as described herein). Thus, in general, a Nanobody® ISV may be defined as an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (wherein FR1-FR4 refer to framework regions 1-4, respectively, CDR1-CDR3 refer to complementarity determining regions 1-3, respectively, and one or more of the hallmark residues are as further defined herein).

[0077] In particular, a Nanobody® ISV can be an immunoglobulin sequence with the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and framework sequences are as further defined herein).

[0078] More specifically, a Nanobody® ISV can be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively; One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from the hallmark residues set out in Table A below).

[0079] [Table 9]

[0080] The term "CD28," as used herein, refers to a transmembrane costimulatory signaling protein expressed on T cells. CD28 is involved in T cell activation, proliferation, cytokine production, and survival. An exemplary wild-type human CD28 amino acid sequence can be found under NCBI Reference Sequence: NP_006130.1; and UniProt Reference Number: P10747.

[0081] The term "tumor-associated antigen" or "TAA," as used herein, refers to any antigen that is highly expressed by tumor cells or in the tumor stroma. The term tumor-associated antigen includes TAAs that are not completely specific to the tumor, but rather are overexpressed on the tumor or its stroma. The term tumor-associated antigen also includes tumor-specific antigens that are expressed exclusively on tumors.

[0082] As used herein, a "CD28-binding polypeptide" or "anti-CD28 antibody" refers to any antigen-binding protein having at least one antigen-binding site that specifically binds to CD28. This encompasses bivalent forms of antibodies (such as native immunoglobulin molecules or F(ab)'2 fragments) having two CD28-binding sites, as well as monovalent forms of antibodies having a single CD28-binding site. As used herein, a CD28-binding polypeptide is typically an immunoglobulin single variable domain antibody (e.g., VHH)-containing polypeptide having at least one immunoglobulin single variable domain (e.g., VHH domain) that specifically binds to CD28. In certain embodiments, an anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain selected from any one of the VHH amino acid sequences in Table 2.

[0083] [Table 10]

[0084] [Table 11]

[0085] [Table 12]

[0086] [Table 13]

[0087] [Table 14]

[0088] [Table 15]

[0089] [Table 16]

[0090] [Table 17]

[0091] [Table 18]

[0092] The CD28-binding polypeptides provided herein include monovalent and multivalent (e.g., bivalent) constructs. In some embodiments, the CD28-binding polypeptides provided herein contain one or two immunoglobulin single variable domains (e.g., VHH domains), each of which individually binds to CD28.

[0093] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain that is at least about 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%, at least 99% or more identical to the amino acid sequence of any one of the VHH sequences listed in Table 2.

[0094] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 regions that are at least about 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%, at least 99% or more identical to any one of the HCDR1, HCDR2, or HCDR3 amino acid sequences listed in Table 1.

[0095] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.

[0096] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0097] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a monospecific antibody.

[0098] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a bispecific antibody.

[0099] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a multispecific antibody. In some embodiments, the multispecific antibody comprises at least one Fab domain. In certain embodiments, the VH and VL domains of the Fab are replaced with any one of the VHH amino acid sequences listed in Table 2. The Fab domain may function as a specific heterodimerization scaffold to which additional binding domains may be linked. The additional binding domain may be present in several different formats, including, but not limited to, another Fab domain, an scFv, or an sdAb (e.g., a VHH).

[0100] As used herein, "administering" or "administration" refers to the act of injecting or otherwise physically delivering an exogenous substance (e.g., an antibody provided herein) to a patient, such as, but not limited to, by pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. Where a disease or a symptom thereof is being managed or treated, administration of the substance typically occurs after the onset of the disease or a symptom thereof. Where a disease or a symptom thereof is being prevented, administration of the substance typically occurs before the onset of the disease or a symptom thereof and may be continued chronically to postpone or reduce the appearance or magnitude of disease-related symptoms.

[0101] "Effective amount" means an amount of an active pharmaceutical agent (e.g., an isolated binding polypeptide of the present disclosure) sufficient to achieve a desired physiological outcome in an individual in need thereof. The effective amount may vary from individual to individual depending on the health and condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, an assessment of the individual's medical condition, and other relevant factors.

[0102] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, the term "subject" as used herein refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, feral animals, farm animals, sport animals, and pets.

[0103] As used herein, the term "treatment" refers to any protocol, method, and / or agent that can be used to prevent, manage, treat, and / or ameliorate a disease or its associated symptoms. In some embodiments, the term "treatment" refers to any protocol, method, and / or agent that can be used to modulate an immune response to an infection or its associated symptoms in a subject. In some embodiments, the terms "therapies" and "therapy" refer to biological therapies, supportive therapies, and / or other therapies known to one of skill in the art, such as a healthcare professional, that are useful for preventing, managing, treating, and / or ameliorating a disease or its associated symptoms. In other embodiments, the terms "therapies" and "therapy" refer to biological therapies, supportive therapies, and / or other therapies known to one of skill in the art, such as a healthcare professional, that are useful for modulating an immune response to an infection or its associated symptoms in a subject.

[0104] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or symptoms associated therewith resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, e.g., an isolated binding polypeptide provided herein). The term "treating," as used herein, can also refer to altering the course of the disease in the subject being treated. Therapeutic effects of treatment include, but are not limited to, prevention of the onset or recurrence of the disease, alleviation of symptoms, reduction of the direct or indirect pathological effects of the disease, slowing the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0105] The terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm," as used herein, unless otherwise specified, are used interchangeably and refer to a cell (or cells) that exhibit uncontrolled proliferation and / or an abnormal increase in cell survival and / or an inhibition of apoptosis that interferes with the normal function of bodily organs and systems. This definition includes benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases. The terms "cancer" and "tumor" encompass solid cancers and hematologic / lymphatic cancers, and also encompass malignant, premalignant, and benign growths, such as dysplasias. This definition also includes cells with abnormal growth that is not prevented (e.g., immune evasion and immune evasion mechanisms) by the immune system (e.g., virally infected cells).

[0106] As used herein, "immune disease" refers to any disease associated with the development of an immune response in an individual, including a cellular and / or humoral immune response. Examples of immune diseases include, but are not limited to, inflammation, allergies, autoimmune diseases, transplant-related diseases, cancer, and viral infections.

[0107] As used herein, "autoimmune disease" refers to disease states and conditions in which an individual's immune response is directed against the individual's own components, resulting in an undesirable and often debilitating condition. As used herein, "autoimmune disease" is intended to further include autoimmune conditions, syndromes, and the like.

[0108] Expression of antigen-binding proteins In one aspect, nucleic acid molecules encoding the antibodies and antigen-binding fragments thereof disclosed herein are provided. Methods of making binding proteins comprising expressing these nucleic acid molecules are also provided.

[0109] Nucleic acid molecules encoding the antibodies disclosed herein are typically inserted into expression vectors for introduction into host cells, which can be used to produce desired quantities of the antibody. Thus, in certain aspects, the disclosure provides expression vectors comprising the nucleic acid molecules disclosed herein, as well as host cells comprising these vectors and nucleic acid molecules.

[0110] The term "vector" or "expression vector" is used herein to mean a vector used in accordance with the present disclosure as a vehicle for introducing and expressing a desired gene in a cell. As known to those skilled in the art, such vectors may be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors compatible with the present disclosure will contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0111] Numerous expression vector systems can be utilized for the purposes of this disclosure. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Furthermore, cells into which the DNA has integrated into the chromosome can be selected by introducing one or more markers that allow for selection of transfected host cells. Markers can provide prototrophy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or can be introduced into the same cell by cotransformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include signal sequences, splice signals, and transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with a heavy chain constant region gene (eg, a human constant region gene) synthesized as discussed above.

[0112] In other embodiments, antibodies can be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as antibody heavy and light chains, can be produced from a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRES) to produce relatively high levels of polypeptides in eukaryotic host cells. Suitable IRES sequences are described in U.S. Patent No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those skilled in the art will understand that such expression systems can be used to effectively produce the full range of polypeptides disclosed in the present application.

[0113] More generally, once a vector or DNA sequence encoding an antibody or fragment thereof has been prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Plasmid introduction into host cells can be accomplished by a variety of techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See Ridgway, AAG, "Mammalian Expression Vectors," Chapter 24.2, pp. 470-472, in Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Plasmid introduction into the host can also be by electroporation. Transformed cells are grown under conditions appropriate for the production of light and heavy chains, and assayed for the synthesis of heavy and / or light chain proteins. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0114] As used herein, the term "transformation" refers broadly to the introduction of DNA into a recipient host cell, resulting in a change in the genotype.

[0115] Similarly, a "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA techniques and encoding at least one heterologous gene. In describing the process of isolating a polypeptide from a recombinant host, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the antibody, unless clearly specified otherwise. In other words, recovery of polypeptide from the "cells" can mean either from spun down whole cells, from the supernatant of a lysed cell culture, or from the cell culture fluid containing both the medium and suspended cells.

[0116] In one embodiment, the host cell line used for antibody expression is of mammalian origin. One skilled in the art can determine the particular host cell line most suitable for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 carrying the SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HEK (human kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). In one embodiment, the cell line provides altered glycosylation, e.g., afucosylation, of the antibody expressed therefrom (e.g., PER.C6® (Crucell) or a FUT8-knockout CHO cell line (POTELLIGENT® cells) (Biowa, Princeton, NJ)). In one embodiment, NSO cells may be used. CHO cells are particularly useful. Host cell lines are typically available from commercial services, e.g., the American Tissue Culture Collection, or from authors in the published literature.

[0117] In vitro production allows for scale-up to obtain large quantities of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogenous suspension culture in airlift reactors or continuous stirred reactors, or culturing cells immobilized or entrapped, for example, in hollow fibers, in microcapsules, on agarose microbeads, or on ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immuno)affinity chromatography.

[0118] Genes encoding the antibodies featured in this disclosure can also be expressed in non-mammalian cells, such as bacterial cells or yeast cells or plant cells. In this regard, it will be understood that various unicellular microorganisms other than mammals, such as bacteria, can also be transformed, i.e., can be grown in culture or fermentation. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as Escherichia coli or strains of Salmonella, Bacillaceae family, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. It will further be appreciated that when expressed in bacteria, the binding proteins may become part of inclusion bodies. In some embodiments, the binding proteins are subsequently isolated, purified, and assembled into functional molecules. In some embodiments, the binding proteins of the present disclosure are expressed in bacterial host cells. In some embodiments, the bacterial host cells are transformed with an expression vector comprising a nucleic acid molecule encoding a binding protein of the present disclosure.

[0119] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although many other strains are commonly available. For expression in Saccharomyces, plasmid YRp7, for example (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)), is commonly used. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow on tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0120] Methods of Administering Antigen Binding Proteins Methods for preparing and administering antigen binding proteins (e.g., anti-CD28 antibodies or antigen-binding fragments thereof disclosed herein) to a subject are well known to, or can be readily determined by, those of skill in the art. Routes of administration of the antigen binding proteins of the present disclosure can be oral, parenteral, inhalation, or topical. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all of these modes of administration are expressly contemplated within the scope of the present disclosure, the administration form will be an injectable solution, particularly a solution for intravenous injection, intraarterial injection, or infusion. Typically, a suitable injectable pharmaceutical composition may include a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer (e.g., human albumin), and the like. However, in other methods consistent with the teachings herein, modified antibodies can be delivered directly to the site of harmful cell populations, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0121] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. In the compositions and methods of the present disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M or 0.05 M phosphate buffer, or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases. More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0122] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, can also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0123] In either case, sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide alone or a modified binding polypeptide in combination with other active agents) in the required amount in an appropriate solvent, with one or a combination of ingredients enumerated herein, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods typically include vacuum drying and freeze-drying, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof. Preparations for injection are processed and filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under aseptic conditions according to methods known in the art. Furthermore, preparations can be packaged and sold in the form of kits, such as those described in U.S. Patent Application Publication No. 20020102208 and U.S. Patent No. 6,994,840, each of which is incorporated herein by reference. Such articles of manufacture may include a label or package insert indicating that the associated composition is useful for treating a subject suffering from or predisposed to an autoimmune or neoplastic disorder.

[0124] The effective dose of the compositions of the present disclosure for treating the above conditions will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other pharmaceutical agents administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, although non-human mammals, including transgenic mammals, can also be treated. Treatment dosages can be titrated to optimize safety and efficacy using routine methods known to those skilled in the art.

[0125] As previously discussed, the antigen binding proteins, immunoreactive fragments or recombinant forms thereof of the present disclosure may be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders. In this regard, it will be understood that the disclosed antigen binding proteins are formulated to facilitate administration and to promote stability of the active agent.

[0126] Pharmaceutical compositions according to the present disclosure typically comprise a pharmaceutically acceptable, non-toxic, sterile carrier, such as saline, non-toxic buffers, preservatives, etc. For purposes of this application, a pharmaceutically effective amount of a modified antigen-binding protein, immunoreactive fragment, or recombinant thereof, conjugated or unconjugated to a therapeutic agent, shall be held to mean an amount sufficient to achieve effective binding to the antigen and achieve a benefit, e.g., to ameliorate the symptoms of a disease or disorder, or to detect a substance or cell. In the case of tumor cells, the modified binding polypeptide will typically be able to interact with a selected immunoreactive antigen on tumor cells or immunoreactive cells, resulting in increased death of those cells. Of course, the pharmaceutical compositions of the present disclosure may be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

[0127] In accordance with the scope of the present disclosure, antigen-binding proteins of the present disclosure may be administered to humans or other animals in accordance with the aforementioned treatment methods in an amount sufficient to produce a therapeutic or prophylactic effect. The antigen-binding proteins of the present disclosure may be administered to such humans or other animals in conventional dosage forms prepared by combining an antibody of the present disclosure with a conventional pharmaceutically acceptable carrier or diluent in accordance with known techniques. It will be recognized by those skilled in the art that the form and characteristics of the pharmaceutically acceptable carrier or diluent will be dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables. Those skilled in the art will further appreciate that cocktails comprising one or more binding polypeptides described in the present disclosure may prove particularly effective.

[0128] The biological activity of the pharmaceutical compositions defined herein can be determined, for example, by cytotoxicity assays as described in the Examples below, WO 99 / 54440, or Schlereth et al. (Cancer Immunol. Immunother. 55 (2006), 503-514). Biological activity can also be determined by T cell activation assays, for example, through the detection of pro-inflammatory or anti-inflammatory cytokine expression. "Efficacy" or "in vivo efficacy," as used herein, refers to the response to therapy with the pharmaceutical compositions of the present invention, for example, using standardized NCI efficacy criteria. The success or in vivo efficacy of therapy using the pharmaceutical compositions of the present invention refers to the effectiveness of the composition for its intended purpose, i.e., the ability of the composition to cause its desired effect, i.e., the depletion of pathological cells, e.g., tumor cells, or the suppression of activated immune cells. In vivo efficacy can be monitored by established standard methods for each disease entity, including, but not limited to, white blood cell count, differential, fluorescently activated cell sorting, and bone marrow aspirate. In addition, various disease-specific clinical chemistry parameters and other established standard methods may be used. [Example]

[0129] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions featured in this invention, and are not intended to limit the scope of what the inventors regard as their invention. While attempts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0130] Example 1 Materials and Methods Cell lines and cells derived from human donors Buffy coats from healthy donors were obtained from the French blood bank (Etablissement Français du Sang). After purification of peripheral blood mononuclear cells by Ficoll gradient, global CD3 +T cells were enriched using the RoboSep™ Human T Cell Enrichment Kit (StemCell Technologies, 19051) according to the manufacturer's instructions. Dendritic cells (DCs) were generated by culturing monocytes isolated from peripheral blood mononuclear cells using a Monocyte Purification Kit (StemCell Technologies, 17858) in vitro with 200 ng / mL IL-4 (Miltenyi Biotec, 130-093-922) and 200 ng / mL granulocyte-macrophage colony-stimulating factor (GMF) (Miltenyi Biotec, 130-093-866) for 7 days. FreeStyle™ HEK293-FS cells were purchased from Invitrogen, Jurkat cells were purchased from the American Type Culture Collection (ATCC, TIB-152), and Jurkat-IL-2-Luc2P cells were purchased from Promega. All cell lines were grown at 37°C in a humidified atmosphere containing 5% CO2 in the medium recommended by the cell supplier. All cell line culture media and reagents were purchased from Gibco. TGN1412 and 9.3 antibodies were produced and purified in-house.

[0131] Transfection of CD28 expression FreeStyle™ HEK293-FS cells were transfected with a high-quality plasmid preparation encoding human CD28 using 293fectin™ (Gibco, 12347-019) according to the manufacturer's instructions. CD28 expression was assessed 48 hours post-transfection by flow cytometry using AF647-conjugated anti-human CD28 mAb (BD Pharmingen™, 560683).

[0132] Example 2. Llama immunization and library construction Immunization and library construction were performed by the VIB Nanobody Service Facility (Brussels, Belgium). As shown in Figure 1, llamas were immunized with CD28 and TAA (DNA immunization) by four intradermal injections, each with approximately 2 mg of vector harboring the gene of interest (human full-length CD28 and human full-length TAA). After each injection, the animals were electroporated to introduce the DNA construct into the animal cells. Three weeks after the final DNA injection, the animals were subcutaneously boosted with recombinant proteins (human CD28 [Sino Biological, 11524-HCCH], in-house human TAA; adjuvant for protein boost, GERBU LQ 3000). Four days after the protein boost, anticoagulated blood was collected for VHH library construction, as shown in Figure 1. Figure 1 summarizes the general workflow for CD28xTAA bispecific screening: llamas were immunized with CD28 and TAA (DNA immunization), VHH libraries were constructed for phage display selection on recombinant proteins and cells; clones were rapidly screened for binding, sequenced, and cloned in-frame with human construct domains CH1 or Cλ to generate bsFabs by transient transfection in FreeStyle™ HEK293-FS cells. Cell supernatants were functionally screened in primary cell assays, and clones of interest were further characterized for their binding properties and biological function.

[0133] A VHH library was constructed for phage display selection on recombinant proteins and cells as previously described. Total ribonucleic acid was extracted from peripheral blood lymphocytes and used as a template for first-strand complementary DNA synthesis with an oligo(deoxythymine) primer. VHH coding sequences were amplified from the complementary DNA by polymerase chain reaction, digested with PstI and NotI, and cloned between the PstI and NotI sites of the phagemid vector pHEN4 upstream of the human influenza hemagglutinin decapeptide tag. Approximately 108 A VHH library of independent transformants was obtained. Clones were then rapidly screened for binding, sequenced, and cloned in-frame with the human construct domains CH1 or Cλ to generate bsFabs by transient transfection in FreeStyle™ HEK293-FS cells, as shown in Figure 1. Cell supernatants were functionally screened in primary cell assays, and clones of interest were further characterized for their binding properties and biological function.

[0134] Phage display panning Ready-made phage-VHH preparations were obtained as described. Bacterial libraries were grown in 2YTAG medium (2xYT medium, ampicillin 100 μg / mL, glucose 2%) until the optical density at 600 nm (OD600) reached 0.5 and infected with M13K07 helper phage (Invitrogen). After centrifugation, bacteria were resuspended in 2YTAK medium (2xYT medium, ampicillin 100 μg / mL, kanamycin 50 μg / mL) and grown overnight. Phage particles were precipitated from the culture supernatant by adding 20% ​​(weight / volume [w / v]) polyethylene glycol 8000 (PEG8000) and 2.5% NaCl, centrifuged, and resuspended in phosphate-buffered saline (PBS). The phages were subjected to another washing and precipitation step and finally resuspended in cold PBS / glycerol 15% (volume / volume [v / v]).

[0135] Panning against recombinant proteins Panning against recombinant proteins was performed as described. M-450 epoxy beads (Dynabeads, Invitrogen) were coated with His-tagged CD28 or TAA-recombinant according to the manufacturer's recommendations. Phage-VHH libraries (10 11Phages (selection round) and beads (coated and naked) were saturated for 1 h at room temperature (RT) in PBS / milk 2% (w / v). First, the phage-VHH library was depleted twice by 30 min incubation on naked beads to remove nonspecific clones. Unbound phage-VHHs were collected and incubated with target-bound beads in PBS / milk 2% (w / v) for 2 h at RT. After 10 washes with PBS / Tween 0.1% (v / v) and two washes with PBS, bound phage-VHHs were resuspended in PBS (output selection), added to exponentially growing TG1 bacteria, and amplified overnight in 2YTAG medium or plated on 2YTAG plates for a new round of panning.

[0136] Panning against cells Panning was performed at 4°C on CD28- or TAA-transfected FreeStyle™ HEK293-FS cells. After two washes with PBS, the cell pellet was resuspended in PBS and loaded onto a fetal bovine serum / Percoll gradient as previously described. After centrifugation, the cell layer was collected and washed twice with PBS. The recovered cells with bound phage were added to a second fetal bovine serum / Percoll gradient and washed before mechanical lysis using beads (Dynabeads, Invitrogen). The recovered phage-VHHs were used to infect exponentially growing Escherichia coli TG1 bacteria and were either amplified overnight in 2YTAG medium or plated onto 2YTAG plates for a new round of panning.

[0137] Fab-like construction, production, and purification After amplification by polymerase chain reaction, complementary DNA of anti-CD28 VHHs, anti-TAA VHHs, or anti-foot-and-mouth disease virus (FMDV) VHHs (Harmsen et al. Veterinary microbiology. 2007;120(3-4):193-206) was cloned in frame with human influenza hemagglutinin and either a human CL domain or a human IgG1 CH1 domain fused to a 6-His tag into a proprietary mammalian expression vector. Plasmids were purified using a NucleoBond Macherey-Nagel kit and Sanger sequenced. Bispecific (bsFab) or bivalent Fab-like (bvFab) antibodies were produced by cotransfecting FreeStyle™ HEK293-FS cells with a mixture of two plasmids encoding two separate (bsFab) or two identical (bvFab) VHHs fused to each of the Fab constant domains. Supernatants were collected after 7 days, purified on a nickel affinity column, and analyzed on a CALIPER CXII (Perkin Elmer).

[0138] Flow cytometry binding and competition assays All flow cytometry assays were performed on a MACSQuant cytometer (Miltenyi Biotec, Germany) using V-bottom 96-well microtiter plates. Cells were gated on viable single cells (Dapi staining) and analyzed at 10 4 Events were collected for each sample. Data were analyzed using MACSQuant software and results were expressed as median fluorescence intensity.

[0139] Jurkat cells were first incubated with serial dilutions of bvFab for 1 h at 4° C., followed by incubation with human CD80-Fc fusion at its effective concentration of 90% (EC90) for 30 min at 4° C. Bound ligand was detected with an anti-human IgG (Fc-specific) mAb (Sigma, I2136) followed by an Alexa647-conjugated goat anti-mouse mAb (Invitrogen, A11013).

[0140] Competitive binding assay (TGN1412 and 9.3 mAb competition using enzyme-linked immunosorbent assay [ELISA]) Phage-VHH generation in 96-well plates Individual TG1 colonies of the CD28 VHH of interest were grown in 2YTA medium at 37° C. until an OD600 of 0.5 was reached. Cells were then infected with M13K07 helper phage and grown overnight in 2YTAK at 30° C. The supernatant containing the phage-VHH was collected and used for testing.

[0141] ELISA ELISA was performed in PBS at 4°C on Nunc® MaxiSorp™ 96-well plates (Sigma) pre-coated overnight with 1 μg / mL human CD28 recombinant protein and further saturated with PBS / milk 2% (w / v) for 1 h at RT. Next, serial dilutions of competitor mAb (TGN1412 or 9.3) were incubated for 1 h at RT, and phage-VHHs at their EC90 were added for an additional 30 min at RT. After multiple washes in PBS / Tween 0.1% (v / v), anti-M13 horseradish peroxidase-conjugated mAb (Santa Cruz Biotechnology, sc-53004) was added to detect bound phage-VHHs. Peroxidase activity was detected using 3,3',5,5'-tetramethylbenzidine substrate (Thermo Scientific, 34029). Absorbance was measured at OD 450 nm on a SpectraMax microplate reader (Molecular Devices) after addition of sulfuric acid stop solution.

[0142] Reporter assay For conditions involving CD3 preactivation Wells were coated (plates Costar 3917) with 50 μL of anti-CD3 (UCHT-1 clone, BioLegend, BLE300414) and stored overnight at 4° C., followed by washing twice with 100 μL of PBS / well. Jurkat-IL-2-Luc2P cells were harvested during their exponential growth phase, and 25 μL of the cell suspension was added to a 96-well plate (50,000 cells / well) with 25 μL of test compound.

[0143] Cross-linking experiments Test compounds were preincubated with saturating concentrations of anti-human Fab (Sigma, I5260) or anti-human Fc (Sigma, I2136) for 30 min at RT. Jurkat-IL-2-Luc2P cells were harvested during their exponential growth phase, and 25 μL of the cell suspension was added to a 96-well plate (50,000 cells / well) with 25 μL of crosslinked or non-crosslinked test compound.

[0144] For conditions involving TAA-expressing cells Jurkat-IL-2-Luc2P cells were harvested during their exponential growth phase and mixed with TAA-expressing cells to obtain a final ratio of 1:1 between reporter and accessory cells. 25 μL of the cell suspension was added to a 96-well plate (50,000 cells / well) along with 25 μL of test compound.

[0145] For all three conditions (CD3 preactivation, crosslinking, and TAA-expressing cells), plates were incubated for 6 hours in a humidified incubator at 37°C with 5% CO2. Next, 50 μL of Bio-Glo™ (Promega, G7941) reagent, prepared according to the manufacturer's instructions, was added to each well and mixed. Complete cell lysis was allowed to occur for at least 5 minutes, after which luminescence was measured using an EnVision multimode plate reader (Perkin Elmer).

[0146] T cell activation assay U-bottom 384-well plates were coated overnight at 4°C with 5 μg / mL anti-human CD3 antibody (eBioscience, 15288347, OKT3 clone). Plates were washed with PBS, and 50,000 T cells were added in complete X-VIVO™ 15 culture medium (Lonza, BE02-060F) in the presence of 10, 30, and 100 nM negative (isotype or FMDV bvFab) and positive (TGN1412 or 9.3 mAb) control antibodies or test compounds (CD28×FMDV bsFab and CD28 bvFab) and incubated at 37°C in a 5% CO2 incubator. After 6 days of incubation, supernatants were collected and stored at -20°C until cytokine measurement. Promega CellTiter-Glo® reagent was added to the cells for cell counting. Cytokine levels were measured using a homogeneous time-resolved fluorescent human IFNγ / tumor necrosis factor (TNF)α cytokine kit according to the manufacturer's instructions (Cisbio). Samples were read on a PHERAstar FSX multimode reader (BMG Labtech). Data were expressed as a percentage effect compared to the negative control.

[0147] mixed leukocyte reaction (MLR) Carboxyfluorescein succinimidyl ester (CFSE)-labeled CD3 + T cells (1×10 5 ) and allogeneic DC (1 × 10 4) were co-cultured with or without 10 nM negative (FMDV bvFab) and positive (TGN1412 or 9.3 mAb) control antibodies or test compounds (CD28xTAA bsFab and CD28 bvFab) at the beginning of the assay. After 4 days, supernatants were collected and cytokine levels were measured using the CBA Human Th1 / Th2 / Th17 Kit (BD Biosciences, 550749) according to the manufacturer's instructions. Cells were stained with antibody cocktails for CD4 (BD Biosciences, 563550), CD8 (BD Biosciences, 560662), CD25 (BD Biosciences, 555434), and CD69 (BD Biosciences, 562617). T cell proliferation was measured using CFSE dilutions. Samples were analyzed on a Fortessa X-20 flow cytometer (BD Biosciences).

[0148] Epitope binning Tandem epitope binning of CD28 antibodies was performed by surface plasmon resonance (SPR) using a BIAcore T200 (updated T100, Cytiva Life Sciences, France) instrument with HBS EP+ (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM ethylenediaminetetraacetic acid, 0.005% [v / v] surfactant P20; Cytiva Life Sciences Biacore BR100826) as the running buffer. Anti-human Fc antibodies (Human Antibody Capture Kit, Cytiva Life Sciences BR-1008-39) were covalently coupled to a sensor chip CM5 (Cytiva Life Sciences, Biacore BR100530).

[0149] Each of the four flow cells was prepared independently. First, all four flow cells were activated with a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide mixture (75-11.5 mg / mL) for 7 minutes using a flow rate of 5 μL / min (amine coupling kit, Cytiva LifeSciences, BR100050). Anti-human Fc antibodies were diluted to 25 μg / mL in 10 mM acetate, pH 5.0, and coupled for 7 minutes using a flow rate of 5 μL / min. Next, unbound sites were inactivated with a 7-minute burst of 1 M methanolamine, pH 8.5, using a flow rate of 5 μL / min. Approximately 10,000 resonance units (RU) of antibody were typically observed in all spots.

[0150] After surface preparation, huCD28-huFc (Sino Biological, 11524-H02H) was diluted to 0.5 μg / mL in running buffer and captured at 5 μL / min for 120 seconds to 200 RU (only on FC2 or FC4). Duplicate injections were performed with a first injection of crude HEK293-FS supernatant containing the first test bsFab for 120 seconds at 30 μL / min (saturation and stability conditions), followed by a second injection of crude HEK293-FS supernatant containing the second test bsFab for 60 seconds (both FC1-FC2 or FC3-FC4). Dissociation was then monitored for 60 seconds. The surface was regenerated with a 30-second burst of 3M MgCl2 using a flow rate of 10 μL / min.

[0151] Sensorgrams were double-referenced by subtracting a reference spot (without huCD28-huFc) and buffer injection. Normalized competition signals (C = normalized signal SN2 - normalized signal SN1) were calculated for each antibody pair. Normalized signals correspond to signals normalized to huCD28-Fc capture. For quantitative data, C<+5 represented no binding of SN2 and competition; C>+5 represented no binding of SN2 and competition. For quantitative data based on normalized signals (full or partial competition), the formula C*100 / (theoretical NormB SN2 - NormB dissociation SN1) was used for analysis.

[0152] statistical analysis Two-way hierarchical clustering using a Euclidean distance matrix and Ward's aggregation method was performed using statistical software R3.2.3 to regroup binders with the same profile when each binder was injected first or second into clusters. The number of clusters was selected using a graphical representation of the number of classes as a function of the inertial jump of the dendrogram (where inertia is a dispersion measure). Pearson correlation coefficient p-values ​​were calculated using a two-tailed t-test.

[0153] Example 3 result VHH selection, screening, and reformatting This study aimed to investigate the use of VHH-based bsFabs in activating T cells via the CD28 receptor to kill tumor cells and provide a detailed characterization of a panel of CD28 constructs in terms of binding, epitope diversity, agonistic and antagonistic properties.

[0154] VHHs against CD28 and TAAs were isolated using phage display. A highly diverse VHH sequence library was constructed, and biopanning was performed in two rounds of selection against purified recombinant ectodomains of each antigen or target-expressing cells. After selection, one plate (95 clones) was screened for each round using ELISA against the recombinant antigen (data not shown). For each selection, more than 80% of the clones tested were specific for their target. All specific clones were further sequenced, and sequence analysis revealed high diversity and low redundancy in the isolated CD28 VHHs, as shown in Figure 2. Briefly, sequence analysis consisted of aligning the VHH sequences of all hits identified by phage display selection using Clustal Omega; subsequently, the sequence tree shown in Figure 2 was constructed using the guide tree output obtained with Clustal Omega. The scale bar in the figure corresponds to the distance between sequences. The rectangular tags represent the 48 VHHs tested in epitope binning, color-coded by bin (blue = D, orange = D*, green = E, red = F, gray = undetermined). Black stars highlight compounds 2–18, which were further tested in cellular assays. Non-statistical analysis (binning by second injection) clearly identified subgroups in group D or 16; however, no statistically significant differences were observed.

[0155] Cluster diversity analysis based on 95% sequence identity revealed that among 277 CD28-specific clones, 66 clusters of VHHs were isolated on recombinant proteins and 56 on CD28-transfected cells. Similar results were found for TAA-specific VHHs (data not shown).

[0156] Subsequently, 47 anti-CD28 VHHs with high sequence diversity (colored tags in Figure 2 ) were cloned fused to the C-lambda IgG domain, and 39 anti-TAA VHHs were cloned fused to the CH1 IgG domain for eukaryotic expression.

[0157] Example 4. Generation and functional screening of CD28xTAA bsFab CD28 × TAA bsFabs were generated using the compact, linker-free Fab-like format shown in Figure 1 (33). More precisely, a matrix combination of 47 anti-CD28 (plus one negative control) × 39 anti-TAA (plus one negative control) bsFabs (i.e., 24 96-well plates) was produced at a 1 mL scale. Quality control was performed on the supernatants of 288 randomly selected wells across the 24 96-well plates, for a total of three quality control plates. The quality control consisted of three components: sufficient antibody quantity was confirmed by titrating antibody production (approximately 20 μg / mL) in HEK293-FS supernatants; calipers were performed to confirm the presence of dimers in the supernatants; and ELISA binding capacity of the Fab-like antibodies produced in HEK293-FS supernatants on the two targets, CD28 and TAA, was analyzed, revealing efficient binding of nearly all of the 288 tested antibodies.

[0158] Supernatants from 24 96-well plates of bsFabs were then tested in a functional assay to assess the ability of the bsFabs to induce killing of TAA-expressing tumor cells by purified T cells and IL-2 and IFNg secretion at 72 hours. Despite experimental verification of the expected results obtained with positive (CD3 × TAA) and negative (CD3 × irrelevant target and CD28 × irrelevant target) control bsAbs, none of the 1,800 bsFabs demonstrated tumor cell killing or IFNg secretion (data not shown). Replicate experiments with one-quarter of the constructs confirmed the initial results. Finally, a set of 34 bsFabs was purified and tested in the same assay in a dose-ranging experiment (0.1–300 nM) (data not shown). Even at the highest tested dose of 300 nM, no tumor cell killing or IFNg secretion was observed. Little IL-2 secretion was detected for some clones at the highest dose.

[0159] Example 5. Detailed characterization of anti-CD28 VHHs Epitope diversity To test the hypothesis that the lack of killing properties of all tested bsFabs may be due to a lack of diversity in the epitopes and functions of the selected CD28 VHHs, we first characterized the epitope diversity of the anti-CD28 VHHs.

[0160] Epitope binning experiments using SPR for 48 CD28 VHHs (including the 47 tested above) revealed three statistically significant epitope clusters (two-way hierarchical clustering using a Euclidean distance matrix and Ward's aggregation method) that could be further refined into six subclusters, as shown in Figure 3 and the table in Figure 11. Briefly, Figure 3 shows the epitope binning of CD28 bsFabs, i.e., competition between anti-CD28 VHHs by SPR. Figure 3A shows the following experimental scheme: human CD28-Fc was captured on a sensor chip CM5 using an anti-human Fc antibody, followed by the addition of the first CD28 x TAA bsFab, followed by the second one for binning analysis, ultimately testing 48 x 48 constructs. Figure 3B shows the collected competition data in a heat map showing whether each VHH pair competed (green indicates blocked, red indicates unblocked). The heatmap was processed using a dendrogram and two-way hierarchical clustering to generate three significantly different epitope clusters (A, B, C, and D, and E and F depending on the injection sequence). As shown in Figure 3B, asymmetry depending on the injection sequence was observed.

[0161] A limited set of 17 CD28 VHHs (compounds 2–18, identified by stars in Figure 2) spanning different epitope bins and maximizing sequence diversity was selected to perform an orthogonal analysis of epitope diversity in a fluorescently labeled cell sorting competition assay with CD80 or an ELISA competition assay with TGN1412 or 9.3 mAb. This analysis revealed that the CD28 VHHs could be classified as competitors and non-competitors of CD80, as shown in Figure 8, which shows the competition of CD28 bvFab with CD80 using flow cytometry, and independently as full, partial, or non-competitors of TGN1412 or 9.3 mAb. In summary, Figure 8 shows serial dilutions of CD28 bvFab incubated with Jurkat cells before the addition of recombinant human CD80-Fc protein at its EC90. Ligand binding was detected via flow cytometry using an Alexa 647-conjugated anti-human Fc mAb. Results are expressed as median fluorescence intensity (MFI). Epitope binning and all competition data for the VHH subsets are reported in the table shown in Figure 7, which is a summary table of results from epitope binning and competition with the TGN1412 and 9.3 benchmark antibodies or CD80, one of the natural ligands of CD28.

[0162] Example 6 Agonist properties: Reporter assay To evaluate the agonist potential of CD28 compounds, a bioluminescent reporter cell-based assay was used, which included a genetically engineered Jurkat T cell line expressing a luciferase reporter gene driven by the IL-2 promoter.

[0163] First, a series of CD28xTAA bsFabs were tested in the presence of TAA-expressing HCT116 cells, both with and without suboptimal TCR activation of engineered Jurkat cells via anti-CD3-coated mAb. All bsFabs enabled activation of the IL-2 promoter when the TCR was preactivated (Figure 9). This means that the bsFabs can simultaneously bind to two cells, each expressing either CD28 or a TAA. Figure 9 shows that the CD28xTAA bsFabs can simultaneously bind to two cells expressing either CD28 or a TAA. Briefly, a 1:1 ratio of Jurkat-IL-2-Luc2P cells and TAA-expressing HCT116 cells were added to a 96-well microplate, either pre-coated with anti-CD3 mAb or not. The cells were then incubated with 30 nM of test compound at 37°C for 6 hours before luminescence was measured. Luciferase luminescence signals normalized to those of cells treated with a negative control (IRR = FMDV bvFab) are shown (S / B = signal to background). Each bar on the graph in Figure 9 represents the average value of two independent experiments.

[0164] Eleven CD28 VHHs (compounds 2–12 in the table in Figure 7 ), ensuring maximum epitope coverage and sequence diversity, were retained for detailed functional characterization. They were produced and purified in a Fab-like format, either as CD28×FMDV bsFab (monovalent on CD28) or as CD28 bvFab (bivalent Fab-like, in which the same VHH is fused to both CH1 and CL). Evaluation of these bsFabs and bvFabs without suboptimal TCR activation by anti-CD3-coated mAbs revealed that, in the absence of cross-linking, none of the constructs were active, regardless of valency, whereas TGN1412 and 9.3 mAbs were highly active, as shown in Figures 10A and 10B. Figures 4 and 10 show the evaluation of the CD28 bsFabs and bvFabs in the following IL-2 luciferase reporter assay. Jurkat-IL-2-Luc2P cells were added to 96-well microplates precoated with anti-CD3 mAb, as shown in Figures 4 and 10C-F, or as shown in Figures 10A and B. Cells were then incubated with 100 nM of pre-crosslinked or uncrosslinked bsFab and bvFab at 37°C for 6 hours, after which luminescence was measured. Luciferase luminescence signals normalized to those of cells treated with a negative control (IRR = FMDV bvFab) are shown (S / B = signal to background). These assays were performed in triplicate and are plotted in Figures 4 and 10.

[0165] However, when cross-linked, several bvFabs in bins E and F were active at levels lower than those seen with TGN1412 and 9.3 mAb, as shown in Figure 10B. The ability of 9.3 mAb to induce luciferase expression without CD3 coactivation was unexpected in light of its description as an agonist rather than a superagonist. This suggests some discrepancy between T cell activation and the Jurkat reporter assay.

[0166] Upon CD3 coactivation in the absence of cross-linking, one bvFab from bin E and three of four bvFabs from bin F were able to induce IL-2 pathway activation, whereas all other bsFabs and bvFabs were inactive in soluble conditions, as shown in Figure 4C, which shows monovalent CD28xFMDV bsFabs versus bivalent CD28 bvFabs without cross-linking, and Figure 10E, which shows monovalent CD28xFMDV bsFabs versus bivalent CD28 bvFabs without cross-linking after CD3 preactivation.

[0167] When cross-linked, the IL-2 pathway was induced for all bvFabs, as shown in Figure 4B, which shows bivalent CD28 bvFabs both with and without cross-linking, and Figure 10D, which shows bivalent CD28 bvFabs both with and without cross-linking after CD3 preactivation, and for all bin E bsFabs and one bin F bsFab, as shown in Figure 4A, which shows monovalent CD28xFMDV bsFabs both with and without cross-linking, and Figure 10C, which shows monovalent CD28xFMDV bsFabs both with and without cross-linking after CD3 preactivation. Superior activity was observed for most CD28 clones under the bvFab than bsFab format, revealing the greatest impact on IL-2 induction when bivalency and cross-linking were combined, as shown in Figure 4B, which shows bivalent CD28 bvFab both with and without cross-linking, and Figure 4D, which shows monovalent CD28xFMDV bsFab versus bivalent CD28 bvFab with cross-linking, and Figure 10D, which shows bivalent CD28 bvFab both with and without cross-linking after CD3 pre-activation, and Figure 10F, which shows monovalent CD28xFMDV bsFab versus bivalent CD28 bvFab with cross-linking after CD3 pre-activation.

[0168] Clones 5 and 7 from bin E demonstrated unique behavior in that their cross-linking enabled CD28 activation, as shown in the plots in Figure 4A, which shows monovalent CD28xFMDV bsFab with and without cross-linking, and Figure 4B, which shows bivalent CD28 bvFab with and without cross-linking, but did not enable increased valency, as shown in Figure 4C, which shows monovalent CD28xFMDV bsFab versus bivalent CD28 bvFab without cross-linking, and Figure 4D, which shows monovalent CD28xFMDV bsFab versus bivalent CD28 bvFab with cross-linking. In both bsFab and bvFab formats, these two clones were inactive and active in the absence and presence of cross-linking, respectively; however, the bvFab was less active than the bsFab.

[0169] Agonistic properties: T cell activation The agonistic properties of the constructs were then evaluated using a primary T cell activation assay. Compounds were tested in soluble conditions without cross-linking, which resulted in suboptimal TCR activation via anti-CD3-coated mAbs. Some, but not all, CD28 bvFabs demonstrated the ability to increase IFNg secretion, as shown in Figure 5. Figure 5 shows the evaluation of CD28 bsFabs and bvFabs in a T cell activation assay. Figure 5A shows the following experimental setup: purified human CD3+ T cells were added to 384-well microplates pre-coated with anti-CD3 mAbs in the presence of 10, 30, and 100 nM negative (IRR = FMDV bvFab) and positive (TGN1412 and 9.3 mAbs) control antibodies or test compounds (CD28 × FMDV bsFab and CD28 bvFab). After 6 days of incubation, INFγ and TNFα levels were determined in the cell culture supernatants, and T cells were counted. (B) IFNγ secretion on day 6 after treatment with 100 nM IRR. Data are expressed as the ratio of IFNγ levels in treated cells relative to cells treated with the IRR negative control. Each bar represents ± the standard error of the mean of five independent assays (five independent donors). A strong correlation was observed between IFNγ secretion and two other readouts: T cell proliferation (r = 0.63 and p-value = 8.10-41) and TNFα secretion (r = 0.87 and p-value = 7.81-110), with some inter-donor variability (data not shown).

[0170] Bin D was inactive in both monovalent and bivalent formats. In addition, four of the five Bin E constructs were active in monovalent bsFab formats (compounds 5-8); two were active in bivalent formats (compounds 6 and 8). These findings were inconsistent with the reporter assays. All Bin F monovalent bsFabs were inactive, but all bivalent constructs were agonists (with a range of potencies). These findings were consistent with the reporter assays.

[0171] Although the T cell activation assay conditions were comparable to some of those of the reporter assay as shown by the bsFab / bvFab in Figure 4C, the correlation between T cell activation and the Jurkat reporter assay was not strong. Most bsFabs from Bin E unexpectedly exhibited T cell activation despite monovalency and lack of cross-linking.

[0172] Antagonist properties CD28 bsFab and bvFab were evaluated in an MLR assay as shown in Figure 6. This is a two-cell system in which monocyte-derived DCs known to express CD28 ligands (CD80 / 86) from a first donor induce TCR activation of T cells from a second donor. Figure 6A shows the experimental setup in which CFSE-labeled CD3+ T cells and allogeneic monocyte-derived DCs were cocultured with and without negative (IRR = FMDV bvFab) and positive (TGN1412 and 9.3 mAb) control or test compounds (CD28 bvFab) at 10 nM and 100 nM at the beginning of the assay. After 4 days, cytokine levels in cell culture supernatants and proliferating T cells were determined. Figure 6B shows IFNg secretion on day 4 after treatment with 10 nM. Each bar represents the mean ± SEM of three independent assays (three independent donors).

[0173] All bvFabs from bin F increased T cell proliferation (data not shown) and cytokine secretion as shown in Figure 6, and were more active than TGN1412 and 9.3 mAbs. All other bvFabs in the figure blocked T cell proliferation and cytokine secretion as shown in Figure 6. In contrast, all bsFabs from bin F were inactive, while bsFabs from bins D and E remained antagonists (data not shown). Overall, the results revealed a strong association between CD80-competing bins and antagonist properties in the MLR assay.

[0174] T cell signaling is primarily determined by costimulatory and co-inhibitory receptors that regulate TCR function (Chen et al. Nat Rev Immunol (2013) 13(4):227-42.). These receptors are diverse, and their function is recognized to be largely context-dependent (Chen, supra). Costimulatory receptors such as CD27, OX40 (CD134), 4-1BB (CD137), or GITR (glucocorticoid-inducible tumor necrosis factor receptor-related protein or CD357) with their corresponding activating ligands have become a major research focus within the biologics community, with efforts directed at their activation or inhibition for cancer and inflammatory immunotherapy, respectively (Edner et al. Nat Rev Drug Discov (2020) 19 (12): 860-83; Blanco et al. Clin Cancer Res (2021) 27 (20): 5457-64; Kraehenbuehl et al. Nat Rev Clin Oncol (2022) 19 (1): 37-50).

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CD28, comprising an immunoglobulin single variable domain (IVSD) comprising a CDR-H1 sequence, a CDR-H2 sequence, and a CDR-H3 sequence; a) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 1, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 2, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 3; b) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:4, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:5, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:6; c) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:7, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:8, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:9; d) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 10, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 11, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 12; e) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 13, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 14, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 15; f) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 16, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 17, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 18; g) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 19, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 20, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 21; h) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:22, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:23, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:24; i) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:25, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:26, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:27; j) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:28, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:29, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:30; k) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:31, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:32, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:33; l) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:34, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:35, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:36; m) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:37, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:38, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:39; n) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:40, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:41, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:42; o) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:43, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:44, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:45; p) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:46, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:47, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:48; q) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:49, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:50, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:51; r) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:52, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:53, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:54; s) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:55, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:56, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:57; t) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:58, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:59, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:60; u) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:61, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:62, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:63; v) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:64, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:65, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:66; w) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:67, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:68, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:69; x) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:70, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:71, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:72; y) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:73, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:74, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:75; z) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:76, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:77, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:78; aa) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:79, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:80, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:81; ab) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 82, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 83, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 84; ac) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 85, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 86, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 87; ad) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 88, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 89, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 90; ae) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 91, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 92, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 93; af) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:94, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:95, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:96; ag) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:97, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:98, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:99; ah) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 100, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 101, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 102; ai) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 103, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 104, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 105; aj) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 106, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 107, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 108; ak) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 109, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 110, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 111; al) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 112, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 113, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 114; am) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 115, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 116, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 117; an) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 118, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 119, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 120; ao) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 121, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 122, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 123; ap) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 124, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 125, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 126; aq) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 127, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 128, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 129; ar) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 130, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 131, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 132; as) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 133, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 134, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 135; at) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 136, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 137, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 138; au) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 139, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 140, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 141; av) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 142, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 143, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 144; aw) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 145, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 146, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 147; ax) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 148, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 149, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 150; ay) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 151, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 152, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 153; az) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 154, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 155, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 156; ba) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 157, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 158, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 159; bb) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 160, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 161, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 162; bc) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 163, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 164, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 165; bd) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 166, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 167, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 168; be) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 169, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 170, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 171; bf) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 172, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 173, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 174; bg) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 175, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 176, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 177; bh) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 178, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 179, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 180; bi) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 181, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 182, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 183; bj) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 184, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 185, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 186; bk) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 187, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 188, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 189; bl) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 190, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 191, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 192; bm) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 193, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 194, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 195; bn) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 196, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 197, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 198; bo) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 199, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 200, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 201; bp) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:202, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:203, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:204; bq) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:205, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:206, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:207; br) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:208, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:209, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:210; bs) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:211, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:212, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:213; bt) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:214, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:215, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:216; bu) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:217, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:218, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:219; bv) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:220, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:221, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:222; bw) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 223, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 224, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 225; bx) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:226, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:227, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:228; by) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 229, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 230, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 231; bz) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:232, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:233, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:234; ca) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:235, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:236, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:237; cb) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:238, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:239, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:240; cc) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:241, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:242, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:243; cd) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:244, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:245, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:246; ce) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:247, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:248, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:249; cf) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 250, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 251, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 252; cg) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:253, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:254, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:255; ch) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:256, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:257, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:258; ci) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:259, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:260, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:261; cj) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:262, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:263, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:264; ck) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:265, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:266, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:267; cl) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:268, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:269, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:270; cm) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:271, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:272, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:273; cn) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:274, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:275, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:276; co) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:277, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:278, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:279; cp) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:280, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:281, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:282; cq) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:283, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:284, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:285; cr) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:286, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:287, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:288; cs) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:289, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:290, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:291; ct) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:292, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:293, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:294; cu) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:295, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:296, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:297; cv) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:298, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:299, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:300; cw) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 301, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 302, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 303; cx) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 304, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 305, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 306; cy) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 307, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 308, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 309; cz) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 310, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 311, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 312; da) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 313, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 314, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 315; db) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 316, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 317, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 318; dc) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 319, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 320, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 321; dd) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 322, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 323, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 324; de) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 325, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 326, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 327; df) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 328, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 329, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 330; dg) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:331, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:332, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:333; dh) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 334, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 335, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 336; di) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 337, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 338, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 339; dj) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 340, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 341, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 342; dk) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:343, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:344, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:345; dl) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 346, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 347, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 348; dm) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 349, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 350, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 351; dn) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 352, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 353, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 354; do) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 355, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 356, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 357; dp) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 358, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 359, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 360; dq) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:361, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:362, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:363; dr) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:364, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:365, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:366; ds) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:367, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:368, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:369; dt) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 370, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 371, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 372; du) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:373, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:374, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:375; dv) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:376, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:377, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:378; dw) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 379, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 380, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 381; dx) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 382, ​​the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 383, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 384; dy) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 385, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 386, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 387; dz) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 388, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 389, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 390; a) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 391, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 392, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 393; eb) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:394, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:395, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:396; ec) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:397, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:398, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:399; ed) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 400, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 401, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 402; ee) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:403, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:404, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:405; ef) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:406, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:407, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:408; e.g., the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 409, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 410, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 411; eh) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:412, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:413, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:414; ei) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:415, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:416, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:417; ej) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:418, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:419, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:420; ek) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:421, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:422, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:423; el) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:424, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:425, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:426; <em>the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 427, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 428, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 429; en) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:430, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:431, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:432; eo) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:433, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:434, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:435; ep) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 436, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 437, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 438; eq) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:439, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:440, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:441; er) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:442, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:443, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:444; es) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:445, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:446, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:447; et) the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO:448, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO:449, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:450; or eu) An antibody or antigen-binding fragment thereof, wherein the CDR-H1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 451, the CDR-H2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 452, and the CDR-H3 sequence comprises the amino acid sequence set forth in SEQ ID NO:

453.

2. the ISVD comprises a VHH domain, a) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 454; b) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 455; c) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 456; d) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 457; e) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 458; f) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 459; g) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 460; h) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 461; i) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 462; j) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 463; k) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 464; l) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 465; m) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 466; n) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 467; o) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 468; p) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 469; q) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 470; r) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 471; s) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 472; t) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 473; u) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 474; v) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 475; w) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 476; x) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 477; y) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 478; z) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 479; aa) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 480; ab) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 481; ac) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 482; ad) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 483; ae) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 484; af) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 485; ag) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 486; ah) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 487; ai) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 488; aj) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 489; ak) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 490; al) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 491; am) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 492; an) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 493; ao) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 494; ap) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 495; aq) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 496; ar) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 497; as) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 498; at) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 499; au) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 500; av) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 501; aw) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 502; ax) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 503; ay) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 504; az) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 505; a) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 506; bb) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 507; bc) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 508; bd) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 509; be) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 510; bf) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 511; bg) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 512; bh) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 513; bi) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 514; bj) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 515; bk) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 516; bl) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 517; bm) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 518; bn) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 519; bo) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 520; bp) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 521; bq) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 522; br) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 523; bs) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 524; bt) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 525; bu) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 526; bv) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 527; bw) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 528; bx) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 529; by) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 530; bz) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 531; ca) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 532; cb) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 533; cc) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 534; cd) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 535; ce) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 536; cf) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 537; cg) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 538; ch) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 539; ci) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 540; cj) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 541; ck) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 542; cl) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 543; cm) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 544; cn) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 545; co) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 546; cp) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 547; cq) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 548; cr) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 549; cs) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 550; ct) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 551; cu) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 552; cv) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 553; cw) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 554; cx) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 555; cy) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 556; cz) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 557; a) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 558; db) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 559; dc) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 560; dd) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 561; de) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 562; df) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 563; dg) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 564; dh) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 565; di) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 566; dj) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 567; dk) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 568; dl) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 569; dm) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 570; dn) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 571; do) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 572; dp) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 573; dq) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 574; dr) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 575; ds) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 576; dt) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 577; du) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 578; dv) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 579; dw) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 580; dx) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 581; dy) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 582; dz) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 583; a) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 584; eb) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 585; ec) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 586; ed) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 587; ee) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 588; ef) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 589; e.g.) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 590; eh) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 591; ei) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 592; ej) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 593; ek) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 594; el) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 595; em) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 596; en) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 597; eo) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 598; ep) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 599; eq) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 600; er) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 601; es) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 602; et) the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 603; or eu) The antibody or antigen-binding fragment thereof of claim 1, wherein the VHH domain comprises the amino acid sequence set forth in SEQ ID NO:

604.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody or an antigen-binding fragment thereof.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is a bispecific antibody.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the bispecific antibody comprises an antigen-binding domain that comprises binding affinity for a tumor-associated antigen (TAA).

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is operably linked to a CH domain and / or a CL domain.

8. The antibody or antigen-binding fragment thereof of claim 6 or 7, wherein the antibody having binding affinity for CD28 is operably linked to a CH1 domain, and the antigen-binding domain having binding affinity for the TAA is operably linked to a CL domain.

9. The antibody or antigen-binding fragment thereof of claim 6 or 7, wherein the antibody having binding affinity for CD28 is operably linked to a CL domain, and the antigen-binding domain having binding affinity for the TAA is operably linked to a CH1 domain.

10. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof is operably linked to an Fc region.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the Fc region is a human IgG1 Fc region.

12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, comprising an antagonist antibody or antigen-binding fragment thereof.

13. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 12.

14. An expression vector comprising the nucleic acid molecule of claim 13.

15. A host cell comprising the expression vector of claim 14.

16. 13. A method for inhibiting CD28 activity in a subject, comprising administering to the subject an antibody or antigen-binding fragment thereof described in any one of claims 1 to 12, thereby inhibiting CD28 activity in the subject.

17. A method for treating a disease associated with CD28 activity in a subject, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described in any one of claims 1 to 12.

18. 18. The method of claim 17, wherein the disease is an autoimmune disease.

19. 18. The method of claim 17, wherein the disease is cancer.

Images