Anti-CD28 antibodies

Abstract

Antibodies and antigen-binding fragments thereof that bind to CD28 are provided, as well as methods of using these antibodies and antigen-binding fragments thereof.

Description

Cross - Reference to Related Applications

[0001] This application claims priority to EP Application No. 22315222.4, filed on September 30, 2022, the disclosure of which is hereby incorporated by reference in its entirety. Reference to a Sequence Listing Submitted Electronically

[0002] The content of the sequence listing (name: 746135_SA9-345PC_SL.xml; size: 541,509 bytes; and creation date: September 26, 2023) electronically submitted in XML format is hereby incorporated by reference in its entirety herein. Technical Field

[0003] This disclosure relates to novel antibodies that specifically bind to CD28 and antigen-binding fragments thereof, as well as methods of using these antibodies and antigen-binding fragments. Background Art

[0004] Cluster of Differentiation 28 (CD28) is a member of the co-stimulatory proteins expressed on the surface of T receptor cells. Since co-stimulatory receptors can regulate T cell activation and thus regulate specific immune response processes, co-stimulatory receptors have been tested to control T cell responses in both oncological and inflammatory settings. Thus, CD28 antagonists have been investigated for the treatment of autoimmune and inflammatory diseases, and CD28 agonists have been investigated for oncological diseases. However, the administration of agonistic anti-CD28 antibodies is associated with severe systemic inflammatory responses, including cytokine storms. These dangerous inflammatory side effects severely 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

[0005] This specification provides anti-CD28 antibodies (e.g., anti-CD28 VHH antibodies) and antigen-binding fragments thereof. Also provided are methods of inhibiting the activity of CD28 or treating CD28-related diseases.

[0006] In one aspect, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to CD28, the antibody or antigen-binding fragment 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 of Table 1.

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

[0008] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to CD28, the antibody or antigen-binding fragment thereof comprising an immunoglobulin single variable domain (ISVD) containing a CDR-H1 sequence, a CDR-H2 sequence, and a CDR-H3 sequence, wherein:

[0009] 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;

[0010] 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;

[0011] 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;

[0012] 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;

[0013] 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;

[0014] 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;

[0015] 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;

[0016] h) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:22, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:23, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:24;

[0017] i) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:25, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:26, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:27;

[0018] j) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:28, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:29, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:30;

[0019] k) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:31, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:32, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:33;

[0020] l) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:34, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:35, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:36;

[0021] m) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:37, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:38, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:39;

[0022] n) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:40, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:41, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:42;

[0023] o) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:43, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:44, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:45;

[0024] p) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:46, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:47, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:48;

[0025] q) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:49, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:50, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:51;

[0026] r) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:52, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:53, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:54;

[0027] s) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:55, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:56, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:57;

[0028] t) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:58, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:59, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:60;

[0029] u) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:61, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:62, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:63;

[0030] v) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:64, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:65, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:66;

[0031] w) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:67, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:68, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:69;

[0032] x) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:70, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:71, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:72;

[0033] y) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:73, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:74, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:75;

[0034] z) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:76, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:77, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:78;

[0035] aa) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:79, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:80, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:81;

[0036] ab) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:82, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:83, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:84;

[0037] ac) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:85, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:86, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:87;

[0038] ad) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:88, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:89, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:90;

[0039] ae) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:91, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:92, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:93;

[0040] af) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:94, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:95, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:96;

[0041] ag) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:97, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:98, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:99;

[0042] ah) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:100, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:101, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:102;

[0043] ai) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:103, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:104, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:105;

[0044] aj) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:106, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:107, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:108;

[0045] (ak) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:109, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:110, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:111;

[0046] al) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:112, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:113, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:114;

[0047] am) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:115, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:116, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:117;

[0048] an) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:118, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:119, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:120;

[0049] ao) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:121, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:122, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:123;

[0050] ap) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:124, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:125, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:126;

[0051] aq) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:127, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:128, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:129;

[0052] (ar) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 130, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 131, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 132;

[0053] (as) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 133, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 134, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 135;

[0054] (at) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 136, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 137, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 138;

[0055] (au) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 139, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 140, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 141;

[0056] (av) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 142, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 143, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 144;

[0057] (aw) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 145, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 146, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 147;

[0058] (ax) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 148, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 149, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 150;

[0059] ay) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO:151, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO:152, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO:153;

[0060] az) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO:154, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO:155, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO:156;

[0061] ba) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO:157, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO:158, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO:159;

[0062] bb) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO:160, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO:161, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO:162;

[0063] bc) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO:163, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO:164, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO:165;

[0064] bd) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO:166, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO:167, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO:168;

[0065] be) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO:169, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO:170, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO:171;

[0066] bf) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:172, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:173, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:174;

[0067] bg) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:175, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:176, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:177;

[0068] bh) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:178, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:179, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:180;

[0069] bi) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:181, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:182, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:183;

[0070] bj) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:184, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:185, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:186;

[0071] bk) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:187, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:188, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:189;

[0072] bl) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:190, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:191, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:192;

[0073] bm) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 193, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 194, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 195;

[0074] bn) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 196, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 197, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 198;

[0075] bo) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 199, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 200, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 201;

[0076] bp) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 202, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 203, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 204;

[0077] bq) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 205, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 206, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 207;

[0078] br) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 208, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 209, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 210;

[0079] bs) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 211, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 212, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 213;

[0080] bt) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 214, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 215, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 216;

[0081] bu) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 217, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 218, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 219;

[0082] bv) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 220, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 221, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 222;

[0083] bw) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 223, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 224, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 225;

[0084] bx) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 226, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 227, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 228;

[0085] by) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 229, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 230, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 231;

[0086] bz) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 232, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 233, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 234;

[0087] (ca) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:235, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:236, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:237;

[0088] cb) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:238, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:239, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:240;

[0089] cc) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:241, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:242, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:243;

[0090] cd) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:244, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:245, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:246;

[0091] ce) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:247, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:248, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:249;

[0092] cf) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:250, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:251, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:252;

[0093] cg) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:253, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:254, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:255;

[0094] ch) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:256, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:257, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:258;

[0095] ci) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:259, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:260, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:261;

[0096] cj) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:262, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:263, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:264;

[0097] ck) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:265, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:266, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:267;

[0098] cl) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:268, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:269, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:270;

[0099] cm) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:271, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:272, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:273;

[0100] cn) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:274, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:275, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:276;

[0101] co) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:277, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:278, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:279;

[0102] cp) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:280, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:281, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:282;

[0103] cq) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:283, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:284, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:285;

[0104] cr) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:286, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:287, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:288;

[0105] cs) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:289, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:290, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:291;

[0106] ct) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:292, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:293, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:294;

[0107] cu) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:295, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:296, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:297;

[0108] 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;

[0109] 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;

[0110] 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;

[0111] 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;

[0112] 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;

[0113] 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;

[0114] 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;

[0115] 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;

[0116] 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;

[0117] 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;

[0118] 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;

[0119] 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;

[0120] 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;

[0121] 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;

[0122] dj) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 340, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 341, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 342;

[0123] dk) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 343, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 344, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 345;

[0124] dl) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 346, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 347, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 348;

[0125] dm) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 349, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 350, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 351;

[0126] dn) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 352, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 353, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 354;

[0127] do) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 355, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 356, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 357;

[0128] dp) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 358, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 359, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 360;

[0129] dq) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:361, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:362, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:363;

[0130] dr) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:364, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:365, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:366;

[0131] ds) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:367, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:368, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:369;

[0132] dt) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:370, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:371, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:372;

[0133] du) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:373, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:374, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:375;

[0134] dv) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:376, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:377, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:378;

[0135] dw) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:379, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:380, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:381;

[0136] dx) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 382, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 383, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 384;

[0137] dy) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 385, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 386, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 387;

[0138] dz) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 388, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 389, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 390;

[0139] ea) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 391, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 392, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 393;

[0140] eb) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 394, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 395, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 396;

[0141] ec) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 397, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 398, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 399;

[0142] ed) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 400, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 401, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 402;

[0143] (ee) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 403, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 404, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 405;

[0144] (ef) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 406, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 407, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 408;

[0145] (eg) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 409, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 410, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 411;

[0146] (eh) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 412, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 413, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 414;

[0147] (ei) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 415, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 416, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 417;

[0148] (ej) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 418, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 419, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 420;

[0149] (ek) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 421, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 422, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 423;

[0150] el) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:424, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:425, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:426;

[0151] em) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:427, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:428, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:429;

[0152] en) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:430, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:431, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:432;

[0153] eo) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:433, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:434, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:435;

[0154] ep) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:436, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:437, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:438;

[0155] eq) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:439, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:440, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:441;

[0156] er) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:442, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:443, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:444;

[0157] es) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 445, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 446, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 447;

[0158] et) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 448, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 449, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 450; or

[0159] eu) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 451, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 452, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 453.

[0160] In certain embodiments,

[0161] a) The ISVD domain comprises the amino acid sequence shown in SEQ ID NO: 454;

[0162] b) The ISVD domain comprises the amino acid sequence shown in SEQ ID NO: 455;

[0163] c) The ISVD domain comprises the amino acid sequence shown in SEQ ID NO: 456;

[0164] d) The ISVD domain comprises the amino acid sequence shown in SEQ ID NO: 457;

[0165] e) The ISVD domain comprises the amino acid sequence shown in SEQ ID NO: 458;

[0166] f) The ISVD domain comprises the amino acid sequence shown in SEQ ID NO: 459;

[0167] g) The ISVD domain comprises the amino acid sequence shown in SEQ ID NO: 460;

[0168] h) The ISVD domain comprises the amino acid sequence shown in SEQ ID NO: 461;

[0169] i) The ISVD domain comprises the amino acid sequence shown in SEQ ID NO: 462;

[0170] j) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 463;

[0171] k) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 464;

[0172] l) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 465;

[0173] m) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 466;

[0174] n) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 467;

[0175] o) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 468;

[0176] p) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 469;

[0177] q) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 470;

[0178] r) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 471;

[0179] s) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 472;

[0180] t) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 473;

[0181] u) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 474;

[0182] v) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 475;

[0183] w) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 476;

[0184] x) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 477;

[0185] y) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 478;

[0186] z) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 479;

[0187] aa) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 480;

[0188] ab) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 481;

[0189] ac) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 482;

[0190] ad) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 483;

[0191] ae) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 484;

[0192] af) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 485;

[0193] ag) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 486;

[0194] ah) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 487;

[0195] ai) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 488;

[0196] aj) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 489;

[0197] ak) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 490;

[0198] al) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 491;

[0199] am) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 492;

[0200] an) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 493;

[0201] ao) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 494;

[0202] ap) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 495;

[0203] aq) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 496;

[0204] ar) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 497;

[0205] as) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 498;

[0206] at) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 499;

[0207] au) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 500;

[0208] av) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 501;

[0209] aw) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 502;

[0210] ax) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 503;

[0211] ay) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 504;

[0212] az) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 505;

[0213] ba) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 506;

[0214] bb) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 507;

[0215] bc) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 508;

[0216] bd) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 509;

[0217] be) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 510;

[0218] bf) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 511;

[0219] bg) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 512;

[0220] bh) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 513;

[0221] bi) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 514;

[0222] bj) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 515;

[0223] bk) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 516;

[0224] bl) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 517;

[0225] bm) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 518;

[0226] bn) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 519;

[0227] bo) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 520;

[0228] bp) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 521;

[0229] bq) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 522;

[0230] br) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 523;

[0231] bs) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 524;

[0232] bt) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 525;

[0233] bu) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 526;

[0234] bv) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 527;

[0235] bw) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 528;

[0236] bx) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 529;

[0237] by) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 530;

[0238] bz) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 531;

[0239] ca) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 532;

[0240] cb) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 533;

[0241] cc) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 534;

[0242] cd) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 535;

[0243] ce) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 536;

[0244] cf) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 537;

[0245] cg) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 538;

[0246] ch) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 539;

[0247] ci) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 540;

[0248] cj) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 541;

[0249] ck) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 542;

[0250] (cl) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 543;

[0251] (cm) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 544;

[0252] (cn) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 545;

[0253] (co) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 546;

[0254] (cp) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 547;

[0255] (cq) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 548;

[0256] (cr) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 549;

[0257] (cs) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 550;

[0258] (ct) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 551;

[0259] (cu) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 552;

[0260] (cv) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 553;

[0261] (cw) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 554;

[0262] (cx) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 555;

[0263] (cy) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 556;

[0264] (cz) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 557;

[0265] (da) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 558;

[0266] db) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 559;

[0267] dc) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 560;

[0268] dd) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 561;

[0269] de) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 562;

[0270] df) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 563;

[0271] dg) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 564;

[0272] dh) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 565;

[0273] di) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 566;

[0274] dj) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 567;

[0275] dk) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 568;

[0276] dl) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 569;

[0277] dm) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 570;

[0278] dn) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 571;

[0279] do) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 572;

[0280] dp) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 573;

[0281] dq) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 574;

[0282] dr) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 575;

[0283] ds) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 576;

[0284] dt) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 577;

[0285] du) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 578;

[0286] dv) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 579;

[0287] dw) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 580;

[0288] dx) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 581;

[0289] dy) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 582;

[0290] dz) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 583;

[0291] ea) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 584;

[0292] eb) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 585;

[0293] ec) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 586;

[0294] ed) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 587;

[0295] ee) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 588;

[0296] ef) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 589;

[0297] eg) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 590;

[0298] eh) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 591;

[0299] ei) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 592;

[0300] ej) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 593;

[0301] ek) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 594;

[0302] el) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 595;

[0303] em) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 596;

[0304] en) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 597;

[0305] eo) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 598;

[0306] ep) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 599;

[0307] eq) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 600;

[0308] er) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 601;

[0309] es) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 602;

[0310] et) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 603; or

[0311] eu) The ISVD domain contains the amino acid sequence shown in SEQ ID NO: 604.

[0312] In certain embodiments, the antibody or its antigen-binding fragment is a chimeric or humanized antibody or its antigen-binding fragment.

[0313] In certain embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment.

[0314] In certain embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody.

[0315] In certain embodiments, the bispecific antibody comprises an antigen-binding domain having binding affinity for a tumor-associated antigen (TAA).

[0316] In certain embodiments, the ISVD is operably linked to the CH1 domain and / or the CL domain.

[0317] In certain embodiments of the bispecific antibody, the ISVD having binding affinity for CD28 is operably linked to the CH1 domain, and the antigen-binding domain having binding affinity for the TAA is operably linked to the CL domain.

[0318] In certain embodiments of the bispecific antibody, the ISVD having binding affinity for CD28 is operably linked to the CL domain, and the antigen-binding domain having binding affinity for the TAA is operably linked to the CH1 domain.

[0319] In certain embodiments, the antibody or antigen-binding fragment thereof is operably linked to an Fc region.

[0320] In certain embodiments, the Fc region is a human IgG1 Fc region.

[0321] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an antagonistic antibody or antigen-binding fragment thereof.

[0322] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding the above antibody or antigen-binding fragment thereof or the above bispecific antibody.

[0323] In one aspect, the present disclosure provides an expression vector comprising the above nucleic acid molecule.

[0324] In one aspect, the present disclosure provides a host cell comprising the above expression vector.

[0325] In one aspect, the present disclosure provides a method for inhibiting CD28 activity in a subject, the method comprising administering the above antibody or antigen-binding fragment thereof to the subject, thereby inhibiting CD28 activity in the subject.

[0326] In one aspect, the present disclosure provides a method for treating a disease associated with CD28 activity in a subject, the method comprising administering the above antibody or antigen-binding fragment thereof to a subject in need thereof.

[0327] In certain embodiments, the disease is an autoimmune disease.

[0328] In certain embodiments, the disease is cancer.

[0329] The foregoing summary of the present disclosure is non-limiting, and other features and advantages of the disclosed antigen-binding proteins and methods will be apparent from the following drawings description, detailed description, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0330] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of the illustrative embodiments in conjunction with the drawings. This patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fee.

[0331] Figure 1 Shows the general workflow for CD28 x TAA bispecific screening.

[0332] Figure 2 Shows the sequence diversity of all CD28 binders identified in the primary screen.

[0333] Figure 3 shows epitope binning of CD28 bispecific Fabs (bsFabs). Competition between anti-CD28 VHHs was explored using SPR. Figure 3A Shows the experimental protocol for exploring competition between anti-CD28 VHHs using SPR. Figure 3B Shows the competition data collected in the heatmap.

[0334] Figure 4 shows the evaluation of CD28 bsFabs and bivalent Fabs (bvFabs) in an IL-2 luciferase reporter assay with CD3 pre-activation. Figure 4A Shows monovalent anti-CD28 x anti-foot-and-mouth disease virus (FMDV) bsFabs with and without crosslinking. Figure 4B Shows bivalent CD28 bvFabs with and without crosslinking. Figure 4C Shows a comparison of monovalent CD28×FMDV bsFab without crosslinking with bivalent CD28 bvFab. Figure 4D Shows a comparison of monovalent CD28×FMDV bsFab with crosslinking with bivalent CD28 bvFab.

[0335] Figure 5 shows the evaluation of CD28 bsFabs and bvFabs in a T cell activation assay. Figure 5A Shows the experimental setup. Figure 5B Shows IFNγ secretion on day 6 after treatment at 100 nM.

[0336] Figure 6 shows the evaluation of CD28 bvFab in the MLR assay. Figure 6A The experimental setup of the MLR assay is shown. Figure 6B The IFNγ secretion on day 4 after treatment with 10 nM is shown.

[0337] Figure 7 A table containing the results of full epitope binning for 48 CD28 VHHs is shown.

[0338] Figure 8 The competition between CD28 bvFab and CD80 using flow cytometry is shown.

[0339] Figure 9 The CD28 xTAA bsFab that simultaneously binds two cells expressing CD28 or TAA is shown.

[0340] Figure 10 shows the evaluation of CD28 bsFab and bvFab in the IL-2 luciferase reporter assay. Figure 10A The monovalent CD28 x FMDV bsFab with and without crosslinking in the absence of CD3 pre-activation is shown. Figure 10B The bivalent CD28 bvFab with and without crosslinking in the absence of CD3 pre-activation is shown. Figure 10C The monovalent CD28 x FMDV bsFab with and without crosslinking after CD3 pre-activation is shown. Figure 10D The bivalent CD28 bvFab with and without crosslinking after CD3 pre-activation is shown. Figure 10E The comparison between the monovalent CD28 xFMDV bsFab and the bivalent CD28 bvFab without crosslinking after CD3 pre-activation is shown. Figure 10F The comparison between the monovalent CD28 xFMDV bsFab with crosslinking and the bivalent CD28 bvFab after CD3 pre-activation is shown. Figure 11 The results of full epitope binning for 48 CD28 VHHs are shown. Detailed Description

[0341] Before describing this disclosure, it should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure will be limited only by the appended claims.

[0342] 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.

[0343] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, exemplary methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

[0344] The terms “about” or “approximately” mean within about 20% (such as about 10%, about 5%, or about 1% or less) of a given value or range.

[0345] As used herein, the term “antibody” or “antigen-binding protein” refers to an immunoglobulin molecule that specifically binds to an antigen or epitope or is immunoreactive with an antigen or epitope, and includes both polyclonal antibodies 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, VHH). The term “antibody” includes genetically engineered or other modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, meditope-enabled antibodies, heteroconjugate antibodies (e.g., multispecific antibodies, bispecific antibodies, diabodies, triabodies, tetra-bodies, tandem bis-scFv, tandem tris-scFv), etc.

[0346] 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 of the parent antibody from which the fragment is derived.

[0347] As used herein, the term “multispecific antibody” refers to bispecific antibodies, trispecific antibodies, or multispecific antibodies and their antigen-binding fragments. Multispecific antibodies may be specific for multiple different epitopes of a target polypeptide, or may contain antigen-binding domains that are specific for epitopes of more than one target polypeptide. Multispecific antibodies can be a single multifunctional polypeptide, or they can be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with each other. The term “multispecific antibody” includes the antibodies of the present disclosure that can be linked or co-expressed with another functional molecule (such as another peptide or protein). For example, an antibody or its fragment can be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent association, or other means) to one or more other molecular entities, such as a protein or its fragment, to produce a bispecific or multispecific antibody with a second binding specificity. In certain exemplary embodiments, the antibodies of the present disclosure are functionally linked to another antibody or its antigen-binding fragment to produce a bispecific antibody with a second binding specificity. In certain embodiments, the second binding specificity is directed against a tumor-associated antigen (TAA).

[0348] As used herein, "monovalent" with respect to an antibody refers to an antibody having a single antigen recognition site that is specific for a target antigen. Examples of monovalent antibodies include monovalent immunoglobulin single variable domain antibodies (e.g., VHH) or monovalent antibody fragments. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single-chain Fv fragments (scFv). In addition, a multispecific antibody can have multiple antigen-binding sites, each antigen-binding site recognizing a different target antigen. Thus, each antigen-binding site will be monovalent with respect to the target antigen.

[0349] As used herein, "multivalent" with respect to an antibody refers to an antibody having multiple (more than one) antigen recognition sites that are specific for a target antigen.

[0350] As used herein, the term "complementary determining region" or "CDR" refers to the amino acid sequences within the variable region of an antibody that confer antigen specificity and binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region. "Framework region" or "FR" is known in the art and refers to the non-CDR portion of the variable region of the heavy chain. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each heavy chain variable region.

[0351] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described in the following references: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland ("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 M P et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, January 2003; 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, June 8, 2001; 309(3):657-70 (AHo numbering scheme).

[0352] 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 in both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, provides insertions by inserting letters (e.g., "30a"), and deletions occur in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects.

[0353] The "CDR" or "complementary determining region" of a given antibody or its region (such as its variable region), or a separately specified CDR (e.g., "CDR-H1", "CDR-H2", "CDR-H3") should be understood to cover the complementary determining region as defined by (or specific to) any known scheme. Similarly, the "FR" or "framework region" of a given antibody or its region (such as its variable region), or a separately specified FR (e.g., "FR-H1", "FR-H2") should be understood to cover the framework region as defined by (or specific to) any known scheme. In some cases, the scheme used to identify a specific CDR or FR is specified, such as the CDR defined by the IMGT, Kabat, Chothia, AbM, or Contact methods. In other cases, the specific amino acid sequence of the CDR or FR is given. Unless otherwise specified, all specific CDR amino acid sequences mentioned in this disclosure are IMGT CDRs. However, this disclosure also covers alternative CDRs defined by other schemes, such as those determined by abYsis Key Annotation (website: abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi). Exemplary heavy chain CDR (HCDR) sequences of anti-CD28 VHH antibodies are listed in Table 1 below. Table 1. Antibody HCDR amino acid sequences for each VHH antibody.

[0354] The "humanized" form of a non-human antibody is a chimeric antibody that contains a minimal sequence derived from a non-human antibody. A humanized antibody is typically a human antibody (the recipient antibody) in which the residues from one or more CDRs are replaced with the residues from one or more CDRs of a non-human antibody (the donor antibody). The donor antibody can be any suitable non-human antibody having the desired specificity, affinity, or biological effect, such as a mouse, rat, rabbit, chicken, camelid, or non-human primate antibody. In some cases, the selected framework region residues of the recipient antibody are replaced with the corresponding framework region residues from the donor antibody. A humanized antibody can also contain residues not found in the recipient antibody or the donor antibody. Such modifications can be made to further improve antibody function. A humanized sequence can be identified by its primary sequence and does not necessarily represent the process by which the antibody was produced.

[0355] As used herein, the terms "specifically binds", "specifically binding", "binding specificity", or "specifically recognizes" refer to an antigen-binding protein or an antigen-binding fragment thereof that exhibits a distinct affinity for an antigen (e.g., the CD28 antigen) and does not exhibit significant cross-reactivity with non-CD28 protein targets. As used herein, the term "affinity" refers to the strength of the interaction between the antigen-binding site of an antigen-binding protein or an antigen-binding fragment thereof and the epitope to which it binds. In certain exemplary embodiments, the affinity is measured by surface plasmon resonance (SPR), e.g., in a Biacore instrument. As will be readily understood by those skilled in the art, the antigen-binding protein affinity can be reported as a dissociation constant (KD) expressed in molar concentration (M).

[0356] Specific binding can be determined according to any means recognized in the art for determining such binding. In some embodiments, specific binding is determined by a competitive binding assay (e.g., ELISA) or a Biacore assay. In certain embodiments, the assay is performed at about 20°C, 25°C, 30°C, or 37°C.

[0357] As used herein, the term "agonist" with respect to an antibody means that, upon binding to a target protein expressed on the cell surface, the antibody stimulates or activates signal transduction via the target protein.

[0358] As used herein, the term "antagonist" with respect to an antibody means that, upon binding to a target protein expressed on the cell surface, the antibody inhibits signal transduction via the target protein. Immunoglobulin Single Variable Domain (ISVD)

[0359] The term "immunoglobulin single variable domain" (ISV or ISVD) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which the antigen-binding site resides in and is formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (such as monoclonal antibodies) or their fragments (such as Fab, Fab', F(ab') 2 , scFv, di-scFv), in which two immunoglobulin domains (specifically, 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, the complementarity-determining regions (CDRs) of both V H and V L will contribute to the antigen-binding site, i.e., a total of 6 CDRs will participate in the formation of the antigen-binding site.

[0360] Given the above definition, the antigen-binding domains of conventional 4-chain antibodies or Fab fragments, F(ab') 2 fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments), or diabodies (all known in the art) derived from such conventional 4-chain antibodies are generally not considered immunoglobulin single variable domains, because in these cases, binding to the corresponding epitope in the antigen typically does not occur through one (single) immunoglobulin domain, but rather through a pair of (associated) immunoglobulin domains that jointly bind to the epitope of the corresponding antigen (such as the light chain variable domain and the heavy chain variable domain, i.e., through the V H -V L pair) of immunoglobulin domains.

[0361] In contrast, an immunoglobulin single variable domain is capable of specifically binding to an antigen epitope without pairing with another immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single V H , a single V HH or a single V L domain.

[0362] Thus, a single variable domain can be a light chain variable domain sequence (e.g., a V L sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a V H sequence or a V HHa sequence) or a suitable fragment thereof; provided that 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 a single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0363] An immunoglobulin single variable domain (ISV) can be, for example, a heavy chain ISV, such as V H , V HH , including camelized V H or humanized V HH . In one embodiment, it is V HH , including camelized V H or humanized V HH . The heavy chain ISV can be derived from a conventional four-chain antibody or a heavy chain antibody.

[0364] For example, the immunoglobulin single variable domain can be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb) or ISV (as defined herein and including, but not limited to, V HH ); other single variable domains, or any suitable fragment of any of them.

[0365] In particular, the immunoglobulin single variable domain can be ISV (such as V HH , including humanized V HH or camelized V H ) or a suitable fragment thereof. [Note: and are registered trademarks of Ablynx N.V.]

[0366] The "V HH domain", also known as V HH s, V HH antibody fragments and V HH antibodies, was originally described as the antigen-binding immunoglobulin variable domain of "heavy chain antibodies" (i.e., "antibodies lacking light chains"; Hamers-Casterman et al., Nature 363:446-448, 1993). The term "V HH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains present in conventional four-chain antibodies (which are referred to herein as "V H domains") and from the light chain variable domains present in conventional four-chain antibodies (which are referred to herein as "V L domains"). For V HHFor further description, refer to the review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0367] The generation of immunoglobulin sequences such as VHHs has been widely described in a variety of publications, 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. VHHs that bind the target antigen are further screened from the VHH library obtained from the immunization.

[0368] In these cases, the generation of antibodies requires purified antigen for immunization and / or screening. The antigen can be purified from natural sources or during recombinant production. Peptide fragments of such antigens can be used for immunization and / or screening of immunoglobulin sequences.

[0369] Immunoglobulin sequences from different sources (including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences) can be sequenced in the methods described herein. In addition, fully human, humanized, or chimeric sequences can be sequenced in 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, for example, WO 94 / 04678 and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531-537, 1996)) can be sequenced in the methods described herein. In addition, multiple ISVs can be fused to form multivalent and / or multispecific constructs (for multivalent and multispecific polypeptides containing one or more V HH For the preparation of multivalent and multispecific polypeptides containing one or more V domains, also refer to Conrath et al., J. Biol. Chem., Vol. 276, 10.7346-7350, 2001, and refer to, for example, WO 96 / 34103 and WO 99 / 23221).

[0370] "Humanized V HH " includes an amino acid sequence corresponding to a naturally occurring V HH domain, but the amino acid sequence has been "humanized" by replacing the naturally occurring V HHOne or more amino acid residues in the amino acid sequence of the sequence (and in particular in the framework sequence) are replaced by one or more amino acid substitutions present at one or more corresponding positions in the V H domain of a conventional four-chain antibody from humans (such as those described above). This can be done in a manner known per se, which is clear to the person skilled in the art, for example based on the prior art (such as WO 2008 / 020079). In addition, it should be noted that such humanized V HH can be obtained in any suitable manner known per se and is thus not strictly limited to polypeptides that have been obtained using a polypeptide comprising a naturally occurring VHH domain as starting material.

[0371] "Camelized V H " comprises an amino acid sequence corresponding to the naturally occurring V H domain, but an amino acid sequence that has been "camelized" by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring V H domain of a conventional four-chain antibody with one or more amino acid residues present at one or more corresponding positions in the V HH domain of a heavy-chain antibody (from camelids). This can be done in a manner known per se, which is clear to the person skilled in the art, for example based on the description in the prior art (such as Davies and Riechman (1994 and 1996), supra). Such "camelizing" substitutions are inserted at positions where V H -V L interface formation and / or presence occurs, and / or at so-called camelid signature residues as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996), supra). In one embodiment, the V H sequence used as starting material or starting point for generating or designing camelized V H is a V H sequence from a mammal, such as a human V H sequence, such as a V H 3 sequence. However, it should be noted that such camelized V H can be obtained in any suitable manner known per se and is thus not strictly limited to polypeptides that have been obtained using a polypeptide comprising a naturally occurring V H domain as starting material.

[0372] The structure of an immunoglobulin single variable domain sequence can be considered to consist of four framework regions (“FR”), 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”); and these framework regions are interrupted by three complementarity determining regions (“CDR”), 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”).

[0373] In such an immunoglobulin sequence, the framework sequence can be any suitable framework sequence, and examples of suitable framework sequences will be apparent to the skilled person, for example based on standard manuals and additional disclosures and prior art mentioned herein.

[0374] The framework sequence is an immunoglobulin framework sequence or a framework sequence (a suitable combination thereof) derived from an immunoglobulin framework sequence (e.g., by humanization or camelization). For example, the framework sequence can be derived from a light chain variable domain (e.g., V L sequence) and / or from a heavy chain variable domain (e.g., V H sequence or V HH sequence). In a particular aspect, the framework sequence is a framework sequence derived from a V HH sequence (wherein the framework sequence may optionally have been partially or fully humanized) or a conventional V H sequence that has been camelized (as defined herein).

[0375] Specifically, the framework sequence present in the ISV sequence used in the methods described herein may contain one or more signature residues (as defined herein) such that the ISV sequence is ISV, such as V HH , including humanized V HH or camelized V H . Non-limiting examples of such framework sequences (suitable combinations thereof) will become apparent in light of further disclosures herein.

[0376] V H domain and V HH domain will generally have a total number of amino acid residues in the range of 110 to 120, typically between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0377] Note that in the method of the present invention, the ISV contained in the multivalent ISV polypeptide being sequenced is not limited to the source of the ISV sequence (or the nucleotide sequence used to express it), nor to the manner in which the ISV sequence or nucleotide sequence is produced or obtained (or has been produced or obtained). Thus, the ISV sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In certain non-limiting aspects, the ISV sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized murine or rabbit immunoglobulin sequences, particularly partially or fully humanized V HH sequences), "camelized" (as defined herein) immunoglobulin sequences (particularly camelized V H sequences), and ISVs obtained by techniques such as affinity maturation (starting, for example, from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, surface veneering, combining fragments from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques known to those skilled in the art for engineering immunoglobulin sequences; or any suitable combination of any of the foregoing techniques.

[0378] Similarly, the nucleotide sequence can be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and can be, for example, a sequence isolated by PCR from a suitable naturally occurring template (such as DNA or RNA isolated from cells), a nucleotide sequence isolated from a library (particularly 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 DNA synthesis techniques known per se.

[0379] Generally, ISV (particularly the V HH sequence, including (partial) humanized V HH sequences and camelized V H sequences) can be characterized by the presence of one or more "signature residues" (as described herein) in one or more framework sequences (also further described herein). Thus, generally, ISV can be defined as an immunoglobulin sequence having the following (general) structure:

[0380] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0381] Wherein FR1 to FR4 respectively refer to framework regions 1 to 4, and wherein CDR1 to CDR3 respectively refer to complementarity-determining regions 1 to 3, and wherein one or more of the signature residues are as further defined herein.

[0382] In particular, The ISV can be an immunoglobulin sequence having the following (general) structure:

[0383] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0384] Wherein FR1 to FR4 respectively refer to framework regions 1 to 4, and wherein CDR1 to CDR3 respectively refer to complementarity-determining regions 1 to 3, and wherein these framework sequences are as further defined herein.

[0385] More particularly, The ISV can be an immunoglobulin sequence having the following (general) structure:

[0386] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0387] Wherein FR1 to FR4 respectively refer to framework regions 1 to 4, and wherein CDR1 to CDR3 respectively refer to complementarity-determining regions 1 to 3, and wherein:

[0388] One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to Kabat numbering are selected from the signature residues mentioned in Table A below. Table A: Signature residues in ISV

[0389] As used herein, the term "CD28" refers to a transmembrane co-stimulatory signaling protein expressed on T cells. CD28 is involved in T cell activation, proliferation, cytokine production, and survival. Exemplary wild-type human CD28 amino acid sequences can be found under NCBI reference sequence: NP_006130.1; and Uniprot reference number: P10747.

[0390] As used herein, the term "tumor-associated antigen" or "TAA" refers to any antigen highly expressed by tumor cells or in the tumor stroma. The term tumor-associated antigen includes TAAs that are not entirely tumor-specific but are overexpressed in the tumor or its stroma. The term tumor-associated antigen also includes tumor-specific antigens expressed only on tumors.

[0391] As used herein, "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. It encompasses antibodies in bivalent form having two CD28-binding sites (such as native immunoglobulin molecules or F(ab)'2 fragments), as well as antibodies in monovalent form having a single CD28-binding site. In the present context, a CD28-binding polypeptide is typically a polypeptide containing an immunoglobulin single variable domain antibody (such as a VHH), which polypeptide has at least one immunoglobulin single variable domain (such as a VHH domain) that specifically binds to CD28. In certain embodiments, the anti-CD28 antibody or its antigen-binding fragment comprises a VHH domain selected from any of the VHH amino acid sequences of Table 2. Table 2. VHH Amino Acid Sequences.

[0392] 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 (such as VHH domains) that each independently bind CD28.

[0393] In certain embodiments, the anti-CD28 antibody or its antigen-binding fragment comprises a VHH domain having 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 higher identity to the amino acid sequence of any of the VHH sequences listed in Table 2.

[0394] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises an HCDR1 region, an HCDR2 region, and an HCDR3 region having 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 higher identity to any one of the HCDR1, HCDR2, or HCDR3 amino acid sequences listed in Table 1.

[0395] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.

[0396] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0397] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a monospecific antibody.

[0398] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a bispecific antibody.

[0399] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a multispecific antibody. In an embodiment, 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 can serve as a specific heterodimerization scaffold and can be linked to additional binding domains. The additional binding domains can be in several different forms, including but not limited to another Fab domain, scFv, or sdAb (e.g., VHH).

[0400] As used herein, "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance as it exists in vitro (e.g., an antibody provided herein) into a patient's body, such as by, but not limited to, 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. When treating or managing a disease or its symptoms, administration of the substance typically occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, administration of the substance typically occurs before the onset of the disease or its symptoms and can be continued for a long time to delay or reduce the appearance or severity of disease-related symptoms.

[0401] "Effective amount" means an amount of an active agent (e.g., an isolated binding polypeptide of the present disclosure) sufficient to achieve a desired physiological outcome in an individual in need of the agent. The effective amount can vary between individuals depending on, among other things, the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the formulation of the composition, the assessment of the medical condition of the individual, and other relevant factors.

[0402] 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., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). In certain embodiments, as used herein, the term "subject" refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non - human primates, wild animals, untamed animals, farm animals, racing animals, and pets.

[0403] As used herein, the term "therapy" refers to any protocol, method, and / or agent that can be used in the prevention, management, treatment, and / or amelioration of a disease or its associated symptoms. In some embodiments, the term "therapy" refers to any protocol, method, and / or agent that can be used in the modulation of an immune response against an infection or its associated symptoms in a subject. In some embodiments, the terms "therapies" and "therapy" refer to biotherapies, supportive therapies, and / or other therapies known to those of skill in the art (such as medical personnel) that are useful in the prevention, management, treatment, and / or amelioration of a disease or its associated symptoms. In other embodiments, the terms "therapies" and "therapy" refer to biotherapies, supportive therapies, and / or other therapies known to those of skill in the art (such as medical personnel) that are useful in the modulation of an immune response against an infection or its associated symptoms in a subject.

[0404] As used herein, the terms "treat", "treatment", and "treating" refer to a decrease or improvement in the progression, severity, and / or duration of a disease or its associated symptoms caused by the administration of one or more therapies, including but not limited to the administration of one or more prophylactic or therapeutic agents, such as an isolated binding polypeptide provided herein. As used herein, the term "treatment" can also refer to altering the course of the subject being treated. Therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviation of one or more symptoms, attenuation of the direct or indirect pathological consequences of a disease, reduction in the rate of disease progression, improvement or alleviation of a disease state, and remission or improved prognosis.

[0405] Unless otherwise specified, the terms "tumor cell", "cancer cell", "cancer", "tumor" and / or "neoplasm" are used interchangeably herein and refer to a cell (or cells) that exhibits uncontrolled growth and / or increased abnormal cell survival and / or inhibition of apoptosis that interferes with the normal function of the body's organs and systems. This definition includes benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases. The terms "cancer" and "tumor" encompass solid cancers and blood / lymph cancers and also encompass malignant growths, premalignant growths, and benign growths such as dysplasia. This definition also includes cells with abnormal proliferation that are not hindered by the immune system (e.g., immune evasion and immune escape mechanisms) (e.g., virus-infected cells).

[0406] As used herein, "immune disease" refers to any disease associated with the development of an individual's immune response (including cellular and / or humoral immune responses). Examples of immune diseases include, but are not limited to, inflammation, allergy, autoimmune disease, graft-related disease, cancer, and viral infection.

[0407] As used herein, "autoimmune disease" refers to disease conditions and states in which an individual's immune response targets the individual's own components, resulting in an undesirable and often debilitating condition. As used herein, "autoimmune disease" is intended to further include autoimmune disorders, syndromes, etc. Expression of Antigen - Binding Protein

[0408] In one aspect, nucleic acid molecules encoding the antibodies and antigen-binding fragments thereof disclosed herein are provided. Methods of making binding proteins are also provided, which methods include expressing these nucleic acid molecules.

[0409] The nucleic acid molecules encoding the antibodies disclosed herein are typically inserted into an expression vector for introduction into a host cell, whereby the desired amount of antibody can be produced. Thus, in certain aspects, the present disclosure provides expression vectors comprising the nucleic acid molecules disclosed herein and host cells comprising these vectors and nucleic acid molecules.

[0410] The term "vector" or "expression vector" is used herein to mean a vector used according to the present disclosure that serves as a vehicle for introducing a desired gene into a cell and expressing the gene in the cell. As is known to those of skill in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors compatible with the present disclosure will include selectable markers, appropriate restriction sites (to facilitate cloning of the desired gene), and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0411] For the purposes of this disclosure, a variety of expression vector systems can be employed. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV), or SV40 virus. Other vectors involve the use of polycistronic systems with internal ribosome entry sites. Additionally, cells that have integrated DNA into their chromosomes can be selected by introducing one or more markers that permit selection of transfected host cells. The markers can provide prototrophy for auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. Selectable marker genes can be ligated directly to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Optimal synthesis of mRNA may also require additional elements. These elements can include signal sequences, splice signals, and transcriptional promoter, enhancer, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector together with the synthetic heavy chain constant region genes (e.g., human constant region genes) as described above.

[0412] In other embodiments, polycistronic constructs can be used to express antibodies. In such expression systems, multiple gene products of interest, such as the heavy and light chains of an antibody, can be produced from a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRESs) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is hereby incorporated by reference in its entirety for all purposes. Those skilled in the art will appreciate that such expression systems can be used to effectively produce the full range of polypeptides disclosed in this application.

[0413] More generally, once a vector or DNA sequence encoding an antibody or a fragment thereof has been prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Introduction of a plasmid into a host cell can be accomplished by a variety of techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electroporation and electrotransfer), protoplast fusion, calcium phosphate precipitation, cell fusion with envelopment DNA, microinjection, and infection with intact virus. See Ridgway, A.A.G. “Mammalian Expression Vectors” Chapter 24.2, pages 470 - 472, Vectors, edited by Rodriguez and Denhardt (Butterworths, Boston, Massachusetts, 1988). The plasmid can be introduced into the host by electroporation. The transformed cells are grown under conditions suitable for the production of the light and heavy chains, and the synthesis of the heavy and / or light chain proteins is assayed. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and the like.

[0414] As used herein, the term “transformation” is used in a broad sense to refer to the introduction of DNA into a recipient host cell, thereby altering the genotype.

[0415] By analogy, a “host cell” is a cell that has been transformed with a vector constructed using recombinant DNA techniques and encoding at least one heterologous gene. In the description of methods for isolating polypeptides from recombinant hosts, unless otherwise expressly stated, the terms “cell” and “cell culture” are used interchangeably to denote the source of the antibody. In other words, recovering a polypeptide from a “cell” can mean recovering from whole cells pelleted by spinning, from the supernatant of a lysed cell culture, or from a cell culture containing both the culture medium and the suspended cells.

[0416] In one embodiment, the host cell line for antibody expression is of mammalian origin. One skilled in the art can determine the specific host cell line that is most suitable for expressing the desired gene product. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary line, DHFR-), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 with SV40 T antigen), R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HEK (human kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney). In one embodiment, the cell line provides altered glycosylation of the antibody expressed therefrom, such as afucosylation (e.g., PER. (Crucell) or FUT8 knockout CHO cell line ( cells) (Biowa, Princeton, NJ)). In one embodiment, NS0 cells can be used. CHO cells are particularly useful. Host cell lines are generally available from commercial services (e.g., American Type Culture Collection) or from the authors of the published literature.

[0417] In vitro production allows for scale-up to produce large amounts of the desired polypeptide. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture (e.g., in an airlift reactor or continuous stirred reactor), or immobilized or encapsulated cell culture (e.g., in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges). If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatography (e.g., gel filtration, ion exchange chromatography, DEAE-cellulose chromatography, and / or (immuno)affinity chromatography).

[0418] Genes of the characteristic antibodies in the encoding book disclosure can also be expressed in non-mammalian cells (such as bacteria or yeast or plant cells). In this regard, it should be understood that various single-celled non-mammalian microorganisms (such as bacteria), that is, those capable of growing in culture or fermentation, can also be transformed. Bacteria that are easily transformable include members of the following: Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. It should be further recognized that when expressed in bacteria, the binding protein can become part of inclusion bodies. In some embodiments, the binding protein is subsequently isolated, purified and assembled into a functional molecule. 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 containing a nucleic acid molecule encoding the binding protein of the present disclosure.

[0419] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, but generally many other strains can also be used. For expression in the genus Saccharomyces, for example, the plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is usually used. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutant strains lacking the ability to grow in tryptophan (such as ATCC No. 44076 or PEP4-1) (Jones, Genetics, 85:12 (1977)). Then, the presence of a trpl lesion, which is characteristic of the yeast host cell genome, provides an effective environment for detecting transformation by growth in the absence of tryptophan. Methods of Administering Antigen - Binding Protein

[0420] Methods for preparing an antigen-binding protein (e.g., an anti-CD28 antibody or antigen-binding fragment thereof disclosed herein) and administering it to a subject are well known or readily determinable to those skilled in the art. The route of administration of the antigen-binding proteins disclosed herein can be oral, parenteral, by inhalation or topical. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Although all of these forms of administration are expressly considered to be within the scope of this disclosure, one form of administration will be a solution for injection, particularly a solution for intravenous or intraarterial injection or infusion. Generally, suitable pharmaceutical compositions for injection may contain a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer (e.g., human albumin), etc. However, in other methods compatible with the teachings herein, the modified antibody can be directly delivered to the site of the undesirable cell population, thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0421] 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 this 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 solution or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases. More specifically, pharmaceutical compositions suitable for injection use include sterile aqueous solutions (where 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 should be fluid to the extent that easy injectability exists. It should be stable under the conditions of manufacture and storage and should also be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by the use of surfactants.

[0422] The action of preventing microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). Isotonic agents such as sugars, polyols (e.g., mannitol, sorbitol) or sodium chloride can also be included in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0423] In any case, sterile injectable solutions can be prepared by incorporating the active compound (e.g., the modified binding polypeptide itself or in combination with other active agents) in the required amount into a suitable solvent, followed by filtration sterilization, the solvent having, as required, one or a combination of the ingredients listed herein. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation generally include vacuum drying and freeze drying to yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution. The injectable formulations are processed, filled into containers (such as ampoules, bags, bottles, syringes or vials) and sealed under sterile conditions according to methods known in the art. In addition, these formulations can be packaged and sold in the form of kits, such as those described in US20020102208 and US 6994840, which are hereby incorporated by reference in their respective entireties. Such articles can include labels or package inserts indicating that the relevant composition can be used to treat subjects suffering from or susceptible to autoimmune disorders or tumor disorders.

[0424] The effective dose of the compositions of the present disclosure for treating the above-mentioned conditions varies according to many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other agents being administered, and whether the treatment is prophylactic or therapeutic. Generally, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. The treatment dose can be adjusted step by step using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0425] As previously mentioned, the antigen-binding proteins, immunoreactive fragments or recombinants of the present disclosure can be administered in a pharmaceutically effective amount for in vivo treatment of mammalian disorders. In this regard, it should be recognized that the disclosed antigen-binding proteins will be formulated to facilitate the administration of the active agent and to promote the stability of the active agent.

[0426] The pharmaceutical compositions according to the present disclosure generally comprise a pharmaceutically acceptable non-toxic sterile carrier such as physiological saline, non-toxic buffers, preservatives, etc. For the purposes of this application, a pharmaceutically effective amount of a modified antigen-binding protein, an immunoreactive fragment thereof or a recombinant, conjugated or unconjugated with a therapeutic agent, should be understood to mean an amount sufficient to effectuate effective binding to an antigen and to achieve a benefit (e.g., ameliorating the symptoms of a disease or disorder or detecting a substance or cell). In the case of tumor cells, the modified binding polypeptide is generally capable of interacting with a selected immunoreactive antigen on neoplastic or immunoreactive cells and increasing the death of those cells. Of course, the pharmaceutical compositions of the present disclosure may be administered in a single dose or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

[0427] Consistent with the scope of the present disclosure, the antigen-binding proteins of the present disclosure may be administered to a human or other animal in an amount sufficient to produce a therapeutic or prophylactic effect according to the above-described methods of treatment. The antigen-binding proteins of the present disclosure may be administered to the human or other animal in a conventional dosage form prepared according to known techniques by combining the antibodies of the present disclosure with a conventional pharmaceutically acceptable carrier or diluent. Those skilled in the art will recognize that the form and characteristics of the pharmaceutically acceptable carrier or diluent will depend on the amount of the active ingredient with which it is combined, the route of administration and other well-known variables. Those skilled in the art will further understand that mixtures comprising one or more species of the binding polypeptides described in the present disclosure may be shown to be particularly effective.

[0428] The biological activity of the pharmaceutical compositions defined herein may be determined, for example, by cytotoxicity assays as described in the following examples, WO 99 / 54440 or Schlereth et al. (Cancer Immunol. Immunother. [Cancer Immunology and Immunotherapy] 55 (2006), 503 - 514). The biological activity may also be determined by T cell activation assays, such as by detecting pro-inflammatory cytokine expression or anti-inflammatory cytokine expression. As used herein, "efficacy" or "in vivo efficacy" refers to the response to the therapy of the pharmaceutical compositions of the present invention, e.g., using standardized NCI response criteria. The success or in vivo efficacy of the 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., depleting pathological cells (e.g., tumor cells) or repressing activated immune cells). In vivo efficacy may be monitored by established standard methods for the respective disease entity, including but not limited to white blood cell count, differential, fluorescence-activated cell sorting, bone marrow aspiration. In addition, various disease-specific clinical chemistry parameters and other established standard methods may be used. Examples

[0429] The following examples are provided to give a complete disclosure and description to those of ordinary skill in the art of how to make and use the methods and compositions described in the present invention and are not intended to limit the scope of the invention as recognized by the inventors. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are atmospheric or near atmospheric pressure. Example 1. Materials and Methods Cell Lines and Cells from Human Donors

[0430] Buffy coats from healthy donors were obtained from the French Blood Bank (Etablissement du Sang). After purification of peripheral blood mononuclear cells using a Ficoll gradient, total CD3 TM T cells were enriched using the RoboSep + Human T Cell Enrichment Kit (StemCell Technologies, 19051). Dendritic cells (DC) were generated by culturing monocytes isolated from peripheral blood mononuclear cells with 200 ng / mL IL-4 (Miltenyi Biotec, 130-093-922) and 200 ng / mL granulocyte macrophage colony-stimulating factor (Miltenyi Biotec, 130-093-866) in vitro for 7 days using a Monocyte Isolation Kit (StemCell Technologies, 17858). FreeStyle TM 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% CO 2 in the medium recommended by the cell supplier. All cell line media and reagents were purchased from Gibco. TGN1412 and 9.3 antibodies were generated and purified in-house. Transfection for CD28 Expression

[0431] FreeStyle was transfected with a high-quality plasmid preparation encoding human CD28 using 293fectin TM (Gibco, 12347-019) according to the manufacturer's instructions TMHEK293-FS cells. At 48 h post-transfection, CD28 expression was evaluated by flow cytometry using AF647-conjugated anti-human CD28 mAb (BD Pharmingen TM , 560683). Example 2. Llama immunization and library construction

[0432] Immunization and library construction were performed by the VIB Nanobody Service Facility (Brussels, Belgium). As Figure 1 shown, llamas were immunized (DNA immunization) four times intradermally against CD28 and TAA, with each injection containing approximately 2 mg of a vector encoding 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 last DNA injection, the animals were boostered subcutaneously with recombinant proteins (human CD28 [Sino Biological, 11524-HCCH], internally generated human TAA; adjuvant for protein boost, GERBU LQ 3000). Four days after the protein boost, anticoagulated blood was collected for VHH library construction, as Figure 1 shown. Figure 1 Summarized is the general workflow for CD28xTAA bispecific screening: llamas are immunized (DNA immunization) against CD28 and TAA, and a VHH library is constructed for phage display selection against recombinant proteins and cells; clones for binding pairs are rapidly screened, these clones are sequenced, and these clones are cloned in-frame with the human constant domain CH1 or Cλ to generate bsFabs in FreeStyle HEK293-FS cells by transient transfection. The cell supernatants are functionally screened in primary cell assays, and the binding properties and biological functions of the clones of interest are further characterized.

[0433] As described previously, a VHH library was constructed for phage display selection against recombinant proteins and cells. Total ribonucleic acid was extracted from peripheral blood lymphocytes and used as a template for first-strand complementary DNA synthesis with oligo(deoxythymidine) primers. The VHH-encoding sequences were amplified from the complementary DNA by polymerase chain reaction, digested with PstI and NotI, and cloned between the PstI and NotI sites upstream of the human influenza hemagglutinin decapeptide tag in the phagemid vector pHEN4. A VHH library of approximately 10 8 independent transformants was obtained. As Figure 1as shown, followed by rapid screening of the clones for the combination, sequencing of these clones, and in-frame cloning of these clones together with the human constant domain CH1 or Cλ to generate bsFab in FreeStyle by transient transfection TM in HEK293-FS cells. Functional screening of the cell supernatants was performed in primary cell assays, and the binding properties and biological functions of the target clones were further characterized. Phage display panning

[0434] As described, a ready-to-use phage-VHH preparation was obtained. The bacterial library was grown in 2YTAG medium (2×YT medium, 100 μg / mL ampicillin, 2% glucose) until the optical density at 600 nm (OD600) reached 0.5, and the bacterial library was infected with M13K07 helper phage (Invitrogen). After centrifugation, the bacteria were resuspended in 2YTAK medium (2×YT medium, 100 μg / mL ampicillin, 50 μg / mL kanamycin) and grown overnight. The phage particles were precipitated from the culture supernatant by adding 20% (weight / volume [w / v]) polyethylene glycol 8000 (PEG8000) and 2.5 M NaCl, centrifuged, and resuspended in phosphate-buffered saline (PBS). The phage was subjected to another washing and precipitation step and finally resuspended in cold PBS / glycerol 15% (volume / volume [v / v]). Panning against recombinant proteins

[0435] Panning against recombinant proteins was performed as described. According to the manufacturer's recommendation, M-450 epoxy beads (Dynabeads, Invitrogen) were coated with His-tagged CD28 or TAA-recombinant protein. The phage-VHH library (10 11 phages / round of selection) and the beads (coated beads and naked beads) were saturated in PBS / milk 2% (w / v) and kept at room temperature (RT) for 1 hour. The phage-VHH library was first depleted twice by incubating on the naked beads for 30 minutes to eliminate non-specific clones. The unbound phage-VHH was recovered and incubated with the target-coupled beads in PBS / milk 2% (w / v) at room temperature for 2 hours. After washing 10 times with PBS / Tween 0.1% (v / v) and twice with PBS, the bound phage-VHH was resuspended in PBS (output selection), added to exponentially growing TG1 bacteria, and amplified overnight in 2YTAG medium for a new round of panning, or plated on 2YTAG plates. Panning against cells

[0436] At 4°C against CD28 or TAA-transfected FreeStyle TMHEK293-FS cells were subjected to panning. After washing twice with PBS, the cell pellet was resuspended in PBS and loaded onto a fetal bovine serum / Percoll gradient as described previously. 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, then mechanically lysed using beads (Dynabeads, Invitrogen). The recovered phage-VHH was used to infect exponentially growing Escherichia coli TG1 bacteria and amplified overnight in 2YTAG medium for a new round of panning, or plated on 2YTAG plates. Fab-like construction, production, and purification

[0437] After amplification by polymerase chain reaction, the complementary DNA of anti-CD28 VHH or anti-TAA VHH or anti-foot-and-mouth disease virus (FMDV) VHH (Harmsen et al. Veterinary microbiology. 2007;120(3-4):193-206) was cloned in-frame with the human CL domain or human IgG1 CH1 domain fused to human influenza hemagglutinin and a 6-His tag into a proprietary mammalian expression vector. The plasmid was purified using the NucleoBond Macherey-Nagel kit and subjected to Sanger sequencing. Bispecific (bsFab) or bivalent Fab-like (bvFab) antibodies were generated by co-transfecting HEK293-FS cells with a mixture of two plasmids, each encoding two different (bsFab) or two identical (bvFab) VHHs fused to each Fab constant domain. The supernatant was harvested after 7 days, purified on a nickel affinity column, and analyzed on a CALIPER GXII (PerkinElmer). TM HEK293-FS cells were co-transfected with a mixture of two plasmids, each encoding two different (bsFab) or two identical (bvFab) VHHs fused to each Fab constant domain. The supernatant was harvested after 7 days, purified on a nickel affinity column, and analyzed on a CALIPER GXII (PerkinElmer). Flow cytometry binding and competitive assays

[0438] All flow cytometry assays were performed on a MACSQuant cell counter (Miltenyi Biotec, Germany) using V-bottom 96-well microtiter plates. Cells were gated against live single cells (Dapi staining), and 10 4 events were collected per sample. Data were analyzed using MACSQuant software, and the results were expressed as the median fluorescence intensity.

[0439] First, Jurkat cells were incubated with serial dilutions of bvFab at 4 °C for 1 h and then with human CD80-Fc fusion at its 90% effective concentration (EC90) at 4 °C for 30 min. Bound ligands were detected with anti-human IgG (Fc-specific) mAb (Sigma, I2136), followed by detection with Alexa647-conjugated goat anti-mouse mAb (Invitrogen, A11013). Competitive binding assay (competition of TGN1412 and 9.3 mAb using enzyme-linked immunosorbent assay [ELISA]) Phage-VHH production in 96-well plates

[0440] A single TG1 colony of the CD28 VHH of interest was grown in 2YT medium at 37 °C until the OD600 reached 0.5. The cells were then infected with M13K07 helper phage and grown overnight at 30 °C in 2YTK. The supernatant containing phage-VHH was harvested and used for testing. ELISA

[0441] In MaxiSorp TM ELISA was performed on 96-well plates (Sigma) pre-coated overnight at 4 °C with 1 μg / mL human CD28 recombinant protein in PBS and further saturated with PBS / milk 2% (w / v) for 1 h at room temperature. Serial dilutions of the competing mAb (TGN1412 or 9.3) were then incubated for 1 h at room temperature, and EC90 of phage-VHH was further added and kept for 30 min at room temperature. After several washes in PBS / Tween 0.1% (v / v), anti-M13 horseradish peroxidase-conjugated mAb (Santa Cruz Biotechnology, sc-53004) was added to detect the bound phage-VHH. 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 adding the sulfuric acid stop solution. Reporter assay For the condition with CD3 pre-activation

[0442] The wells were coated with 50 μL of anti-CD3 (UCHT-1 clone, BioLegend, BLE300414) (Costar 3917 plates) and stored overnight at 4 °C, then washed twice with 100 μL of PBS / well. These cells were harvested during the exponential growth phase of Jurkat-IL-2-Luc2P cells, and 25 μL of the cell suspension was added to a 96-well plate (50,000 cells / well) with 25 μL of the test compound. For cross-linking experiments

[0443] The test compound was pre-incubated with saturated concentrations of anti-human Fab (Sigma, I5260) or anti-human Fc (Sigma, I2136) for 30 minutes at room temperature. These cells were harvested during the exponential growth phase of Jurkat-IL-2-Luc2P cells, and 25 μL of the cell suspension was added to a 96-well plate (50,000 cells / well) with 25 μL of cross-linked or non-cross-linked test compound. For conditions with cells expressing TAA

[0444] These cells were harvested during the exponential growth phase of Jurkat-IL-2-Luc2P cells and mixed with cells expressing TAA to obtain a final ratio of 1:1 between reporter cells and accessory cells. 25 μL of the cell suspension was added to a 96-well plate (50,000 cells / well) with 25 μL of the test compound.

[0445] For all three conditions (CD3 pre-activation, cross-linking, and with cells expressing TAA), the plates were incubated at 37 °C in a humidified incubator with 5% CO 2 for 6 hours. Then 50 μL of Bio-GloTM (Promega, G7941) reagent, prepared according to the manufacturer's instructions, was added to each well and mixed. At least 5 minutes were allowed for complete cell lysis, followed by measurement of luminescence using an Envision multimode plate reader (PerkinElmer). T cell activation assay

[0446] At 4 °C, U-bottom 384-well plates were coated overnight with 5 μg / mL anti-human CD3 antibody (eBioscience, 15288347, OKT3 clone). The plates were washed with PBS and in the presence of 10 nM, 30 nM, and 100 nM of negative (isotype or FMDV bvFab) and positive (TGN1412 or 9.3 mAb) control antibodies or test compounds (CD28×FMDV bsFab and CD28 bvFab) in complete X-VIVO TM50,000 T cells were added to the medium (Lonza, BE02 - 060F) and incubated at 37 °C in a 5% CO 2 incubator. After 6 days of incubation, the supernatant was collected and stored at -20 °C until cytokine measurement. Promega's CellTiter- reagent was added to the cells for cell counting. Cytokine levels were measured using a homogeneous time-resolved fluorescence 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 are presented as the percentage of effect compared to the negative control. Mixed leukocyte reaction (MLR)

[0447] At the start of the assay, carboxyfluorescein succinimidyl ester (CFSE)-labeled CD3 + T cells (1×10 5 cells) and allogeneic DCs (1×10 4 cells) were co-cultured with or without 10 nM negative (FMDV bvFab) and positive (TGN1412 or 9.3 mAb) control antibodies or test compounds (CD28×TAA bsFab and CD28 bvFab). After 4 days, the supernatant was collected and cytokine levels were measured using a CBA human Th1 / Th2 / Th17 kit (BD Biosciences, 550749) according to the manufacturer's instructions. The cells were stained with a mixture of antibodies against CD4 (BD Biosciences, 563550), CD8 (BD Biosciences, 560662), CD25 (BD Biosciences, 555434), and CD69 (BD Biosciences, 562617). T cell proliferation was measured using CFSE dilution. Samples were analyzed on a Fortessa X-20 flow cytometer (BD Biosciences). Epitope binning

[0448] Using a BIAcore T200 (upgraded T100, Cytiva Life Sciences, France) instrument, with HBS EP+ as the running buffer (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), the tandem epitope binning of CD28 antibodies was performed by surface plasmon resonance (SPR). An anti-human Fc antibody (human antibody capture kit, Cytiva Life Sciences BR-1008-39) was covalently coupled to the sensor chip CM5 (Cytiva Life Sciences, Biacore BR100530).

[0449] Each of the four flow cells was prepared independently. First, using a flow rate of 5 μL / min (amine coupling kit, Cytiva Life Sciences, BR100050), all four flow cells were activated for 7 minutes with a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide mixture (75 - 11.5 mg / mL). The anti-human Fc antibody was 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. Then, using a flow rate of 5 μL / min, the uncoupled sites were inactivated with a 7-minute pulse of 1 M methanolamine (pH 8.5). Antibody of approximately 10,000 resonance units (RU) was typically observed at all points.

[0450] After surface preparation, huCD28-huFc (Beijing Sino Biological Inc., 11524-H02H) was diluted to 0.5 μg / mL in the running buffer and captured for 120 seconds at 5 μL / min until 200 RU (only on FC2 or FC4). A dual injection was performed, where the first injection at 30 μL / min was the crude HEK293-FS supernatant containing the first test bsFab for 120 seconds (saturation condition and stability condition), followed by the second injection at 30 μL / min of the crude HEK293-FS supernatant containing the second test bsFab for 60 seconds (on both FC1-FC2 or FC3-FC4). Then dissociation was monitored for 60 seconds. Using a flow rate of 10 μL / min, the surface was regenerated with a 30-second pulse of 3M MgCl 2 2.

[0451] The sensorgrams were double calibrated by subtracting the reference point (no huCD28-huFc) and buffer injection. The normalized competition signal (C = normalized signal SN2 - normalized signal SN1) was calculated for each antibody pair. The normalized signal corresponded to the signal normalized by capture with huCD28-Fc. For qualitative data, C < +5 indicated no binding and competition of SN2; C > +5 indicated binding of SN2 but no competition. For quantitative data based on normalized signals (complete or partial competition), the equation C*100 / (theoretical NormB SN2 - NormB dissociation SN1) was used for analysis. Statistical analysis

[0452] Two-way hierarchical clustering using the Euclidean distance matrix and Ward's agglomeration method was performed using the statistical software R 3.2.3 to regroup binders with the same spectra in the cluster when each binder was injected first or second. The number of clusters was selected using a graphical representation of the number of clusters varying according to the inertia jump of the dendrogram (where inertia is the variance criterion). The p-value of the Pearson correlation coefficient was calculated using a two-tailed t-test. Example 3. Results Selection, screening, and reformatting of VHHs

[0453] The current study aimed to explore the use of VHH-based bsFabs in activating T cells via the CD28 receptor to kill tumor cells and to provide in-depth characterization of a panel of CD28 constructs in terms of binding, epitope diversity, agonist, and antagonist properties.

[0454] VHHs specific for CD28 and TAAs were isolated using phage display. A large and highly diverse library of VHH sequences was constructed and biopanned in two rounds of selection against the purified recombinant extracellular domains of each antigen or target-expressing cells. After selection, one plate (95 clones) was screened for the recombinant antigen using ELISA in each round (data not shown). For each selection, more than 80% of the tested clones were specific for their target. All specific clones were further sequenced, and sequence analysis revealed a high diversity and low redundancy of the isolated CD28 VHHs, as Figure 2 shown. Briefly, sequence analysis consisted of aligning all hit VHH sequences identified by phage display selection performed by Clustal Omega; then a bootstrap tree output obtained with Clustal Omega was used to construct Figure 2The sequence tree shown in []. The scale bar in the figure corresponds to the distance between sequences. The rectangular labels represent the 48 VHHs tested in the epitope binning, one color code per bin (blue = D, orange = D*, green = E, red = F, gray = undetermined), and the black stars highlight Compounds 2 to 18 that were further tested in the cellular assay. Non-statistical analysis (with the second injection binning) clearly identified subgroups or 16 subgroups in group D; however, no statistically significant differences were observed.

[0455] Cluster diversity analysis based on 95% sequence identity revealed that among 277 CD28-specific clones, 66 VHH clusters were isolated against the recombinant protein and 56 clusters were isolated against CD28-transfected cells. Similar results were found for TAA-specific VHHs (data not shown).

[0456] Forty-seven highly sequence-diverse anti-CD28 VHHs ( Figure 2 the colored labels in []) were then fusion-cloned with the Cλ IgG domain, and 39 anti-TAA VHHs were fusion-cloned with the CH1 IgG domain for eukaryotic expression. Example 4. Generation and functional screening of CD28×TAA bsFab

[0457] Using Figure 1 the compact and linker-free Fab-like format depicted in

[33] to generate CD28×TAA bsFab. More precisely, a matrix combination of 47 anti-CD28 (plus 1 negative control) × 39 anti-TAA (plus 1 negative control) bsFab (i.e., twenty-four 96-well plates) was generated on a 1 mL scale. Quality control was performed on the supernatants of 288 randomly selected wells in the twenty-four 96-well plates, for a total of three quality control plates. The quality control consisted of three components: confirmation of sufficient antibody amount by titration antibody production in HEK293-FS supernatant (approx. 20 μg / mL); caliper measurement to confirm the presence of dimers in the supernatant; and analysis of the ELISA binding ability of the Fab-like antibodies produced in HEK293-FS supernatant to the two targets CD28 and TAA to reveal effective binding in almost all 288 tested antibodies.

[0458] Then, the supernatants of the bsFabs from twenty-four 96-well plates were tested in a functional assay to evaluate the ability of the bsFabs to trigger the killing of tumor cells expressing TAA by purified T cells and the secretion of IL-2 and IFNg over 72 hours. Despite experimental validation with the expected results obtained with positive (CD3×TAA) and negative (CD3×irrelevant target and CD28×irrelevant target) control bsAbs, none of the 1800 bsFabs showed tumor cell killing and IFNg secretion (data not shown). Replicate experiments performed with a quarter of the constructs confirmed the initial results. Finally, a set of 34 bsFabs was purified and tested in a dose range experiment (0.1 - 300 nM) in the same assay (data not shown). Even at the highest tested dose of 300 nM, no tumor cell killing and IFNg secretion were observed. At the highest dose, slight IL-2 secretion was detected for some clones. Example 5. In-depth characterization of anti-CD28 VHH Epitope diversity

[0459] To test the hypothesis that the lack of killing properties in all tested bsFabs might be due to a lack of epitope diversity and functionality of the selected CD28 VHH, the epitope diversity of the anti-CD28 VHH was first characterized.

[0460] Epitope binning experiments of 48 CD28 VHHs (including 47 of those tested above) using SPR revealed three statistically significant epitope clusters (two-way hierarchical clustering using Euclidean distance matrix and Ward agglomeration method), which could be further subdivided into six sub-clusters, as shown in Figure 3 and Figure 11 in the table. Briefly, Figure 3 shows the epitope binning of CD28 bsFabs, i.e., the competition between anti-CD28 VHH and SPR. Figure 3A The following experimental protocol is depicted: Human CD28-Fc was captured on a sensor chip CM5 using an anti-human Fc antibody, followed by the addition of a first CD28×TAA bsFab, followed by the addition of a second CD28×TAA bsFab for binning analysis, and finally 48×48 constructs were tested. Figure 3B The competition data collected in a heatmap is depicted, which indicates whether each VHH pair competes (green indicates blocking and red indicates non-blocking). The heatmap was processed using two-way hierarchical clustering, yielding a dendrogram and 3 significantly different epitope clusters (A, B, C and D, E, F, depending on the injection order). Asymmetry depending on the injection order was observed, as Figure 3B shown.

[0461] A limited set of 17 CD28 VHHs (compounds 2 - 18) that span different epitope bins and maximize sequence diversity was selected (inFigure 2 Orthogonal analysis of epitope diversity was performed for the CD28 VHHs (indicated by the star in) in a fluorescence-activated cell sorting competition assay using CD80 or in an ELISA competition assay using TGN1412 or 9.3 mAb. This analysis revealed that the CD28 VHHs could be classified as competitors and non-competitors of CD80 (shown in Figure 8 which depicts the competition of CD28 bvFab with CD80 using flow cytometry), and independently classified as full competitors, partial competitors, or non-competitors of TGN1412 or 9.3 mAb. In summary, Figure 8 Serial dilutions of CD28 bvFab incubated with Jurkat cells were depicted prior to the addition of recombinant human CD80-Fc protein at EC90. Ligand binding was detected via flow cytometry using Alexa647-conjugated anti-human Fc mAb. Results were expressed as the median fluorescence intensity (MFI). Epitope binning of the VHH subsets and all competition data are reported in Figure 7 the table depicted in which is a summary table from epitope binning and results of competition with TGN1412 and 9.3 benchmark antibodies or CD80 (one of the natural ligands of CD28). Example 6. Agonist properties: Reporter assay

[0462] To evaluate the agonist capacity of CD28 compounds, a bioluminescence 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.

[0463] First, a series of CD28×TAA bsFabs were tested in the presence of HCT116 cells expressing TAA with and without suboptimal TCR activation of the engineered Jurkat cells by anti-CD3-coated mAb. In Figure 9 if the TCR was pre-activated, all bsFabs allowed activation of the IL-2 promoter, indicating that the bsFabs were able to bind both cells, each of which expressed CD28 or TAA. Figure 9 showed that CD28xTAA bsFabs could bind both cells expressing CD28 or TAA. In summary, Jurkat-IL-2-Luc2P cells and HCT116 cells expressing TAA were added to 96-well microplates pre-coated or not pre-coated with anti-CD3 mAb at a ratio of 1:1. These cells were then incubated with 30 nM of the test compound at 37 °C for 6 hours, and then luminescence was measured. The luciferase luminescence signal (S / B = signal-to-background ratio) normalized to cells treated with a negative control (IRR = FMDV bvFab) was shown. Figure 9Each bar on the graph represents the average of two independent experiments.

[0464] Eleven CD28 VHHs that ensured maximum epitope coverage and sequence diversity ( Figure 7 compounds 2 - 12 in the table) were used for in - depth functional characterization. They were generated and purified as Fab - like forms as CD28×FMDV bsFab (monovalent for CD28) or CD28 bvFab (divalent Fab - like where the same VHH is fused to both CH1 and CL). Evaluation of these bsFabs and bvFabs in the absence of sub - optimal TCR activation by anti - CD3 - coated mAb revealed that in the absence of cross - linking, all constructs were inactive regardless of valency, while TGN1412 and 9.3 mAb were highly active, as Figure 10A and Figure 10B shown. Figures 4 and 10 show the evaluation of CD28 bsFabs and bvFabs in the following IL - 2 luciferase reporter assay: Jurkat - IL - 2 - Luc2P cells were added to 96 - well microplates pre - coated with anti - CD3 mAb, as shown in Figures 4 and Figures 10C to 10F shown, rather than as shown in Figure 10A and Figure 10B shown; then these cells were incubated with 100 nM of previously cross - linked or non - cross - linked bsFabs and bvFabs at 37 °C for 6 hours, and then luminescence was measured, and the luciferase luminescence signal (S / B = signal - to - background ratio) normalized to cells treated with a negative control (IRR = FMDV bvFab) was shown. These assays were performed three times and plotted in Figures 4 and 10.

[0465] However, when cross - linked, the activity levels of some bvFabs in bins E and F were lower than the activity levels observed for TGN1412 and 9.3 mAb, as Figure 10B shown. Based on the description of 9.3 mAb as an agonist rather than a super - agonist, the ability of 9.3 mAb to induce luciferase expression in the absence of CD3 co - activation was unexpected. This indicates a certain deviation between T - cell activation and the Jurkat reporter assay.

[0466] In the presence of CD3 co - activation, in the absence of cross - linking, one bvFab from bin E and three out of four bvFabs from bin F were able to induce IL - 2 pathway activation, while all other bsFabs and bvFabs were inactive under soluble conditions, as Figure 4C (which shows the comparison of monovalent CD28×FMDV bsFabs and divalent CD28 bvFabs in the absence of cross - linking) and Figure 10E(It is shown in the comparison of monovalent CD28xFMDVbsFab and bivalent CD28 bvFab without crosslinking after CD3 pre-activation).

[0467] When crosslinked, all of the following induce the IL-2 pathway: all bvFabs, as Figure 4B (which shows bivalent CD28 bvFab with and without crosslinking) and Figure 10D (which shows bivalent CD28bvFab with and without crosslinking after CD3 pre-activation), as shown; all bin E bsFabs and one bin F bsFab, as Figure 4A (which shows monovalent CD28×FMDV bsFab with and without crosslinking) and Figure 10C (which shows monovalent CD28xFMDV bsFab with and without crosslinking after CD3 pre-activation). Most CD28 clones were observed to have better activity under bvFab relative to the bsFab form, revealing the greatest effect on IL-2 induction when combining crosslinking with bivalency, as Figure 4B (which shows bivalent CD28 bvFab with and without crosslinking) and Figure 4D (which shows the comparison of monovalent CD28×FMDV bsFab with crosslinking and bivalent CD28bvFab) and Figure 10D (which shows bivalent CD28 bvFab with and without crosslinking after CD3 pre-activation) and Figure 10F (which shows the comparison of monovalent CD28xFMDV bsFab with crosslinking and bivalent CD28 bvFab after CD3 pre-activation), as shown.

[0468] Clones 5 and 7 from bin E revealed a unique behavior in that their crosslinking allowed CD28 activation, as Figure 4A (which shows monovalent CD28×FMDV bsFab with and without crosslinking) and Figure 4B (which shows bivalent CD28bvFab with and without crosslinking), as shown in the insets, while an increase in potency did not allow CD28 activation, as Figure 4C (which shows the comparison of monovalent CD28×FMDV bsFab without crosslinking and bivalent CD28 bvFab) and Figure 4D (which shows the comparison of monovalent CD28×FMDV bsFab with crosslinking and bivalent CD28 bvFab), as shown. In both the bsFab and bvFab forms, these 2 clones were inactive and active, respectively, in the absence and presence of crosslinking; however, the activity of bvFab was not higher than that of bsFab. Agonist properties: T cell activation

[0469] Then, the agonist properties of the constructs were evaluated using a primary T cell activation assay, where the compounds were tested under soluble conditions without crosslinking in the context of suboptimal TCR activation by anti-CD3 coated mAbs. Some but not all CD28 bvFabs showed the ability to increase IFNγ secretion, as shown in Figure 5. Figure 5 shows the evaluation of CD28 bsFabs and bvFabs in the T cell activation assay. Figure 5A The following experimental setup is shown: Purified human CD3+ T cells were added to 384-well microplates pre-coated with anti-CD3 mAb in the presence of 10 nM, 30 nM, and 100 nM negative (IRR = FMDV bvFab) and positive (TGN1412 and 9.3 mAb) control antibodies or test compounds (CD28×FMDV bsFab and CD28 bvFab). After 6 days of incubation, the levels of IFNγ and TNFα in the cell culture supernatant were determined, and the T cells were counted. (B) IFNγ secretion on day 6 after treatment with 100 nM. Data are represented as the ratio of IFNγ levels in treated cells to cells treated with the IRR negative control. Each bar represents the mean ± standard error of the mean of 5 independent assays (5 independent donors). A strong correlation was observed between IFNγ secretion and two other readouts, namely T cell proliferation (r = 0.63 and p-value = 8.10-41) and TNFα secretion (r = 0.87 and p-value = 7.8.10-110), with some donor-to-donor variability (data not shown).

[0470] Bin D was inactive in both the monovalent and bivalent forms. In addition, four out of five Bin E constructs were active in the monovalent bsFab form (compounds 5 - 8); two were active in the bivalent form (compounds 6 and 8). These findings were inconsistent with the reporter assay. All monovalent bsFabs of Bin F were inactive, while all bivalent constructs were agonists (with variable potency). These findings were consistent with those of the reporter assay.

[0471] Some of the conditions of the T cell activation assay were comparable to those of the reporter assay, as Figure 4C shown by the bsFab / bvFab in Antagonist properties

[0472] CD28 bsFab and bvFab were evaluated in MLR assays, as shown in Figure 6. This is a two-cell system in which monocyte-derived DCs from a first donor known to express CD28 ligands (CD80 / 86) trigger TCR activation of T cells from a second donor. Figure 6A The experimental setup is shown: at the start of the assay, CFSE-labeled CD3+ T cells and allogeneic monocyte-derived DCs were co-cultured with or without 10 nM and 100 nM negative (IRR = FMDV bvFab) and positive (TGN1412 and 9.3 mAb) controls or the test compound (CD28 bvFab). After 4 days, cytokine levels and T cell proliferation in the cell culture supernatant were determined. Figure 6B IFNg secretion on day 4 after treatment with 10 nM is shown. Each bar represents the mean ± SEM of 3 independent assays (3 independent donors).

[0473] All bvFabs from bin F increased T cell proliferation (data not shown) and cytokine secretion (shown in Figure 6), and were more active than TGN1412 and 9.3 mAb. All other bvFabs 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 antagonistic (data not shown). Overall, the results revealed a strong association between the CD80 competition bin and antagonist properties in MLR assays.

[0474] T cell signaling depends mainly on co-stimulatory and co-inhibitory receptors that control TCR function (Chen et al. Nat Rev Immunol (2013) 13(4):227 - 42.). These receptors are diverse, and their functions are largely context-dependent, as has been understood (Chen, ibid.). Co-stimulatory receptors such as CD27, OX40 (CD134), 4-1BB (CD137) or GITR (glucocorticoid-induced TNF receptor-related protein or CD357) and their respective activating ligands have become major research foci in the biopharmaceutical field, with research directions towards their activation or inhibition, respectively, for cancer and inflammatory immunotherapies (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, the antibody or antigen-binding fragment thereof comprising an immunoglobulin single variable domain (IVSD) containing a CDR-H1 sequence, a CDR-H2 sequence, and a CDR-H3 sequence, wherein: a) the CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:1, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:2, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:3; b) the CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:4, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:5, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:6; c) the CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:7, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:9; d) the CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:10, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:11, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:12; e) the CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:13, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:14, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:15; f) the CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:16, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:17, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:18; g) the CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:19, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:20, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:21; h) the CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:22, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:23, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:24; i) the CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:25, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:26, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:27; j) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:28, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:29, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:30; k) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:31, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:32, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:33; l) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:34, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:35, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:36; m) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:37, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:38, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:39; n) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:40, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:41, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:42; o) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:43, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:44, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:45; p) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:46, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:47, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:48; q) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:49, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:50, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:51; r) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:52, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:53, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:54; s) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:55, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:56, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:57; t) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:58, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:59, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:60; u) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:61, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:62, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:63; v) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:64, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:65, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:66; w) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:67, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:68, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:69; x) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:70, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:71, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:72; y) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:73, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:74, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:75; z) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:76, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:77, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:78; aa) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:79, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:80, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:81; ab) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:82, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:83, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:84; ac) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:85, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:86, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:87; ad) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:88, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:89, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:90; ae) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:91, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:92, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:93; af) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:94, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:95, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:96; ag) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:97, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:98, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:99; ah) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:100, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:101, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:102; ai) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:103, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:104, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:105; aj) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:106, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:107, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:108; ak) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 109, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 110, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 111; al) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 112, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 113, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 114; am) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 115, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 116, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 117; an) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 118, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 119, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 120; ao) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 121, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 122, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 123; ap) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 124, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 125, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 126; aq) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 127, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 128, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 129; ar) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 130, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 131, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 132; as) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 133, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 134, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 135; (at) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 136, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 137, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 138; (au) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 139, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 140, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 141; (av) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 142, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 143, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 144; (aw) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 145, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 146, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 147; (ax) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 148, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 149, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 150; (ay) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 151, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 152, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 153; (az) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 154, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 155, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 156; (ba) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 157, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 158, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 159; (bb) The CDR-H1 sequence contains the amino acid sequence shown in SEQ ID NO: 160, the CDR-H2 sequence contains the amino acid sequence shown in SEQ ID NO: 161, and the CDR-H3 sequence contains the amino acid sequence shown in SEQ ID NO: 162; bc) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 163, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 164, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 165; bd) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 166, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 167, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 168; be) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 169, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 170, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 171; bf) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 172, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 173, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 174; bg) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 175, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 176, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 177; bh) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 178, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 179, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 180; bi) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 181, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 182, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 183; bj) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 184, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 185, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 186; bk) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 187, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 188, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 189; bl) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:190, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:191, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:192; bm) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:193, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:194, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:195; bn) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:196, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:197, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:198; bo) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:199, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:200, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:201; bp) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:202, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:203, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:204; bq) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:205, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:206, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:207; br) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:208, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:209, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:210; bs) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:211, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:212, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:213; bt) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:214, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:215, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:216; bu) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 217, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 218, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 219; bv) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 220, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 221, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 222; bw) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 223, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 224, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 225; bx) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 226, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 227, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 228; by) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 229, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 230, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 231; bz) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 232, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 233, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 234; ca) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 235, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 236, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 237; cb) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 238, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 239, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 240; cc) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 241, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 242, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 243; cd) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 244, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 245, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 246; ce) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 247, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 248, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 249; cf) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 250, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 251, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 252; cg) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 253, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 254, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 255; ch) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 256, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 257, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 258; ci) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 259, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 260, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 261; cj) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 262, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 263, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 264; ck) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 265, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 266, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 267; cl) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 268, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 269, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 270; The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 271, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 272, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 273; The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 274, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 275, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 276; The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 277, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 278, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 279; The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 280, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 281, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 282; The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 283, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 284, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 285; The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 286, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 287, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 288; The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 289, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 290, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 291; The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 292, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 293, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 294; The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 295, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 296, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 297; cv) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 298, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 299, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 300; cw) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 301, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 302, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 303; cx) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 304, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 305, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 306; cy) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 307, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 308, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 309; cz) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 310, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 311, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 312; da) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 313, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 314, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 315; db) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 316, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 317, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 318; dc) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 319, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 320, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 321; dd) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 322, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 323, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 324; (de) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 325, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 326, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 327; (df) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 328, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 329, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 330; (dg) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 331, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 332, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 333; (dh) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 334, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 335, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 336; (di) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 337, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 338, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 339; (dj) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 340, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 341, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 342; (dk) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 343, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 344, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 345; (dl) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 346, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 347, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 348; (dm) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 349, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 350, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 351; dn) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:352, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:353, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:354; do) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:355, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:356, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:357; dp) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:358, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:359, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:360; dq) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:361, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:362, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:363; dr) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:364, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:365, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:366; ds) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:367, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:368, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:369; dt) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:370, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:371, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:372; du) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:373, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:374, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:375; dv) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO:376, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO:377, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:378; dw) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 379, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 380, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 381; dx) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 382, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 383, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 384; dy) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 385, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 386, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 387; dz) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 388, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 389, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 390; ea) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 391, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 392, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 393; eb) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 394, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 395, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 396; ec) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 397, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 398, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 399; ed) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 400, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 401, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 402; ee) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 403, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 404, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 405; ef) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 406, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 407, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 408; eg) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 409, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 410, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 411; eh) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 412, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 413, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 414; ei) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 415, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 416, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 417; ej) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 418, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 419, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 420; ek) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 421, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 422, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 423; el) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 424, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 425, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 426; em) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 427, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 428, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 429; en) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 430, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 431, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 432; eo) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 433, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 434, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 435; ep) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 436, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 437, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 438; eq) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 439, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 440, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 441; er) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 442, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 443, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 444; es) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 445, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 446, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 447; et) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 448, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 449, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO: 450; or eu) The CDR-H1 sequence comprises the amino acid sequence shown in SEQ ID NO: 451, the CDR-H2 sequence comprises the amino acid sequence shown in SEQ ID NO: 452, and the CDR-H3 sequence comprises the amino acid sequence shown in SEQ ID NO:

453.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the ISVD comprises a VHH domain, wherein: a) the VHH domain comprises the amino acid sequence shown in SEQ ID NO: 454; b) the VHH domain comprises the amino acid sequence shown in SEQ ID NO: 455; c) the VHH domain comprises the amino acid sequence shown in SEQ ID NO: 456; d) the VHH domain comprises the amino acid sequence shown in SEQ ID NO: 457; e) the VHH domain comprises the amino acid sequence shown in SEQ ID NO: 458; f) the VHH domain comprises the amino acid sequence shown in SEQ ID NO:

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

604.

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

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

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

7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is operably linked to the CH1 domain and / or the CL domain.

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

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

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

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

12. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, the antibody or antigen-binding fragment thereof comprises an antagonistic antibody or antigen-binding fragment thereof.

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

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

15. A host cell comprising the expression vector according to claim 14.

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

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

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

19. The method according to claim 17, wherein the disease is cancer.

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