Novel anti-LILRB4 antibodies and uses thereof

Abstract

The present disclosure provides an anti-LILRB4 antibody or an antigen binding fragment thereof, a pharmaceutical composition comprising the anti-LILRB4 antibody or an antigen binding fragment thereof, and uses thereof.

Description

Technical Field

[0001] The present disclosure generally relates to novel anti-LILRB4 antibodies and antigen-binding fragments thereof, and uses thereof. Background Art

[0002] Acute myeloid leukemia (AML) is one of the most common acute leukemias in adults. In order to effectively treat AML, new molecular targets and treatment methods should be identified. Leukocyte immunoglobulin-like receptors (LILRs) are a family of at least 13 receptors expressed mainly by lymphocytes and myelomonocytic cells (Rachel Thomas et al., "Clin Rev Allergy Immunol., April 2010; 38 (2-3): 159-62). Generally, the LILR family includes LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRB6 and LILRB7. LILRB is known to be expressed on bone marrow cells and certain other hematopoietic cells (Mori et al., "Journal of Immunology (JImmunol.)", October 1, 2008; 181 (7): 4742-51). Several members of the LILRB family are highly expressed on AML cells, and their expression is negatively correlated with the overall survival of human AML patients. In addition, individual inhibition of the expression of several LILRBs can inhibit different human leukemia cell lines in culture and block leukemia development in xenograft mice (see WO 2013181438A2).

[0003] Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) is a protein encoded by the LILRB4 gene in humans. The encoded protein belongs to the subfamily B class of LILR, which contains two or four extracellular immunoglobulin domains, one transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LILRB4 is expressed on monocytes and transduces negative signals that inhibit stimulation of the immune response. LILRB4 may also play a role in antigen capture and presentation. It is believed that LILRB4 controls inflammatory responses and cytotoxicity to help focus the immune response and limit autoreactivity. LILRB4 has been considered a potential target for tumor immunotherapy. It has been shown that LILRB4 is expressed on tumor-associated macrophages and negatively regulates immune responses in tumors. Expression of LILRB4 on monocytic myeloid leukemia cells supports infiltration and inhibits T cell proliferation. IO-202, developed by Immune-Onc Therapeutics, is in Phase I clinical trials for the treatment of AML and chronic myelomonocytic leukemia (CMML).

[0004] There remains a need for novel anti-LILRB4 antibodies. Summary of the invention

[0005] Throughout this disclosure, the articles "a", "an" and "the" are used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, "antibody" means one antibody or more than one antibody.

[0006] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to LILRB4, the antibody or the antigen-binding fragment thereof comprising:

[0007] one or two or three heavy chain complementarity determining regions (HCDR1, HCDR2 and / or HCDR3) contained within any one of a heavy chain variable (VH) region sequence selected from the group consisting of SEQ ID NO: 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 154 and 156; and / or

[0008] One or two or three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3), wherein the one or two or three light chain complementary determining regions are contained within any one of the light chain variable (VL) region sequences selected from the group consisting of: SEQ ID NO: 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 155 and 157.

[0009] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise at least one heavy or light chain complementary determining region (CDR) comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 33, 34, 35, 36, 37, 38, 41, 42, 43, 44, 45, 46, 49, 50, 51, 52, 53, 54, 57, 58, 59, 60, 61, 62, 65, 66, 67, 68, 69, 70, 73, 74, 75, 76, 77, 78, 81, 82, 83, 84, 85, 86, 89, 90, 91, 92 , 93, 94, 97, 98, 99, 100, 101, 102, 105, 106, 107, 108, 109, 110, 113, 114, 115, 116, 117, 118, 121, 122, 123, 124, 125, 126, 129, 130, 131, 132, 133, 134, 137, 138, 139, 140, 141, 142, 145, 146, 147, 148, 149, 150 and 158.

[0010] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise one or two or three of HCDR1, HCDR2 and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, 25, 26, 27, 33, 34, 35, 41, 42, 43, 49, 50, 51, 57, 58, 59, 65, 66, 67, 73, 74, 75, 81, 82, 83, 89, 90, 91, 97, 98, 99, 105, 106, 107, 113, 114, 115, 121, 122, 123, 129, 130, 131, 137, 138, 139, 145, 146, 147 and 158.

[0011] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise one or two or three of LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 21, 22, 28, 29, 30, 36, 37, 38, 44, 45, 46, 52, 53, 54, 60, 61, 62, 68, 69, 70, 76, 77, 78, 84, 85, 86, 92, 93, 94, 100, 101, 102, 108, 109, 110, 116, 117, 118, 124, 125, 126, 132, 133, 134, 140, 141, 142, 148, 149, and 150.

[0012] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise:

[0013] i. HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137 and 145;

[0014] ii. a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, 146 and 158; and

[0015] iii. HCDR3, said HCDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139 and 147.

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise:

[0017] i. LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132, 140 and 148;

[0018] ii. LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141 and 149; and

[0019] iii. LCDR3, said LCDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134, 142 and 150.

[0020] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise:

[0021] i. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 19;

[0022] ii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 27;

[0023] iii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 33, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 34, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 35;

[0024] iv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:42, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43;

[0025] v. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:158, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43;

[0026] vi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:50, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:51;

[0027] vii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 57, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 58, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 59;

[0028] viii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 65, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 66, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 67;

[0029] ix. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 73, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 74, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 75;

[0030] x. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:81, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:82, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:83;

[0031] xi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:89, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:90, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:91;

[0032] xii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 97, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 98, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 99;

[0033] xiii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 106, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 107;

[0034] xiv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 114, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 115;

[0035] xv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 122, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 123;

[0036] xvi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 130, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 131;

[0037] xvii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 138, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 139; or

[0038] xviii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 145, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 146, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 147.

[0039] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0040] i. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 20, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 22;

[0041] ii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:28, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:29, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:30;

[0042] iii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:36, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:37, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:38;

[0043] iv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:44, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:45, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:46;

[0044] v. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:52, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:53, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:54;

[0045] vi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:60, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:61, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:62;

[0046] vii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:68, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:69, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:70;

[0047] viii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 76, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 77, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 78;

[0048] ix. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:84, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:85, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:86;

[0049] x. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:92, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:93, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:94;

[0050] xi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 100, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 101, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 102;

[0051] xii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 108, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 109, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 110;

[0052] xiii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 116, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 118;

[0053] xiv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 124, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 125, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 126;

[0054] xv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 132, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 133, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 134;

[0055] xvi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 140, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 141, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 142; or

[0056] xvii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:148, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:149, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:150.

[0057] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise:

[0058] i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 18, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 19, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 20, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 21, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 22;

[0059] ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:25, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:26, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:27, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:28, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:29, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:30;

[0060] iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:33, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:34, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:35, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:36, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:37, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:38;

[0061] iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:42, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:44, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:45, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:46;

[0062] v. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:158, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:44, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:45, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:46;

[0063] vi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:50, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:51, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:52, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:53, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:54;

[0064] vii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:57, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:58, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:59, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:60, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:61, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:62;

[0065] viii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:65, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:66, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:67, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:68, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:69, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:70;

[0066] ix. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:73, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:74, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:75, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:76, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:77, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:78;

[0067] x. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:81, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:82, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:83, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:84, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:85, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:86;

[0068] xi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:89, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:90, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:91, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:92, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:93, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:94;

[0069] xii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:97, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:98, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:99, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:100, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:101, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:102;

[0070] xiii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 106, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 107, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 108, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 109, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 110;

[0071] xiv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 114, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 115, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 116, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 117, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 118;

[0072] xv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 122, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 123, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 124, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 125, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 126;

[0073] xvi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 130, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 131, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 132, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 133, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 134;

[0074] xvii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 138, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 139, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 140, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 141, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 142; or

[0075] xviii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:145, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:146, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:147, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:148, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:149, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:150.

[0076] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a VH region having an amino acid sequence as shown in SEQ ID NO: 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 154 or 156, or a homologous sequence thereof having at least 80% sequence identity to SEQ ID NO: 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 154 or 156.

[0077] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a VL region having an amino acid sequence as set forth in SEQ ID NO: 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 155 or 157, or a homologous sequence thereof having at least 80% sequence identity thereto.

[0078] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a VH / VL amino acid sequence pair selected from the group consisting of SEQ ID NO: 23 / 24, 31 / 32, 39 / 40, 47 / 48, 55 / 56, 63 / 64, 71 / 72, 79 / 80, 87 / 88, 95 / 96, 103 / 104, 111 / 112, 119 / 120, 127 / 128, 135 / 136, 143 / 144, 151 / 152, 154 / 155, and 156 / 157.

[0079] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise one or more amino acid residue substitutions or modifications, but still retain binding affinity to LILRB4. In some embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences in the VH region or the VL region. In some embodiments, at least one of the substitutions or modifications is in one or more of the non-CDR sequences in the VH region or the VL region. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise one or more non-natural amino acid (NNAA) substitutions. In some embodiments, the NNAA can be conjugated.

[0080] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have one or more binding properties to LILRB4 selected from the group consisting of:

[0081] i. capable of specifically binding to human LILRB4 as measured by FACS assay;

[0082] ii. capable of specifically binding to human LILRB4 and human LILRB3 as measured by FACS assay;

[0083] iii. capable of specifically binding to human LILRB4 and cynomolgus monkey LILRB4 as measured by FACS assay;

[0084] iv. Has effective ADCP effect on human AML cell lines;

[0085] v. Has effective ADCC effect on human AML cell lines;

[0086] vi. being able to block LILRB4-fibronectin interaction to reprogram tolerogenic dendritic cells into mature dendritic cells that stimulate T cell activation as measured by FcγR stimulation assay;

[0087] vii. Able to induce the production of TNF-α;

[0088] viii. Ability to reprogram tolerant DCs to activate T cells;

[0089] ix. Ability to reverse macrophage-mediated T cell suppression;

[0090] x. Ability to reverse THP-1-mediated T cell suppression; and

[0091] xi.Can enhance CD8 + T cell-mediated cytotoxicity against THP-1 cells.

[0092] In another aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof, which competes with the antibody or antigen-binding fragment thereof as described above for binding to LILRB4.

[0093] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are chimeric, humanized, or human antibodies or antigen-binding fragments thereof.

[0094] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are labeled antibodies, bivalent antibodies, anti-idiotypic antibodies, or fusion proteins.

[0095] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), camelized single domain antibodies, nanobodies, domain antibodies or bivalent domain antibodies.

[0096] In some embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises an Fc region. In some embodiments, the Fc region is an Fc region of a human immunoglobulin (Ig). In some embodiments, the Fc region is an Fc region of a human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region comprises an amino acid sequence as shown in SEQ ID NO: 153.

[0097] In some embodiments, the light chain of the antibodies or antigen-binding fragments thereof provided herein is a lambda light chain or a kappa light chain.

[0098] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are bispecific or multispecific antibodies or antigen-binding fragments thereof. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to one or more additional antigens other than LILRB4 or a second epitope on LILRB4.In some embodiments, the one or more additional antigens other than LILRB4 are selected from the group consisting of CD3, CD16a, CD33, CD38, CD45, CD123, CD146, CD228, CLL-1, Flt3, TAF1, TgPRF, HVCN1, IL-6R, IL-11R, IL17A, IL-23R, IL-33, ILDR2, LAP, TSLP, TREM-1, ANGPT2, APOE, IFNAR, CypA, DOG-1, NKp30, CSF-1R, CCR2, LRRC15, mesothelin, Dickkopf2, DLL3, HER-2, C10orf54, TrkA, MEKK1, KRAS, ERK, XPO1, mTORC1 / 2, PAK4, NAMPT, ATR, EGFR, FGFR, VEGF, c-MET, Her2, Her3, CTLA4, GITA, CD112 R, CD2, CD7, CD16, CD19, CD20, CD24, CD27, CD30, CD34, CD37, CD39, CD70, CD73, CD83, CD28, CD80(B7-1), CD86(B7-2), CD40, CD40L(CD154), CD 47. SIRPα, CD122, CD137, CD137L, OX40 (CD134), OX40L (CD252), BCMA (e.g., BCMA02), PSMA, CLDN18 (e.g., CLDN18.2), NKG2C, 4-1BB, LIGHT, PVRI G, SLAMF7, HVEM, BAFFR, ICAM-1, 2B4, LFA-1, GITR, ICOS(CD278), ICOSLG(CD275), LAG3(CD223), A2AR, B7-H3(CD276), B7-H4(VTCN1), B7-H5, BTLA(CD272), BTLA, CD160, CTLA-4(CD152), GPRC5D, IDO1, IDO2, ILT3, TDO, KIR, LAIR-1, NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC-7 (CD328), SIGLEC-9(CD329), SIGLEC-15, TIGIT, PVR(CD155), LILRB2, LILRB3, FLT3, FLT3L, TLR3, CLEC9A, DEC-205, STING, IL-12, IDO and TGFβ.

[0099] In some embodiments, the antibody or antigen binding fragment thereof provided herein is connected to one or more conjugate parts. In some embodiments, the conjugate parts include scavenging modifiers, chemotherapeutic agents, toxins, radioisotopes, lanthanides, detectable labels, DNA alkylating agents, topoisomerase inhibitors, tubulin binding agents, purification parts or other anticancer drugs. In some embodiments, the conjugate parts are covalently linked directly or through a linker.

[0100] On the other hand, the present disclosure provides a chimeric antigen receptor, the chimeric antigen receptor comprises an antibody or antigen binding fragment thereof provided herein, a transmembrane region and an intracellular signal region. In some embodiments, the transmembrane region comprises a transmembrane region of CD3, CD4, CD8 or CD28. In some embodiments, the intracellular signal region is selected from the group consisting of: intracellular signal region sequences of CD3, FcγRI, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR or a combination thereof. In some embodiments, the antigen binding fragment of the chimeric antigen receptor is scFv. In some embodiments, the chimeric antigen receptor is transplanted onto allogeneic cells, autologous cells or xenogeneic cells. In some embodiments, the chimeric antigen receptor is transplanted onto immune effector cells. In some embodiments, the chimeric antigen receptor is transplanted onto T cells, natural killer cells, macrophages or tumor infiltrating lymphocytes.

[0101] In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and / or chimeric antigen receptor of the present disclosure and one or more pharmaceutically acceptable carriers.

[0102] In another aspect, the present disclosure provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof and / or chimeric antigen receptor of the present disclosure.

[0103] In another aspect, the present disclosure provides a vector comprising the isolated polynucleotide of the present disclosure.

[0104] On the other hand, the present disclosure provides a host expression system, the host expression system comprises a vector of the present disclosure or has a polynucleotide of the present disclosure integrated into its genome. In some embodiments, the host expression system of the present disclosure is a microorganism, a yeast or a mammalian cell. In some embodiments, the microorganism is selected from the group consisting of Escherichia coli (E.coli) and Bacillus subtilis (B.subtilis). In some embodiments, the yeast is Saccharomyces. In some embodiments, the mammalian cell is selected from the group consisting of: COS, CHO-S, CHO-K1, HEK-293 and 3T3 cells.

[0105] In another aspect, the present disclosure provides a virus comprising the vector of the present disclosure.

[0106] In another aspect, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof of the present disclosure and / or the chimeric antigen receptor of the present disclosure and / or the pharmaceutical composition of the present disclosure and a second therapeutic agent.

[0107] In another aspect, the present disclosure provides a method for expressing the antibody or antigen-binding fragment thereof and / or the chimeric antigen receptor of the present disclosure, the method comprising culturing the host expression system of the present disclosure under conditions for expressing the antibody or antigen-binding fragment thereof or the chimeric antigen receptor of the present disclosure.

[0108] In another aspect, the present disclosure provides a method for treating, preventing or ameliorating a disease, disorder or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof and / or chimeric antigen receptor and / or pharmaceutical composition of the present disclosure.

[0109] In another aspect, the present disclosure provides use of an antibody or antigen-binding fragment thereof and / or chimeric antigen receptor and / or pharmaceutical composition of the present disclosure in the preparation of a medicament for treating a LILRB4-related disease, disorder or condition in a subject.

[0110] In another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof and / or chimeric antigen receptor and / or pharmaceutical composition of the present disclosure in the preparation of a diagnostic reagent for diagnosing a LILRB4-related disease, disorder or condition.

[0111] In some embodiments, the disease, disorder or condition is an immune disease, an inflammatory disease, a cancer or a neurological disease. In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the disease, disorder or condition is a LILRB4-expressing B-cell cancer. In some embodiments, the disease, disorder or condition is selected from the group consisting of Kawasaki disease, Toxoplasma gondii, multiple sclerosis, systemic Lupus erythematosus, lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lung adenocarcinoma, lung squamous cell carcinoma, Lewis lung carcinoma or radiation therapy resistant Lewis lung cancer), peritoneal cancer, carcinoid, bone cancer, pancreatic cancer, primitive neuroectodermal tumor, skin cancer, gallbladder cancer, head and neck cancer, squamous cell carcinoma, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial carcinoma), anal region cancer (e.g., anal squamous cell carcinoma), gastric or stomach cancer. cancer (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatoma or hepatocellular carcinoma), bile duct cancer, sarcoma, colorectal cancer, fallopian tube cancer, salivary gland cancer, cervical cancer, endometrial cancer or uterine cancer, osteosarcoma, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, nasopharyngeal cancer, soft tissue sarcoma, polycythemia vera, urethral cancer, penile cancer, kidney or urethral cancer (e.g., renal rhabdoid tumor), cutaneous T-cell lymphoma, medulloblastoma, nephroblastoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, choroid plexus papilloma, renal cell carcinoma, renal pelvic cancer, central nervous system (CNS) neoplasms, soft tissue sarcomas (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi's sarcoma (Kaposi's sarcoma), spinal cord axial tumors, gliomas (e.g., ependymomas, astrocytomas, anaplastic astrocytomas, oligodendrogliomas), eye cancers (e.g., retinoblastomas), brain stem gliomas or mixed gliomas such as oligoastrocytomas), brain tumors (e.g., glioblastoma / glioblastoma multiforme (GBM), non-glioblastoma brain tumors, or meningiomas), melanomas (e.g., cutaneous or intraocular melanomas), thrombocytosis, mesothelioma, mycosis fungoides, Sezary syndrome, primary myelofibrosis, solitary plasmacytoma, vestibular schwannoma, Ewing's sarcoma, chondrosarcoma, MYH-associated polyposis, pituitary adenomas, pediatric cancers, such as pediatric sarcomas (e.g., neuroblastoma, rhabdomyosarcoma, and osteosarcoma),Blood cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia (e.g., lymphocytic / lymphoblastic leukemia), chronic or acute leukemia, mast cell leukemia, lymphocytic lymphoma, primary CNS lymphoma, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia (HCL), Burkitt's lymphoma (Burkitt's lymphoma, BL), multiple myeloma (e.g., relapsed or refractory multiple myeloma), T or B cell lymphoma, mantle cell lymphoma (MCL) (e.g., relapsed or refractory mantle cell lymphoma), malignant melanoma, diffuse large B cell lymphoma (DLBCL), DLBCL arising from follicular lymphoma, high-grade B cell lymphoma, primary mediastinal large B cell lymphoma, follicular lymphoma (FL), and primary mediastinal B cell lymphoma. In some embodiments, the disease, disorder or condition is acute myeloid leukemia. In some embodiments, the disease, disorder or condition is chronic myelomonocytic leukemia. ,

[0112] In some embodiments, the subject is a human.

[0113] In some embodiments, the administration is by parenteral routes, including subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection; or non-parenteral routes, including transdermal, oral, intranasal, intraocular, sublingual, rectal, or topical.

[0114] In some embodiments, the method for treating, preventing or alleviating a disease, disorder or condition of a subject further comprises administering an additional therapeutic agent to a subject in need thereof. In some embodiments, the additional therapeutic agent is selected from the group consisting of: active agents, imaging agents, cytotoxic agents and angiogenesis inhibitors, kinase inhibitors, co-stimulatory molecule agonists, co-inhibitory molecule blockers, adhesion molecule blockers, anti-cytokine antibodies or their functional fragments, detectable markers or reporter genes, antimicrobial agents, gene editors, beta agonists, viral RNA inhibitors, polymerase inhibitors, interferons and microRNAs. In some embodiments, the additional therapeutic agent is administered to the subject in need thereof before, after or simultaneously with the administration of the antibody or its antigen-binding fragment and / or pharmaceutical composition and / or chimeric antigen receptor of the present disclosure.

[0115] In another aspect, the present disclosure provides a method of modulating LILRB4 activity in a LILRB4-expressing cell, the method comprising exposing the LILRB4-expressing cell to an antibody or antigen-binding fragment thereof and / or a pharmaceutical composition and / or a chimeric antigen receptor of the present disclosure.

[0116] In some embodiments, the LILRB4-expressing cell is a dendritic cell, a monocyte, a macrophage, a B cell, a Treg, a promast cell, an endothelial cell, or an osteoclast.

[0117] In another aspect, the present disclosure provides a method for inducing phagocytosis of a target cell in vivo or in vitro, the method comprising exposing the target cell to an antibody or antigen-binding fragment thereof and / or a pharmaceutical composition and / or a chimeric antigen receptor of the present disclosure. In some embodiments, the target cell is an antigen presenting cell, a cancer cell, or a cell infected by a pathogen.

[0118] In another aspect, the present disclosure provides a method of inducing TNF-α production, the method comprising exposing tolerogenic dendritic cells to the antibody or antigen-binding fragment thereof and / or pharmaceutical composition and / or chimeric antigen receptor of the present disclosure.

[0119] In another aspect, the present disclosure provides a method of reprogramming tolerogenic dendritic cells into mature dendritic cells, the method comprising exposing the tolerogenic dendritic cells to the antibody or antigen-binding fragment thereof and / or pharmaceutical composition and / or chimeric antigen receptor of the present disclosure.

[0120] In another aspect, the present disclosure provides a method for detecting the presence or amount of LILRB4 in a sample, the method comprising: contacting the sample with the antibody or antigen-binding fragment thereof and / or pharmaceutical composition and / or chimeric antigen receptor of the present disclosure; and determining the presence or the amount of LILRB4 in the sample.

[0121] On the other hand, the present disclosure provides a method for diagnosing a LILRB4-related disease or condition in a subject, the method comprising: a) obtaining a sample from the subject; b) contacting the sample obtained from the subject with the antibody or antigen-binding fragment thereof and / or pharmaceutical composition and / or chimeric antigen receptor of the present disclosure; c) determining the presence or amount of LILRB4 in the sample; and d) correlating the presence or amount of LILRB4 with the presence or status of the LILRB4-related disease or condition in the subject.

[0122] In another aspect, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof and / or pharmaceutical composition and / or chimeric antigen receptor of the present disclosure, which can be used to detect LILRB4, optionally recombinant LILRB4, LILRB4 expressed on the cell surface, or LILRB4-expressing cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1 Shown are the binding affinities of selected chimeric antibodies and benchmark antibodies (IO-202 and NGM831, which were produced as human IgG1 chimeric antibodies) to 293F-hLILRB4 cells as measured by FACS assay.

[0124] Figure 2 AB shows the effects of ch43-D12-F3-G11 and ch42-C8-A12-F3-D11 on 293F-hLILRB3 cells ( Figure 2 A) and 293F-hLILRB4 cells ( Figure 2 B) Binding affinity.

[0125] Figure 3 AB shows the effect of ch44-F10-B6-D4 on CHO-S-cynoLILRB4 cells ( Figure 3 A) and 293F-hLILRB4 cells ( Figure 3 B) Binding affinity.

[0126] Figure 4 Shown is FACS analysis of LILRB4 expression in different human cancer cell lines.

[0127] Figure 5 Shown is the ADCC effect of antibody ch2-H1-D7-E5-D5 compared to benchmark antibodies IO-202 and NGM831 on THP-1 cells.

[0128] Figure 6 Shown is the ADCP effect of antibody ch2-H1-D7-E5-D5 compared to benchmark antibody IO-202 on MV-4-11 cells.

[0129] Figure 7 AD shows the efficacy of selected chimeric antibodies in the treatment of radiation-resistant Lewis lung carcinoma in female hLILRB1 / hLILRB4 transgenic mice ( Figure 7 A: Tumor growth inhibition; Figure 7 C: body weight) and EL4-LILRB4 lymphoma ( Figure 7 B: tumor growth inhibition; Figure 7 D: In vivo efficacy test results of body weight).

[0130] Figure 8 AF show the binding affinity of humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 to LILRB4 compared to benchmark antibodies IO-202 and NGM831.

[0131] Fig. 9 Shown are mixed lymphocyte reaction (MLR) results of humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 compared to benchmark antibodies IO-202 and NGM831.

[0132] Fig.10 Shown are the results of macrophage and T cell co-culture assays of humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 compared to benchmark antibodies IO-202 and NGM831.

[0133] Fig.11 AD show THP-1 and T cell co-culture assay results of chimeric antibodies ch2-H1-D7-E5-D5 and ch8-B3-F6-H8 compared to benchmark antibody IO-202.

[0134] Fig.12 The cytotoxicity of humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 on THP-1 cells compared with the benchmark antibody IO-202 is shown. + T cell killing assay results.

[0135] Fig.13 AB shows the ADCC effect of humanized antibody h2-H1-D7-E5-D5 on THP-1 cells compared with the benchmark antibody IO-202 ( Fig.13 A) and ADCP effect ( Fig.13 B).

[0136] Fig.14 AB shows the efficacy of humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 in treating EL4-LILRB4 lymphoma in female hLILRB1 / hLILRB4 transgenic mice ( Fig.14 A: Tumor growth inhibition; Fig.14 B: In vivo efficacy test results in (body weight). DETAILED DESCRIPTION

[0137] The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. In this way, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that such equivalent embodiments will be included herein. All documents cited in this article, including open publications, patents and patent applications are incorporated herein by reference in their entirety.

[0138] definition

[0139] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen. A natural complete antibody includes two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ and μ, each heavy chain including a variable region (VH) and a first, second, third and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are classified as λ or κ, and each light chain includes a variable region (VL) and a constant region. The antibody is in a "Y" shape, wherein the stem of the Y includes the second constant region and the third constant region of two heavy chains bound together by disulfide bonds. Each arm of the Y includes the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the two chains usually include three highly variable loops, called complementary determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2 and LCDR3, and heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The three CDRs are inserted between the side-joined segments (stretches) called framework regions (FRs) (light chain FRs include LFR1, LFR2, LFR3 and LFR4, and heavy chain FRs include HFR1, HFR2, HFR3 and HFR4), which are more highly conserved than CDRs and form a scaffold that supports highly variable loops. The constant regions of the heavy and light chains do not participate in antigen binding, but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the constant region of the heavy chain of the antibody. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, characterized by the presence of α, δ, ε, γ and μ heavy chains, respectively. The several major antibody classes are divided into subclasses, such as IgGl (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgAl (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain).

[0140] In certain embodiments, the antibodies provided herein encompass any antigen binding fragment thereof. As used herein, the term "antigen binding fragment" refers to an antibody fragment formed by a portion of an antibody including one or more (e.g., 1, 2, 3, 4, 5 or 6) CDRs, or any other antibody fragment that binds to an antigen but does not include a complete native antibody structure. Examples of antigen binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (divalent bifunctional antibodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies or divalent domain antibodies. Antigen binding fragments can bind to the same antigen or epitope that the parent antibody binds to.

[0141] "Fab" of an antibody refers to the portion of an antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by disulfide bonds.

[0142] "Fab'" refers to the Fab fragment including a portion of the hinge region.

[0143] "F(ab')2" refers to a dimer of Fab'.

[0144] The "Fc" of an antibody (e.g., an IgG, IgA or IgD isotype antibody) refers to the portion of the antibody consisting of the second constant domain and the third constant domain of the first heavy chain combined with the second constant domain and the third constant domain of the second heavy chain via a disulfide bond. The Fc of IgM and IgE isotype antibodies further includes a fourth constant domain. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

[0145] The "Fv" of an antibody refers to the smallest fragment of an antibody that carries a complete antigen binding site. The Fv fragment consists of the variable region of a single light chain combined with the variable region of a single heavy chain.

[0146] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region, which are directly linked to each other or linked to each other by a linker (e.g., a peptide sequence) (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0147] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc region of an antibody.

[0148] "Camelized single domain antibody", "heavy chain antibody" or "HCAb" refers to an antibody containing two VH domains without light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Pat. No. 6,005,079). Heavy chain antibodies were originally derived from the Camelidae (camels, dromedaries and llamas). Despite the absence of light chains, camelized antibodies have a realistic antigen binding repertoire (Hamers-Casterman C. et al., Nature. June 3; 363(6428): 446-8 (1993); Nguyen VK. et al., Immunogenetics. April; 54(1): 39-47 (2002); Nguyen VK. et al., Immunology. May; 109(1): 93-101 (2003)). The variable domain of the heavy chain antibody (VHH domain) represents the smallest known antigen binding unit produced by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 June 15 (2007)).

[0149] "Nanobody" refers to an antibody fragment consisting of the VHH domain from a heavy chain antibody and the two constant domains CH2 and CH3.

[0150] "Diabodies" or "dAbs" include small antibody fragments with two antigen binding sites, wherein the fragments include a VH domain and a VL domain connected on the same polypeptide chain (VH-VL or VL-VH) (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA Jul. 15;90(14):6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby generating two antigen binding sites. The antigen binding sites can target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a bifunctional antibody that targets two different antigens (or epitopes).

[0151] "Domain antibodies" refer to antibody fragments that contain only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH domains are covalently joined with a peptide linker to produce a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0152] As used herein, the term "valence" refers to the presence of a specified number of antigen binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen binding fragment having only one single antigen binding site; and the term "multivalent" refers to an antibody or antigen binding fragment having multiple antigen binding sites. Thus, the terms "bivalent", "tetravalent" and "hexavalent" refer to the presence of two antigen binding sites, four antigen binding sites and six antigen binding sites in an antigen binding molecule, respectively. In some embodiments, the antibody or its antigen binding fragment is bivalent.

[0153] As used herein, a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens.

[0154] As used herein, a "multispecific" antibody refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes within the same antigen. A multispecific antibody can, for example, bind to different epitopes within two, three, four, five or more different antigens or the same antigen.

[0155] In certain embodiments, the "scFv dimer" is a bivalent bifunctional antibody or a bispecific scFv (BsFv), which includes VH-VL (connected by a peptide linker), which dimerizes with another VH-VL part, so that the VH of one part coordinates with the VL of the other part and forms two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific bifunctional antibody, which includes VH1-VL2 (connected by a peptide linker) and VL1-VH2 (also connected by a peptide linker) that associate with each other, so that VH1 and VL1 coordinate, VH2 and VL2 coordinate, and each coordination pair has a different antigen specificity.

[0156] "dsFv" refers to a disulfide-stabilized Fv fragment, in which the connection between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, "(dsFv)2" or "(dsFv-dsFv')" includes three peptide chains: two VH parts connected by a peptide linker (e.g., a long flexible linker) and respectively bound to two VL parts by a disulfide bridge. In some embodiments, dsFv-dsFv' is bispecific, in which each pair of heavy and light chains paired by a disulfide bond has different antigenic specificity.

[0157] As used herein, the term "chimeric" means an antibody or antigen-binding fragment having a portion of a heavy chain and / or light chain derived from one species and the remainder of the heavy chain and / or light chain derived from a different species. In an illustrative example, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0158] As used herein, the term "humanized" means that the antibody or antigen-binding fragment includes CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans when applicable. The CDRs of the humanized antibodies provided by the present disclosure may contain mutations compared to the CDRs of their parent antibodies.

[0159] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (ie, antibody) or antigen-binding fragment thereof and an antigen.

[0160] An antibody or antigen-binding fragment thereof that "specifically binds" or "specific binding" to a target (e.g., an epitope) is a term well known in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with a greater affinity than it does with alternative cells or substances. An antibody "specifically binds" to a target if it binds with greater affinity, with a higher avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically binds to a LILRB4 epitope is an antibody that binds to this LILRB4 epitope with greater affinity, with a higher avidity, more readily, and / or for a longer duration than it binds to other LILRB4 epitopes or non-LILRB4 epitopes. It should also be understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" (specifically bind) does not necessarily require (although it can include) exclusive binding. Typically, but not necessarily, references to binding refer to specific binding.

[0161] As used herein, the ability to "compete for binding to LILRB4" refers to the ability of a first antibody or antigen-binding fragment thereof to inhibit the binding interaction between LILRB4 and a second anti-LILRB4 antibody to any detectable extent. In certain embodiments, the antibody or antigen-binding fragment that competes for binding to LILRB4 inhibits the binding interaction between LILRB4 and a second anti-LILRB4 antibody by at least 85% or at least 90%. In certain embodiments, this inhibition may be greater than 95% or greater than 99%.

[0162] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitope within the antigen. An epitope may be linear or conformational (i.e., comprising spaced-apart amino acid residues). For example, if an antibody or antigen-binding fragment blocks at least 85%, or at least 90%, or at least 95% binding of a reference antibody to an antigen, then the antibody or antigen-binding fragment may be considered to bind to the same / closely related epitope as the reference antibody.

[0163] As used herein, the term "amino acid" refers to an organic compound containing amino (-NH2) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. The names of amino acids are also represented in this disclosure by standard single-letter or three-letter codes, which are summarized below.

[0164]

[0165]

[0166] "Conservative substitution" with respect to an amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain of similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As known in the art, conservative substitutions generally do not cause significant changes in protein conformational structure, and therefore the biological activity of the protein can be retained.

[0167] As used herein, the term "homologous" refers to a nucleic acid sequence (or its complementary strand) or an amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to another sequence when optimally aligned.

[0168] "Percentage (%) of sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percentage (%) of sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence to which it is compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. For example, publicly available tools such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol. 215:403-410 (1990); Stephen F et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin et al., Methods in Enzymology, 267:383-402 (1997); MA et al. Bioinformatics (Oxford, UK), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software are used to determine the comparison of amino acid (or nucleic acid) sequence identity percentage. Those skilled in the art can use the default parameters provided by the tool or can appropriately customize the parameters according to the needs of the comparison, for example, by selecting a suitable algorithm.

[0169] As used herein, "effector function" refers to biological activity caused by the binding of the Fc region of an antibody to its effectors, such as the C1 complex and Fc receptors. Exemplary effector functions include complement-dependent cytotoxicity (CDC) mediated by the interaction of the antibody and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by the binding of the Fc region of the antibody to Fc receptors on effector cells; and phagocytosis. Effector function can be evaluated using various assays, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays.

[0170] As used herein, "antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which effector cells expressing Fc receptors (FcRs) recognize bound antibodies or antigen-binding fragments on target cells and subsequently cause lysis of the target cells. "ADCC activity" or "ADCC effector" refers to the ability of an antibody or antigen-binding fragment bound to a target cell to elicit an ADCC reaction as described above.

[0171] As used herein, "complement dependent cytotoxicity" or "CDC" refers to a mechanism by which antibodies can mediate specific target cell lysis by activating the complement system of an organism. In CDC, C1q binds to the antibody and this binding triggers the complement cascade, which results in the formation of a membrane attack complex (MAC) (C5b to C9) at the surface of the target cell as a result of complementary activation of the classical pathway. "CDC activity" or "CDC effect" refers to the ability of an antibody or antigen-binding fragment bound to a target cell to elicit a CDC response as described above.

[0172] As used herein, "antibody-dependent cellular phagocytosis" or "ADCP" refers to an immune mechanism by which tumor cells are targeted with monoclonal antibodies to promote their clearance from the body by phagocytic immune cells. The ADCP effect of an antibody can be measured by methods well known in the art (e.g., methods described in Example 4 of the present disclosure).

[0173] As used herein, "target cell" refers to a cell to which an antibody including an Fc region specifically binds, typically through a protein portion located at the C-terminus that specifically binds to the Fc region. "Effector cell" is a leukocyte that expresses one or more Fc receptors and performs effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; wherein PBMC and NK cells are preferred. Effector cells can be isolated from their natural sources, such as from blood or PBMC as known in the art.

[0174] An "isolated" material has been altered from its natural state by the hand of man. If an "isolated" composition or substance occurs in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated", but if the same polynucleotide or polypeptide is sufficiently separated from the coexisting materials of its natural state so that it exists in a substantially pure state, then the polynucleotide or polypeptide is "isolated". An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that is at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure, as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC).

[0175] As used herein, the term "vector" refers to a medium into which a polynucleotide encoding a protein can be operably inserted to cause the expression of the protein. A vector can be used to transform, transduce or transfect a host cell so that the genetic elements it carries are expressed in the host cell. Examples of vectors include plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages, such as lambda phages or M13 phages; and animal viruses. The categories of animal viruses used as vectors include retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses and papovaviruses (e.g., SV40). A vector can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements and reporter genes. In addition, a vector can contain a replication origin. A vector can also include materials that facilitate its entry into cells, including but not limited to viral particles, liposomes or protein coatings. The vector can be an expression vector or a cloning vector. The disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence encoding an antibody or its antigen-binding fragment provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably connected to the nucleic acid sequence and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), λ phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0176] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced.

[0177] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians and reptiles. Except where noted, the terms "patient," "subject," or "individual" are used interchangeably herein.

[0178] The term "anti-tumor activity" means a decrease in tumor cell proliferation, viability or metastatic activity. For example, compared to a control not using therapy, anti-tumor activity can be represented by a decrease in the growth rate of abnormal cells that appear during treatment or a stability or decrease in tumor size or a longer survival rate due to treatment. Such activity can be evaluated using recognized in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, mouse mammary tumor virus (MMTV) models, and other known models known in the art to study anti-tumor activity.

[0179] As used herein, "treating" or "treatment" of a disease, disorder or condition includes preventing or ameliorating the disease, disorder or condition, slowing the onset or rate of development of a disease, disorder or condition, reducing the risk of developing a disease, disorder or condition, preventing or delaying the development of symptoms associated with the disease, disorder or condition, reducing or ending symptoms associated with the disease, disorder or condition, causing complete or partial regression of the disease, disorder or condition, curing the disease, disorder or condition, or some combination thereof.

[0180] The terms "diagnosis," "diagnose," or "diagnosing" refer to the identification of a pathological state, disease, or condition, such as the identification of a LILRB4-associated disease, or to the identification of a subject with a LILRB4-associated disease who may benefit from a particular treatment regimen. In some embodiments, the diagnosis comprises the identification of an abnormal amount or activity of LILRB4. In some embodiments, the diagnosis refers to the identification of cancer in a subject.

[0181] As used herein, the term "biological sample" or "sample" refers to a biological composition obtained from or derived from a subject of interest, the biological composition containing, for example, cells and / or other molecular entities to be characterized and / or identified based on physical, biochemical, chemical and / or physiological properties. Biological samples include, but are not limited to, cells, tissues, organs and / or biological fluids of a subject obtained by any method known to those skilled in the art. In some embodiments, the biological sample is a body fluid sample. In some embodiments, the body fluid sample is whole blood, plasma, serum, mucus (including nasal drainage and sputum), peritoneal fluid, pleural fluid, pleural effusion, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, peritoneal fluid, ascites or pericardial fluid. In some embodiments, the biological sample is a tissue or cell obtained from the stomach, heart, liver, spleen, lung, kidney, skin or blood vessel of the subject.

[0182] As used herein, the term "LILRB4" refers to leukocyte immunoglobulin-like receptor subfamily B member 4, including any variants, conformations, isoforms and species homologs of LILRB4 expressed naturally by cells or by cells transfected with the LILRB4 gene. For example, LILRB4 described herein may refer to leukocyte immunoglobulin-like receptor subfamily B member 4 derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). For example, an exemplary sequence of a human LILRB4 protein is described in UniProtKB entry number Q8NHJ6 or GenBank accession number AAB68665.1. As used herein, the term "LILRB4" is intended to encompass any form of LILRB4, such as 1) a naturally occurring unprocessed LILRB4 molecule, a "full-length" LILRB4 chain, or a naturally occurring variant of LILRB4, including, for example, a splice variant or an allelic variant; 2) any form of LILRB4 produced by processing in a cell; or 3) full-length, fragments (e.g., truncated forms, extracellular / transmembrane domains), or modified forms (e.g., mutant forms, glycosylated / pegylated, His-tag / immunofluorescence fusion forms) of a LILRB4 subunit produced by recombinant methods.

[0183] The term "anti-LILRB4 antibody" refers to an antibody that specifically binds to LILRB4 (eg, human LILRB4). The term "anti-human LILRB4 antibody" or "anti-hLILRB4 antibody" refers to an antibody that specifically binds to human LILRB4.

[0184] As used herein, a "LILRB4 related / LILRB4-related" disease, disorder, or condition refers to any disease, disorder, or condition caused, exacerbated, or otherwise linked to an increase or decrease in the expression or activity of LILRB4. In some embodiments, a LILRB4-related disease, disorder, or condition is a disorder associated with excessive cell proliferation, such as cancer. In certain embodiments, a LILRB4-related disease or condition is characterized by expression or overexpression of LILRB4 and / or a LILRB4-related gene.

[0185] The term "pharmaceutically acceptable" means that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0186] As used herein, the term "LILRB4-expressing cell" refers to a cell that expresses LILRB4 on the cell surface.

[0187] Anti-LILRB4 Antibody

[0188] The present disclosure provides anti-LILRB4 antibodies and antigen-binding fragments thereof. The anti-LILRB4 antibodies and antigen-binding fragments provided herein are capable of binding (eg, specifically binding) to LILRB4 (eg, human LILRB4).

[0189] The binding affinity of the antibodies or antigen-binding fragments thereof provided herein can be determined by K D The value represents the ratio of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium (k off / k on Antigen binding affinity (e.g., K) can be suitably determined using appropriate methods known in the art, including, for example, flow cytometry assays. D In some embodiments, the binding of an antibody or antigen-binding fragment thereof to different concentrations of an antigen can be determined by flow cytometry, and the determined mean fluorescence intensity (MFI) can first be plotted against the antibody concentration, and then, using Prism version 5 (GraphPad Software, San Diego, CA), the K can be calculated by fitting the dependence of the specific binding fluorescence intensity (Y) and the antibody concentration (X) to a site saturation equation. D Value: Y = B max *X / (K D +X), where B max It refers to the maximum specific binding of the tested antibody to the antigen.

[0190] The binding of the antibodies or antigen-binding fragments thereof provided herein to LILRB4 can also be measured by "half maximal effective concentration" (EC 50 ) value, which refers to the concentration of the antibody at which 50% of its maximal binding is observed. 50 The values ​​can be measured by binding assays known in the art, such as direct or indirect binding assays, such as enzyme-linked immunosorbent assay (ELISA), FACS assays, and other binding assays.

[0191] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to human LILRB4 as measured by a FACS assay. For example, the antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to human LILRB4 as measured by a FACS assay with an EC of no more than 5 nM (e.g., no more than 4 nM, no more than 3 nM, no more than 2 nM, or no more than 1 nM). 50 Binds to human LILRB4.

[0192] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to human LILRB4 and human LILRB3 as measured by a FACS assay. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to human LILRB4 and human LILRB3 as measured by a FACS assay. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to human LILRB4 and human LILRB3 as measured by a FACS assay with an EC of no more than 15 nM (e.g., no more than 10 nM, no more than 9 nM, no more than 8 nM, no more than 7 nM, no more than 6 nM, no more than 5 nM, no more than 4 nM, no more than 3 nM, no more than 2 nM, or no more than 1 nM). 50 In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have an EC of no more than 15 nM (e.g., no more than 10 nM, no more than 9 nM, no more than 8 nM, no more than 7 nM, no more than 6 nM, no more than 5 nM, no more than 4 nM, no more than 3 nM, no more than 2 nM, or no more than 1 nM) as measured by FACS assay. 50 Binds to human LILRB4.

[0193] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to human LILRB4 and cynomolgus monkey LILRB4 as measured by FACS assay, which means that the antibodies or antigen-binding fragments thereof provided herein are cross-reactive to human and cynomolgus monkey LILRB4 as measured by FACS assay.

[0194] As used herein, the term "cross-reactivity" refers to the ability of a binding protein to bind to a target other than the target for which it is produced. Typically, a binding protein will bind to its target tissue / antigen with a suitably high affinity, but will exhibit a suitably low affinity to non-target normal tissues / antigens. Individual binding proteins are typically selected to meet the following two criteria: (1) binding of the antibody to a known expressed tissue suitable for the antibody target, as visualized using staining methods known in the art; and (2) similar staining patterns between tissues from humans and toxicological species (e.g., mice and cynomolgus monkeys) of the same organ. These and other methods for assessing cross-reactivity are known to those skilled in the art (e.g., US20090311253A1).

[0195] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein show potent ADCP effects on human AML cell lines (e.g., measured by FACS assay). In certain embodiments, the ADCP effect is measured by the method as described in Example 4 of the present disclosure.

[0196] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein show potent ADCC effects on human AML cell lines (e.g., measured by FACS assay). In certain embodiments, the ADCC effect is measured by the method as described in Example 3 of the present disclosure.

[0197] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of blocking the LILRB4-fibronectin interaction to reprogram tolerant dendritic cells into mature dendritic cells that stimulate T cell activation as measured by an FcγR stimulation assay. In certain embodiments, the LILRB4-fibronectin interaction is measured by a method as described in Example 5 of the present disclosure. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of inducing TNF-α production. TNF-α secretion from tolerant dendritic cells is used as a readout for evaluating FcγR stimulation.

[0198] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can reprogram tolerant dendritic cells (DC) to activate T cells. DC is the most professional antigen presenting cell and plays a key role in coordinating the immune response between innate immunity and adaptive immunity. Mature DC shows strong co-stimulation and T cell activation ability. Mature DC has high expression levels of antigen presenting molecules (HLA-DR and HLA-ABC) and co-stimulatory molecules (CD80 and CD86) to activate anti-tumor T cells. The interaction between HLA and T cell receptor induces primary signal transduction, and the interaction between CD80 / CD86 and CD28 on naive T cells triggers co-stimulatory signal transduction. Two interactions cause T cell activation and proliferation to resist tumor cells. However, when LILRB4 expression on tolerant DC is upregulated, the antigen presenting molecules and co-stimulatory molecules on tolerant DC are weakened. However, treatment of tolerogenic DCs with anti-LILRB4 antibodies (e.g., anti-LILRB4 antibodies of the present disclosure) can increase the expression of antigen presenting molecules and co-stimulatory molecules (such as HLA-DR, HLA-ABC, and CD86) by blocking LILRB4 signaling. In certain embodiments, the ability of the anti-LILRB4 antibodies that reprogram tolerogenic DCs to activate T cells is measured by the method described in Example 6 of the present disclosure.

[0199] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of reversing macrophage-mediated T cell suppression. In certain embodiments, the ability of anti-LILRB4 antibodies to reverse macrophage-mediated T cell suppression is measured by the method described in Example 9.4 of the present disclosure.

[0200] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of reversing THP-1 mediated T cell suppression. In certain embodiments, the ability of anti-LILRB4 antibodies to reverse THP-1 mediated T cell suppression is measured by the method as described in Example 7 of the present disclosure.

[0201] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can enhance CD8 + T cell-mediated cytotoxicity against THP-1 cells. In certain embodiments, anti-LILRB4 antibodies enhance CD8 + The ability of T cells to mediate cytotoxicity against THP-1 cells was measured by the method as described in Example 9.5 of the present disclosure.

[0202] Exemplary Anti-LILRB4 Antibodies

[0203] In certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to LILRB4, the antibody or antigen-binding fragment thereof comprising:

[0204] one, two or three heavy chain complementarity determining regions (HCDR1, HCDR2 and / or HCDR3) contained within any one of a heavy chain variable (VH) region sequence selected from the group consisting of SEQ ID NO: 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 154 and 156; and / or

[0205] One, two or three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3), wherein the one, two or three light chain complementary determining regions are contained within any one of the light chain variable (VL) region sequences selected from the group consisting of: SEQ ID NO: 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 155 and 157.

[0206] A person skilled in the art can define or identify the CDR boundaries of a VH or VL region by methods known in the art, as long as the amino acid sequence of the VH or VL region is known. For example, the CDR boundaries of an antibody or antigen-binding fragment thereof can be defined or identified by the Kabat, IMGT, Chothia or Al-Lazikani rules (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th Public Health Edition. Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (Second Edition), Chapter 26, 481-514, (2015). In some embodiments, the CDR boundaries of the antibodies or antigen-binding fragments thereof provided herein are determined by the Kabat rule. In some embodiments, the CDR boundaries of the antibodies or antigen-binding fragments thereof provided herein are determined by the IMGT rule. In some embodiments, the CDR boundaries of the antibodies or antigen-binding fragments thereof provided herein are determined by the Chothia rule. In some embodiments, the CDR boundaries of the antibodies or antigen-binding fragments thereof provided herein are determined by the Al-Lazikani rule.

[0207] In certain embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that bind to LILRB4, including anti-LILRB4 antibodies 27-F11-E10-G10, 43-D12-F3-G11, 36-F4-H1-D9, 8-B3-F6-H8, 44-F10-B6-D4, 27-G5-E9-B8, 48-E11-H7-B5, 42-C8-A1 one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of 2-F3-D11, 10-H9-E3-G3, 7-B4-C1-C9, 25-E7-A10-H6, 25-G9-G4-C12, 2-H1-D7-E5-D5, 27-D11-C2-A10, 26-H9-B9-B7, 4-E6-E4-B12, or 2-H8-C9-F7-E2.

[0208] As used herein, antibody "27-F11-E10-G10" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:23 and a light chain variable region having the sequence of SEQ ID NO:24.

[0209] As used herein, antibody "43-D12-F3-G11" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:31 and a light chain variable region having the sequence of SEQ ID NO:32.

[0210] As used herein, antibody "36-F4-H1-D9" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:39 and a light chain variable region having the sequence of SEQ ID NO:40.

[0211] As used herein, antibody "8-B3-F6-H8" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:47 and a light chain variable region having the sequence of SEQ ID NO:48.

[0212] As used herein, antibody "44-F10-B6-D4" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:55 and a light chain variable region having the sequence of SEQ ID NO:56.

[0213] As used herein, antibody "27-G5-E9-B8" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:63 and a light chain variable region having the sequence of SEQ ID NO:64.

[0214] As used herein, antibody "48-E11-H7-B5" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:71 and a light chain variable region having the sequence of SEQ ID NO:72.

[0215] As used herein, antibody "42-C8-A12-F3-D11" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:79 and a light chain variable region having the sequence of SEQ ID NO:80.

[0216] As used herein, antibody "10-H9-E3-G3" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:87 and a light chain variable region having the sequence of SEQ ID NO:88.

[0217] As used herein, antibody "7-B4-C1-C9" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:95 and a light chain variable region having the sequence of SEQ ID NO:96.

[0218] As used herein, antibody “25-E7-A10-H6” refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:103 and a light chain variable region having the sequence of SEQ ID NO:104.

[0219] As used herein, antibody “25-G9-G4-C12” refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:111 and a light chain variable region having the sequence of SEQ ID NO:112.

[0220] As used herein, antibody “2-H1-D7-E5-D5” refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:119 and a light chain variable region having the sequence of SEQ ID NO:120.

[0221] As used herein, antibody "27-D11-C2-A10" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:127 and a light chain variable region having the sequence of SEQ ID NO:128.

[0222] As used herein, antibody "26-H9-B9-B7" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:135 and a light chain variable region having the sequence of SEQ ID NO:136.

[0223] As used herein, antibody "4-E6-E4-B12" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:143 and a light chain variable region having the sequence of SEQ ID NO:144.

[0224] As used herein, antibody “2-H8-C9-F7-E2” refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:151 and a light chain variable region having the sequence of SEQ ID NO:152.

[0225] The specific amino acid sequences of the heavy chain variable region and the light chain variable region of each exemplary antibody as described above are shown in Table 3 and Table 21 below.

[0226] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:23 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:24.

[0227] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:31 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:32.

[0228] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:39 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:40.

[0229] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:47 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:48.

[0230] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:55 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:56.

[0231] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:63 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:64.

[0232] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:71 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:72.

[0233] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:79 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:80.

[0234] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:87 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:88.

[0235] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:95 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:96.

[0236] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:103 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:104.

[0237] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO:111 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO:112.

[0238] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO: 119 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO: 120.

[0239] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO: 127 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO: 128.

[0240] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO: 135 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO: 136.

[0241] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO: 143 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO: 144.

[0242] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO: 151 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO: 152.

[0243] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO: 154 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO: 155.

[0244] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence as shown in SEQ ID NO: 156 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence as shown in SEQ ID NO: 157.

[0245] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include at least one (e.g., 1, 2, or 3) heavy or light chain CDRs comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 33, 34, 35, 36, 37, 38, 41, 42, 43, 44, 45, 46, 49, 50, 51, 52, 53, 54, 57, 58, 59, 60, 61, 62, 65, 66, 67, 68, 69, 70, 73, 74, 75, 76, 77, 78, 81, 82, 83, 84, 85, 86, 89, 90, 91, 92, , 93, 94, 97, 98, 99, 100, 101, 102, 105, 106, 107, 108, 109, 110, 113, 114, 115, 116, 117, 118, 121, 122, 123, 124, 125, 126, 129, 130, 131, 132, 133, 134, 137, 138, 139, 140, 141, 142, 145, 146, 147, 148, 149, 150 and 158.

[0246] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include one or two or three of HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, 25, 26, 27, 33, 34, 35, 41, 42, 43, 49, 50, 51, 57, 58, 59, 65, 66, 67, 73, 74, 75, 81, 82, 83, 89, 90, 91, 97, 98, 99, 105, 106, 107, 113, 114, 115, 121, 122, 123, 129, 130, 131, 137, 138, 139, 145, 146, 147 and 158.

[0247] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include one or two or three of LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 20, 21, 22, 28, 29, 30, 36, 37, 38, 44, 45, 46, 52, 53, 54, 60, 61, 62, 68, 69, 70, 76, 77, 78, 84, 85, 86, 92, 93, 94, 100, 101, 102, 108, 109, 110, 116, 117, 118, 124, 125, 126, 132, 133, 134, 140, 141, 142, 148, 149 and 150.

[0248] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include: a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, and 145; a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, 146, and 158; and a HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 NO: 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139 and 147.

[0249] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include: LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132, 140, and 148; LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, and 149; and LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, and 149. NO: 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134, 142 and 150.

[0250] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include:

[0251] i. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 19;

[0252] ii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 27;

[0253] iii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 33, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 34, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 35;

[0254] iv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:42, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43;

[0255] v. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:158, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43;

[0256] vi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:50, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:51;

[0257] vii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:57, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:58, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:59;

[0258] viii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 65, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 66, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 67;

[0259] ix. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 73, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 74, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 75;

[0260] x. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:81, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:82, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:83;

[0261] xi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:89, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:90, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:91;

[0262] xii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:97, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:98, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:99;

[0263] xiii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 106, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 107;

[0264] xiv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 114, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 115;

[0265] xv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 122, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 123;

[0266] xvi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 130, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 131;

[0267] xvii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 138, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 139; or

[0268] xviii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:145, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:146, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:147.

[0269] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include:

[0270] i. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 20, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 22;

[0271] ii. LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:28, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:29, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:30;

[0272] iii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:36, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:37, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:38;

[0273] iv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:44, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:45, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:46;

[0274] v. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:53, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:54;

[0275] vi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:60, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:61, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:62;

[0276] vii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:68, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:69, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:70;

[0277] viii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:76, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:77, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:78;

[0278] ix. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:84, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:85, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:86;

[0279] x. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:92, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:93, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:94;

[0280] xi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 100, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 101, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 102;

[0281] xii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 108, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 109, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 110;

[0282] xiii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 116, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 118;

[0283] xiv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 124, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 125, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126;

[0284] xv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 132, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 133, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 134;

[0285] xvi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 140, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 141, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 142; or

[0286] xvii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:148, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:149, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:150.

[0287] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include:

[0288] i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 18, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 19, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 20, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 21, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 22;

[0289] ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:25, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:26, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:27, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:28, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:29, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:30;

[0290] iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:33, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:34, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:35, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:36, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:37, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:38;

[0291] iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:42, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:44, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:45, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:46;

[0292] v. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:158, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:44, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:45, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:46;

[0293] vi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:50, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:51, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:52, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:53, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:54;

[0294] vii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:57, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:58, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:59, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:60, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:61, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:62;

[0295] viii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:65, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:66, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:67, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:68, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:69, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:70;

[0296] ix. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:73, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:74, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:75, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:76, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:77, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:78;

[0297] x. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:81, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:82, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:83, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:84, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:85, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:86;

[0298] xi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:89, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:90, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:91, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:92, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:93, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:94;

[0299] xii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:97, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:98, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:99, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:100, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:101, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:102;

[0300] xiii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 106, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 107, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 108, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 109, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 110;

[0301] xiv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 114, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 115, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 116, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 117, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 118;

[0302] xv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 122, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 123, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 124, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 125, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 126;

[0303] xvi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 130, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 131, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 132, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 133, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 134;

[0304] xvii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 138, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 139, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 140, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 141, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 142; or

[0305] xviii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:145, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:146, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:147, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:148, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:149, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:150.

[0306] The SEQ ID NOs of the heavy chain (denoted as "H") variable region, the light chain (denoted as "L") variable region, the HCDR and the LCDR of each of the above 17 monoclonal antibodies are shown in Table 1 below. The amino acid sequence of each CDR of the 17 exemplary monoclonal antibodies is shown in Table 2 below. Unless otherwise indicated, the CDR boundaries as described in Table 2 are defined or identified by the Kabat rule. The amino acid sequences of each VH and VL of the 17 exemplary monoclonal antibodies are shown in Table 3 below.

[0307] Table 1: SEQ ID NOs of VH, VL, HCDR and LCDR of 17 exemplary monoclonal antibodies

[0308]

[0309]

[0310] Table 2: Amino acid sequences of each CDR of 17 exemplary monoclonal antibodies

[0311]

[0312]

[0313]

[0314]

[0315] Table 3: Amino acid sequences of each VH and VL of 17 exemplary monoclonal antibodies

[0316]

[0317]

[0318]

[0319] Considering that each of the 17 exemplary monoclonal antibodies can bind to LILRB4, and that the antigen binding specificity is provided mainly by the CDR1 region, the CDR2 region, and the CDR3 region, the HCDR1 sequence, HCDR2 sequence, and HCDR3 sequence, and the LCDR1 sequence, LCDR2 sequence, and LCDR3 sequence of each of the 17 exemplary monoclonal antibodies can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched, but each antibody must contain HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3) to generate the anti-LILRB4 antibodies or antigen-binding fragments thereof of the present disclosure. The LILRB4 binding of such "mixed and matched" antibodies can be tested using the binding assays described above and in the Examples. Preferably, when VH CDR sequences are mixed and matched, the HCDR1 sequence, HCDR2 sequence, and / or HCDR3 sequence from a specific VH sequence is replaced by a structurally similar CDR sequence. Likewise, when VL CDR sequences are mixed and matched, the LCDR1 sequence, LCDR2 sequence, and / or LCDR3 sequence from a particular VL sequence is replaced with a structurally similar CDR sequence. For example, the HCDR1 of antibodies 25-G9-G4-C12 and 2-H1-D7-E5-D5 share some structural similarities and are therefore easily mixed and matched. It will be apparent to one skilled in the art that novel VH and VL sequences can be generated by replacing one or more VH and / or VL CDR sequences with structurally similar sequences from the CDR sequences disclosed herein of the 17 exemplary monoclonal antibodies.

[0320] It is known that CDRs are responsible for antigen binding. However, it has been found that not all 6 CDRs are essential or unchangeable. In other words, one or more CDRs of each of the 17 exemplary monoclonal antibodies can be replaced or changed or modified, but the specific binding affinity for LILRB4 is substantially retained.

[0321] In certain embodiments, the anti-LILRB4 antibodies and antigen-binding fragments provided herein include anti-LILRB4 antibodies 27-F11-E10-G10, 43-D12-F3-G11, 36-F4-H1-D9, 8-B3-F6-H8, 44-F10-B6-D4, 27-G5-E9-B8, 48-E11-H7-B5, 42-C8- In some embodiments, the anti-LILRB4 antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain CDR3 sequence selected from the group consisting of SEQ ID NO: 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139 and 147. The heavy chain CDR3 region is located at the center of the antigen binding site, and is therefore considered to be in contact with the antigen the most and to provide the most free energy for the affinity of the antibody to the antigen. It is also believed that, through a variety of diversification mechanisms, the heavy chain CDR3 is the most diverse CDR in terms of length, amino acid composition and conformation of the antigen binding site so far (Tonegawa S. "Nature" .302:575-81). The diversity of the heavy chain CDR3 is enough to produce most antibody specificities (Xu JL, Davis MM. "Immunity (Immunity)" .13:37-45) and ideal antigen binding affinity (Schier R et al., "Journal of Molecular Biology" 263:551-67).

[0322] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include a VH region having an amino acid sequence as shown in SEQ ID NO: 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 154 or 156, or a homologous sequence thereof having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 154 or 156.

[0323] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include a VL region having an amino acid sequence as set forth in SEQ ID NO: 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 155 or 157, or a homologous sequence thereof having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 155 or 157.

[0324] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include a VH / VL amino acid sequence pair selected from the group consisting of: SEQ ID NO: 23 / 24, 31 / 32, 39 / 40, 47 / 48, 55 / 56, 63 / 64, 71 / 72, 79 / 80, 87 / 88, 95 / 96, 103 / 104, 111 / 112, 119 / 120, 127 / 128, 135 / 136, 143 / 144, 151 / 152, 154 / 155, and 156 / 157.

[0325] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein include suitable framework region (FR) sequences, as long as the antibodies and antigen-binding fragments thereof can bind to LILRB4. The CDR sequences provided in Table 2 above are obtained from mouse antibodies, but the CDR sequences can be transplanted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, etc. using suitable methods known in the art such as recombinant technology.

[0326] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are humanized. Humanized antibodies or their antigen-binding fragments are desirable in terms of their reduced immunogenicity in humans. Humanized antibodies are chimeric in their variable regions because non-human CDR sequences are transplanted to human or substantially human FR sequences. The humanization of antibodies or antigen-binding fragments can be carried out substantially by replacing the corresponding human CDR genes in human immunoglobulin genes with non-human (such as murine) CDR genes (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0327] Suitable human heavy chain and light chain variable domains can be selected using methods known in the art to achieve this purpose. In an illustrative example, a "best fit" method can be used, wherein the database screening or BLAST non-human (e.g., rodent) antibody variable domain sequences for known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as a human support for transplanting non-human CDR sequences (see, e.g., Sims et al., (1993) Journal of Immunology 151: 2296; Chothia et al., (1987) Journal of Molecular Biology 196: 901). Alternatively, the framework derived from the consensus sequence of all human antibodies can be used for transplanting non-human CDR (see, e.g., Carter et al. (1992) Proceedings of the National Academy of Sciences, 89: 4285; Presta et al. (1993) Journal of Immunology, 151: 2623).

[0328] In some embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein are humanized. In certain embodiments, in addition to the non-human CDR sequences, the humanized antibodies or antigen-binding fragments thereof provided herein are composed of substantially all human sequences. In some embodiments, the variable region FR and the constant region (if present) are completely or substantially derived from human immunoglobulin sequences. Human FR sequences and human constant region sequences can be derived from different human immunoglobulin genes, for example, FR sequences are derived from one human antibody, and the constant region is derived from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment thereof includes human heavy chain HFR1, HFR2, HFR3 and HFR4 and / or light chain LFR1, LFR2, LFR3 and LFR4.

[0329] In some embodiments, the present disclosure provides a humanized antibody or antigen-binding fragment thereof of clone 2-H1-D7-E5-D5 (also referred to as "humanized 2-H1-D7-E5-D5" or "h2-H1-D7-E5-D5" in the present disclosure). In some embodiments, the humanized 2-H1-D7-E5-D5 or antigen-binding fragment thereof provided herein includes one, two or three HCDRs (HCDR1, HCDR2 and / or HCDR3) contained in the heavy chain variable region sequence of SEQ ID NO: 154; and / or includes one, two or three LCDRs (LCDR1, LCDR2 and / or LCDR3) contained in the light chain variable region sequence of SEQ ID NO: 155. In some embodiments, the humanized 2-H1-D7-E5-D5 or antigen-binding fragment thereof provided herein comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region sequence of SEQ ID NO: 154; and comprises LCDR1, LCDR2 and LCDR3 contained in the light chain variable region sequence of SEQ ID NO: 155. In some embodiments, the humanized 2-H1-D7-E5-D5 or antigen-binding fragment thereof provided herein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 114, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 115, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 116, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 117, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 118. The amino acid sequences of SEQ ID NOs: 154 and 155 are shown in Table 21 below, and the amino acid sequences of SEQ ID NOs: 113-118 are shown in Table 2 above and Table 21 below.

[0330] In some embodiments, the present disclosure provides a humanized antibody or antigen-binding fragment thereof of clone 8-B3-F6-H8 (also referred to as "humanized 8-B3-F6-H8" or "h8-B3-F6-H8" in the present disclosure). In some embodiments, the humanized 8-B3-F6-H8 or antigen-binding fragment thereof provided herein includes one, two or three HCDRs (HCDR1, HCDR2 and / or HCDR3) contained in the heavy chain variable region sequence of SEQ ID NO: 156; and / or includes one, two or three LCDRs (LCDR1, LCDR2 and / or LCDR3) contained in the light chain variable region sequence of SEQ ID NO: 157. In some embodiments, the humanized 8-B3-F6-H8 or antigen-binding fragment thereof provided herein includes HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region sequence of SEQ ID NO: 156; and includes LCDR1, LCDR2 and LCDR3 contained in the light chain variable region sequence of SEQ ID NO: 157. In some embodiments, the humanized 8-B3-F6-H8 or antigen-binding fragment thereof provided herein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:42, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:44, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:45, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:46. In some embodiments, the humanized 8-B3-F6-H8 or antigen-binding fragment thereof provided herein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 158, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 43, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 44, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 45, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 46. The amino acid sequences of SEQ ID NOs: 156 and 157 are shown in Table 21 below, and the amino acid sequences of SEQ ID NOs: 41-46 and 158 are shown in Table 2 above and Table 21 below.

[0331] Table 21: Amino acid sequences of HCDR, LCDR, VH region and VL region of humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8

[0332]

[0333] In some embodiments, the FR region derived from people can include the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of people's FR are replaced by corresponding residues from parental non-human antibodies. In some embodiments, this can be desirable, so that humanized antibodies or their fragments are very close to non-human parent antibody structures, so as to optimize binding properties (for example, increase binding affinity). In some embodiments, the humanized antibodies or their antigen-binding fragments provided herein include no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue replacements in each FR sequence in the human FR sequence, or include no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue replacements in all FR sequences of heavy chain or light chain variable domains. In some embodiments, the change of such amino acid residues can be present only in the heavy chain FR region, only in the light chain FR region, or in two chains. In some embodiments, one or more amino acids of the people's FR sequence are randomly mutated to increase binding affinity. In certain embodiments, one or more amino acids of the human FR sequence are backmutated to the corresponding amino acids of the parent non-human antibody to increase binding affinity.

[0334] In some embodiments, the anti-LILRB4 antibodies and antigen-binding fragments thereof provided herein include all or a portion of a heavy chain variable domain and / or all or a portion of a light chain variable domain. In one embodiment, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein are single domain antibodies consisting of all or a portion of a heavy chain variable domain provided herein. More information about such single domain antibodies can be obtained in the art (see, e.g., U.S. Patent No. 6,248,516).

[0335] In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof provided herein further comprises an Fc region. In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof provided herein further comprises an Fc region of a human immunoglobulin (Ig). In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof provided herein further comprises a constant region, and the constant region optionally further comprises a heavy chain constant region and / or a light chain constant region. In certain embodiments, the heavy chain constant region comprises a CH1, a hinge and / or a CH2-CH3 region (or optionally a CH2-CH3-CH4 region). In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgM. In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof provided herein comprises a λ (lambda) light chain or a κ (kappa) light chain. The constant region of the anti-LILRB4 antibody or antigen-binding fragment thereof provided herein may be identical to the wild-type constant region sequence or may differ in one or more mutations.

[0336] In certain embodiments, the heavy chain constant region includes an Fc region. It is known that the Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of antibodies. Fc regions of different Ig isotypes have different abilities to induce effector functions. For example, the Fc regions of IgG1 and IgG3 have been recognized to be more effective than the Fc regions of IgG2 and IgG4 in inducing both ADCC and CDC. In certain embodiments, the anti-LILRB4 antibodies and antigen-binding fragments thereof provided herein include: an Fc region of an IgG1 or IgG3 isotype, which can induce ADCC or CDC; or alternatively, a constant region of an IgG4 or IgG2 isotype, which reduces or depletes effector functions. In some embodiments, the Fc region is derived from human IgG1 with enhanced effector functions. In some embodiments, the Fc region includes an amino acid sequence as shown in SEQ ID NO: 153.

[0337] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:153)

[0338] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have specific binding affinity for human LILRB4 sufficient to provide diagnostic and / or therapeutic uses.

[0339] The antibody or antigen-binding fragment thereof provided herein can be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a multispecific antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein. Recombinant antibodies are antibodies prepared in vitro rather than in an animal using a recombinant method.

[0340] In certain embodiments, the present disclosure provides an anti-LILRB4 antibody or antigen-binding fragment thereof, which competes with the antibodies or antigen-binding fragments provided herein for binding to LILRB4. In certain embodiments, the present disclosure provides an anti-LILRB4 antibody or antigen-binding fragment thereof, which competes with antibodies 27-F11-E10-G10, 43-D12-F3-G11, 36-F4-H1-D9, 8-B3-F6-H8, 44-F10-B6-D4, 27-G5-E9-B8, 48-E11-H7-B5, 4 Any of 2-C8-A12-F3-D11, 10-H9-E3-G3, 7-B4-C1-C9, 25-E7-A10-H6, 25-G9-G4-C12, 2-H1-D7-E5-D5, 27-D11-C2-A10, 26-H9-B9-B7, 4-E6-E4-B12 or 2-H8-C9-F7-E2 competes for binding to human LILRB4. In some embodiments, the present disclosure provides an anti-LILRB4 antibody or antigen-binding fragment thereof that competes for the same epitope as the antibody or antigen-binding fragment provided herein.

[0341] As used herein, the ability to "block binding" or "compete for the same epitope" refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human LILRB4 and anti-LILRB4 antibody) to any detectable extent. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85% or at least 90%. In certain embodiments, this inhibition can be greater than 85% or greater than 90%.

[0342] One skilled in the art will recognize that whether a human monoclonal antibody blocks binding of an antibody of the present disclosure to a LILRB4 antigen polypeptide can be determined without undue experimentation by determining whether the human monoclonal antibody blocks binding of an antibody of the present disclosure to a LILRB4 antigen polypeptide. 9-B8, 48-E11-H7-B5, 42-C8-A12-F3-D11, 10-H9-E3-G3, 7-B4-C1-C9, 25-E7-A10-H6, 25-G9-G4-C12, 2-H1-D7-E5-D5, 27-D11-C2-A10, 26-H9-B9-B7, 4-E6-E4-B12 or 2-H8-C9-F7-E2) bind to the same epitope antigen polypeptide. If the test antibody competes with the antibody of the disclosure, as shown by reduced binding of the antibody of the disclosure to the LILRB4 antigen polypeptide, then the two antibodies bind to the same or closely related epitope. Alternatively, if binding of the test antibody to the LILRB4 antigen polypeptide is inhibited by the antibody of the disclosure, then the two antibodies bind to the same or closely related epitope.

[0343] In certain embodiments, the present disclosure provides an anti-LILRB4 antibody or antigen-binding fragment thereof, which competes with antibody IO-202 for binding to LILRB4. In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment thereof provided herein for binding to LILRB4 is not IO-202. Information on IO-202 is described in, for example, WO 2016144728A2, WO 2018022881A2, Mi Deng et al., Nature. October 2018; 562(7728): 605-609, Xun Gui et al., Cancer Immunol Res 2019; 7: 1244-57.

[0344] In certain embodiments, the present disclosure provides an anti-LILRB4 antibody or antigen-binding fragment thereof that competes with antibody NGM831 for binding to LILRB4. In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment thereof provided herein for binding to LILRB4 is not NGM831. Information on NGM831 is described in, for example, US20210221887A1, Kevin J Paavola et al., Cancer Immunology Research, November 2021; 9(11): 1283-1297.

[0345] As used herein, "IO-202" refers to an antibody or antigen-binding fragment thereof comprising one or two or three heavy chain CDRs contained in a heavy chain variable region having an amino acid sequence of SEQ ID NO: 7 and one or two or three light chain CDRs contained in a light chain variable region having an amino acid sequence of SEQ ID NO: 8. In certain embodiments, IO-202 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises an amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 3, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 4, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 6. The amino acid sequences of each CDR, VH region, and VL region of IO-202 are shown in Table 4 below. The CDR boundaries of IO-202 in Table 4 below were identified by IMGT rules.

[0346] As used herein, "NGM831" refers to an antibody or antigen-binding fragment thereof comprising one or two or three heavy chain CDRs contained in a heavy chain variable region having an amino acid sequence of SEQ ID NO: 15 and one or two or three light chain CDRs contained in a light chain variable region having an amino acid sequence of SEQ ID NO: 16. In certain embodiments, NGM831 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises an amino acid sequence of SEQ ID NO: 9, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 10, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 11, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 12, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 13, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 14. The amino acid sequences of each CDR, VH region, and VL region of NGM831 are shown in Table 4 below. The CDR boundaries of NGM831 in Table 4 below were identified by IMGT rules.

[0347] Table 4: Amino acid sequences of HCDR, LCDR, VH region and VL region of IO-202 and NGM831

[0348]

[0349] Antibody variants

[0350] The antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

[0351] In certain embodiments, the antibody variant comprises one or more amino acid residue substitutions or modifications, and still retains binding affinity for LILRB4. In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences of the VH region or the VL region. In certain embodiments, at least one of the substitutions or modifications is in one or more of the non-CDR sequences of the VH region or the VL region. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure further comprise one or more non-natural amino acid (NNAA) substitutions. In certain embodiments, the NNAA is capable of being conjugated.

[0352] For example, the antibody variant includes one or more of the CDR sequences provided in Table 2 or Table 21 above, one or more of the non-CDR sequences of the heavy chain variable region or light chain variable region provided in Table 3 or Table 21 above, and / or one or more amino acid residues in the constant region (e.g., Fc region) are substituted or modified. Such variants retain the binding specificity of their parent antibody to LILRB4, but have one or more desired properties conferred by the modification or substitution. For example, the antibody variant may have improved antigen binding affinity, improved glycosylation profile, reduced glycosylation risk, reduced deamidation, enhanced effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity and / or compatibility with conjugation (e.g., one or more introduced cysteine ​​residues), etc.

[0353] Parent antibody sequences can be screened using methods known in the art (e.g., "alanine scanning mutagenesis") to identify suitable or preferred residues to be modified or substituted (see, e.g., Cunningham and Wells (1989) Science, 244: 1081-1085). In short, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are generated and screened for properties of interest. If the substitution at a particular amino acid position shows a functional change of interest, the position can be identified as a potential residue for modification or substitution. Potential residues can be further evaluated by replacing with another residue (e.g., a cysteine ​​residue, a positively charged residue, etc.).

[0354] Affinity variants

[0355] The affinity variants of the antibodies may contain modifications or substitutions in one or more CDR sequences provided in Table 2 or Table 21 above, one or more FR sequences, or heavy or light chain variable region sequences provided in Table 3 or Table 21 above. A person skilled in the art can easily identify the FR sequences based on the CDR sequences in Table 2 or Table 21 above and the variable region sequences in Table 3 or Table 21 above, because it is well known in the art that in the variable region, the CDR region is flanked by two FR regions. The affinity variants retain the specific binding affinity of the parent antibody for LILRB4, or even have an improved LILRB4 binding affinity than the parent antibody. In certain embodiments, at least one (or all) of the substitutions in the CDR sequence, FR sequence, or variable region sequence comprises a conservative substitution.

[0356] Those skilled in the art will appreciate that one or more amino acid residues may be substituted in the CDR sequences provided in Table 2 or Table 21 above and the variable region sequences provided in Table 3 or Table 21 above, but the resulting antibody or antigen-binding fragment still retains binding affinity or binding capacity for LILRB4 or even has improved binding affinity or capacity. Various methods known in the art may be used to achieve this goal. For example, a library of antibody variants (such as Fab or scFv variants) may be generated and expressed using phage display technology, and then screened for binding affinity to human LILRB4. For another example, computer software may be used to virtually simulate the binding of antibodies to human LILRB4 and identify the amino acid residues on the antibody that form the binding interface. Such residues may be avoided from substitution to prevent a reduction in binding affinity, or may be targeted for substitution to achieve stronger binding.

[0357] In certain embodiments, the humanized antibody or its antigen-binding fragment provided herein comprises one or more amino acid residues in one or more CDR sequences and / or one or more FR sequences in CDR sequences and / or FR sequences. In certain embodiments, affinity variants comprise no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 substitutions in total in CDR sequences and / or FR sequences.

[0358] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein include 1, 2 or 3 CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with one (or more) sequences listed in Table 2 or Table 21 above, but still retain specific binding affinity for LILRB4 at a similar or even higher level relative to its parent antibody.

[0359] In certain embodiments, an anti-LILRB4 antibody or antigen-binding fragment thereof comprises one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with one (or more) sequence listed in Table 3 or Table 21 above, but still retains specific binding affinity for LILRB4 at a level similar to that of its parent antibody or even higher. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted or deleted in the variable region sequences listed in Table 3 or Table 21 above. In some embodiments, the substitution, insertion or deletion occurs in a region outside of a CDR (e.g., in a FR).

[0360] Glycosylation variants

[0361] The anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein also encompass glycosylation variants, which can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment thereof.

[0362] The antibodies or antigen-binding fragments thereof provided herein may include one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) may be attached. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the connection of a carbohydrate moiety to the side chain of an asparagine residue (e.g., an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid except proline). O-linked glycosylation refers to the connection of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Natural glycosylation sites can be conveniently removed, for example, by altering the amino acid sequence so that one of the above tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence is replaced. New glycosylation sites can be generated in a similar manner by introducing such tripeptide sequences or serine or threonine residues.

[0363] Cysteine ​​engineered variants

[0364] The anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein also encompass cysteine ​​engineered variants comprising one or more introduced free cysteine ​​amino acid residues.

[0365] A free cysteine ​​residue is a cysteine ​​residue that is not part of a disulfide bridge. Cysteine ​​engineered variants can be used to conjugate, for example, with a cytotoxic compound and / or imaging compound, a label or a radioisotope, etc., at the site of the engineered cysteine, for example, via a maleimide or haloacetyl group. Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine ​​residues are known in the art, see, for example, WO2006 / 034488.

[0366] Fc variants

[0367] The anti-LILRB4 antibodies and antigen-binding fragments provided herein also encompass Fc variants comprising one or more amino acid residue modifications or substitutions at the Fc region and / or hinge region thereof.

[0368] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein include one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants can have a prolonged pharmacokinetic half-life because they bind to FcRn at acidic pH, which allows them to be protected from degradation in lysosomes and then translocated and released outside the cell. Methods for engineering antibodies or antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, e.g., Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Vol. 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., Journal of Immunology, 176:346-356 (2006).

[0369] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein include one or more amino acid substitutions that alter ADCC. Certain amino acid residues at the CH2 domain of the Fc region can be substituted to provide enhanced ADCC activity. Alternatively or additionally, the carbohydrate structure on the antibody can be altered to enhance ADCC activity. Methods for altering ADCC activity by antibody engineering have been described in the art, see, for example, Shields RL. et al., J Biol Chem. 2001. 276(9):6591-604; Idusogie EE. et al., J Immunol. 2000. 164(8):4178-84; Steurer W. et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE. et al., J Immunol. 2001, 166(4):2571-5; Lazar GA. et al., Proc. Natl. Acad. Sci. USA, 2006, 103(11):4005-4010; Ryan MC. et al., Mol. Cancer Ther., 2007, 6:3009-3018; Richards J .... JO et al., Mol Cancer Therapy, 2008, 7(8): 2517-27; Shields RL et al., J Biol Chem, 2002, 277: 26733-26740; Shinkawa T. et al., J Biol Chem, 2003, 278: 3466-3473.

[0370] In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof includes one or more amino acid substitutions that alter CDC, for example by improving or reducing C1q binding and / or CDC (see, e.g., WO 99 / 51642; Duncan and Winter, Nature, 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants). One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region can be replaced with a different amino acid residue to alter C1q binding and / or enhance CDC (see U.S. Pat. No. 6,194,551 to Idusogie et al.). One or more amino acid substitutions can also be introduced to alter the ability of the antibody to fix complement (see PCT Publication WO 94 / 29351 to Bodmer et al.).

[0371] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein include one or more amino acid substitutions (according to EU numbering) at positions 234 and / or 235 in a human immunoglobulin (e.g., IgG1). In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein include two amino acid substitutions (according to EU numbering) at positions 234 and 235 in a human immunoglobulin (e.g., IgG1). In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein include L234A and L235A (according to EU numbering) amino acid substitutions.

[0372] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein include one or more amino acid substitutions in the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include introducing a protrusion into a first Fc polypeptide and introducing a cavity into a second Fc polypeptide, wherein the protrusion can be positioned in the cavity to promote the interaction of the first Fc polypeptide with the second Fc polypeptide to form a heterodimer or complex. Methods for producing antibodies with these modifications are known in the art, for example, as described in U.S. Pat. No. 5,731,168.

[0373] Fab

[0374] Anti-LILRB4 antigen-binding fragments are also provided herein. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-LILRB4 antibodies provided herein, including, for example, the CDRs shown in Table 2 or Table 21 above and the exemplary antibodies of the variable sequences shown in Table 3 or Table 21 above, and different variants thereof (such as affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered variants, etc.).

[0375] In certain embodiments, the anti-LILRB4 antigen-binding fragment provided herein is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (dsdiabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), camelized single domain antibody, nanobody, domain antibody or bivalent domain antibody.

[0376] Various techniques can be used to produce such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science 229: 81 (1985)), recombinant expression by host cells such as Escherichia coli (e.g., for Fab, Fv and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., for ScFv) and chemical coupling of two Fab'-SH fragments to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). Other techniques for producing antibody fragments are apparent to those skilled in the art.

[0377] In certain embodiments, the antigen binding fragment is a scFv. The production of scFv is described in, for example, WO 93 / 16185; U.S. Pat. Nos. 5,571,894 and 5,587,458. The scFv can be fused to an effector protein at the amino or carboxyl terminus to provide a fusion protein (see, for example, Antibody Engineering, Borrebaeck, ed.).

[0378] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. Any molecule greater than bivalent is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0379] If both binding sites specifically bind to the same antigen or the same epitope, the bivalent molecule can be monospecific. In some embodiments, this provides stronger binding to the antigen or epitope than the monovalent counterpart. Similarly, multivalent molecules can also be monospecific. In some embodiments, in a bivalent or multivalent antigen binding portion, the first valence of the binding site and the second valence of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences but have the same specificity).

[0380] A bivalent can also be bispecific if the two binding sites are specific for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0381] Bispecific or multispecific antibodies

[0382] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein are bispecific or multispecific. In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein are further linked to a second functional portion having a different binding specificity than the anti-LILRB4 antibodies or antigen-binding fragments thereof. In some embodiments, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein have a first specificity for LILRB4 and a second specificity. In some embodiments, the second specificity is for LILRB4 but for a different epitope. In some embodiments, the second specificity is for a second antigen different from LILRB4.

[0383] In certain embodiments, the second specificity is for a tumor-associated antigen or an epitope thereof. The term "tumor-associated antigen" refers to an antigen that is now or can be presented on the surface of a tumor cell and is located on or within a tumor cell. In some embodiments, tumor-associated antigens can only be presented by tumor cells, but not by normal cells (i.e., non-tumor cells). In some other embodiments, tumor-associated antigens can be expressed exclusively on tumor cells or can represent tumor-specific mutations compared to non-tumor cells. In some other embodiments, tumor-associated antigens can be found in both tumor cells and non-tumor cells, but are overexpressed on tumor cells when compared to non-tumor cells, or can be used for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to non-tumor tissue. In some embodiments, tumor-associated antigens are located on the vasculature of the tumor.

[0384] In certain embodiments, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein are capable of binding to one or more (e.g., 1, 2, 3, 4, 5 or more) additional antigens in addition to LILRB4, or to a second epitope on LILRB4.In certain embodiments, the one or more additional antigens other than LILRB4 are selected from the group consisting of CD3, CD16a, CD33, CD38, CD45, CD123, CD146, CD228, CLL-1, Flt3, TAF1, TgPRF, HVCN1, IL-6R, IL-11R, IL17A, IL-23R, IL-33, ILDR2, LAP, TSLP, TREM-1, ANGPT2, APOE, IFNAR, CypA, DOG-1, NKp30, CSF-1R, CCR2, LRRC15, mesothelin, Dickkopf2, DLL3, HER-2, C10orf54, TrkA, MEKK1, KRAS, ERK, XPO1, mTORC1 / 2, PAK4, NAMPT, ATR, EGFR, FGFR, VEGF, c-MET, Her2, Her3, CTLA4, GITA, CD112 R, CD2, CD7, CD16, CD19, CD20, CD24, CD27, CD30, CD34, CD37, CD39, CD70, CD73, CD83, CD28, CD80(B7-1), CD86(B7-2), CD40, CD40L(CD154), CD 47. SIRPα, CD122, CD137, CD137L, OX40 (CD134), OX40L (CD252), BCMA (e.g., BCMA02), PSMA, CLDN18 (e.g., CLDN18.2), NKG2C, 4-1BB, LIGHT, PVRI G, SLAMF7, HVEM, BAFFR, ICAM-1, 2B4, LFA-1, GITR, ICOS(CD278), ICOSLG(CD275), LAG3(CD223), A2AR, B7-H3(CD276), B7-H4(VTCN1), B7-H5, BTLA(CD272), BTLA, CD160, CTLA-4(CD152), GPRC5D, IDO1, IDO2, ILT3, TDO, KIR, LAIR-1, NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC-7 (CD328), SIGLEC-9(CD329), SIGLEC-15, TIGIT, PVR(CD155), LILRB2, LILRB3, FLT3, FLT3L, TLR3, CLEC9A, DEC-205, STING, IL-12, IDO and TGFβ.

[0385] In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and CD3. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and BCMA. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and CD38. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and CD19. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and FcRH5. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and PD-L1. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and PD-1. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and TIM-3. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and FLT3. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and FLT3L. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and TLR3. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and PD-L1. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and LILRB3. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and VISTA. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and TIGIT. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and 4-1BB. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and B7-H3. In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein bind to LILRB4 and B7-H4.

[0386] Conjugate

[0387] In some embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof provided herein further comprises one or more conjugate moieties. The conjugate moiety can be connected to the antibody or antigen-binding fragment thereof. The conjugate moiety is a moiety that can be connected to the antibody or antigen-binding fragment thereof. It is conceivable that a variety of conjugate moieties can be combined with the antibody or antigen-binding fragment thereof provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties can be connected to the antibody or antigen-binding fragment thereof by covalent bonding (e.g., disulfide bonds), affinity bonding, embedding, coordination bonding, complexing, association, blending or addition. In some embodiments, the antibody or antigen-binding fragment thereof can be connected to one or more conjugates by a linker or cross-linking agent. The linker or cross-linking agent includes a reactive chemical group that can react with the anti-LILRB4 antibody or fragment thereof. The reactive chemical group may be N-succinimidyl ester and N-sulfosuccinimidyl ester. In addition, the linker includes a reactive chemical group, which may be a dithiopyridyl group that may react with the drug to form a disulfide bond. Linker molecules include, for example, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (see, e.g., Carlsson et al., J. Biol. Chem., 173:723-737 (1978)), N-succinimidyl 4-(2-pyridyldithio) butyrate (SPDB) (see, e.g., U.S. Pat. No. 4,563,304), N-succinimidyl 4-(2-pyridyldithio) 2-sulfobutyrate (sulfo-SPDB) (see U.S. Publication No. 20090274713), N-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP) (see, e.g., CAS Reg. No. 341498-08-6), 2-iminothiolane, or acetyl succinic anhydride. For example, an antibody or cell binding agent can be modified with a cross-linking agent, and the resulting antibody or cell binding agent containing free or protected thiol groups is then reacted with a maytansinoid containing a disulfide or thiol to produce a conjugate. The conjugate can be purified by chromatography, including but not limited to HPLC, size exclusion, adsorption, ion exchange and affinity capture, dialysis or tangential flow filtration.

[0388] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can be engineered to contain specific sites other than the epitope binding moiety that can be used to bind to one or more conjugate moieties. For example, such sites can include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent attachment to the conjugate moieties.

[0389] In certain embodiments, the antibody or antigen binding fragment thereof provided herein can be connected to the conjugate portion indirectly or through another conjugate portion. For example, the antibody or antigen binding fragment thereof provided herein can be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin. In some embodiments, the conjugate portion includes a scavenging modifier (e.g., a polymer that extends the half-life, such as PEG), a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a detectable label (e.g., a luminescent label, a fluorescent label, an enzyme substrate label), a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a purification portion or other anticancer drugs (e.g., toll-like receptor 7 (TLR-7), an agonist of TLR-8 and / or TLR-9, siRNA, an antibody or its antigen binding fragment, a peptide (e.g., a short peptide), etc.).

[0390] A "toxin" can be any agent that is harmful to cells or that can damage or kill cells. Examples of toxins include, but are not limited to, paclitaxel, taxanes, CC-1065 and CC-1065 analogs, duocarmycin and duocarmycin analogs, enediynes such as calicheamicin, dolastatin and dolastatin analogs (including auristatin), tomaymycin derivatives, leptomycin derivatives, and the like. derivatives), cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil and morpholino doxorubicin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, DM4, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, chlorambucil), melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and dichlorodiamine platinum (II) (DDP cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g.,dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin-binding agents.

[0391] Examples of detectable labels can include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase, or β-D-galactosidase), radioactive isotopes (e.g., 123 I. 124 I. 125 I. 131 I. 35 S. 3 H. 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y. 88 Y. 90 Y. 177 Lu, 211 At 186 Re, 188 Re, 153 Sm, 212 Bihe 32 P, other lanthanides), luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules or gold for detection.

[0392] In certain embodiments, the conjugate moiety can be a scavenging modifier that helps increase the half-life of the antibody. Illustrative examples include water-soluble polymers such as copolymers of PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol / propylene glycol, etc. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers connected to the antibody can vary, and if more than one polymer is connected, the polymers can be the same or different molecules.

[0393] In certain embodiments, the conjugate moiety may be a purification moiety, such as a magnetic bead.

[0394] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are used as the basis for a conjugate.

[0395] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are conjugated to a single peptide. Signal peptides (sometimes referred to as signal sequences, leader sequences or leader peptides) can be used to promote the secretion and separation of antibodies or antigen-binding fragments thereof provided herein. Signal peptides are generally characterized as a core of hydrophobic amino acids that are typically cleaved from mature proteins during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow the signal sequence to be cleaved from the mature protein as the mature protein passes through the secretory pathway. Therefore, the present invention relates to the described polypeptides with signal sequences and polypeptides (i.e., cleavage products) whose signal sequences have been proteolytically cleaved. In one embodiment, a nucleic acid sequence encoding a signal sequence can be operably connected in an expression vector to a protein of interest, such as a protein that is not usually secreted or otherwise difficult to separate. The signal sequence directs the secretion of the protein, such as from an expression vector transformed into a eukaryotic host therein, and the signal sequence is subsequently or simultaneously cleaved. The protein can then be easily purified from the extracellular medium by methods recognized in the art. Alternatively, the signal sequence can be connected to the protein of interest using a sequence that promotes purification, such as using a GST domain.

[0396] Chimeric Antigen Receptor

[0397] In certain embodiments, the present disclosure provides a chimeric antigen receptor, which comprises an antibody or antigen-binding fragment thereof provided herein, a transmembrane region, and an intracellular signaling region.

[0398] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that is specifically transplanted into a cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, a regulatory T cell, or any combination thereof). CAR is also referred to as an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor. In some embodiments, CAR includes an antigen-specific targeting region (e.g., an antigen-binding fragment of an anti-LILRB4 antibody as provided herein), an extracellular region, a transmembrane region, one or more costimulatory regions, and an intracellular signaling region.

[0399] In some embodiments, the antigen-specific targeting region is a scFv. In some embodiments, the transmembrane region includes the transmembrane region of CD3, CD4, CD8 or CD28. In some embodiments, the costimulatory region includes the costimulatory domains of CD28, ICOS, CD27, 4-1BB, OX40 and CD40L. In some embodiments, the intracellular signaling region is selected from the group consisting of: CD3, FcγRI, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR or a combination thereof.

[0400] CARs can be transplanted onto a variety of cells (eg, allogeneic cells, autologous cells, or xenogeneic cells).

[0401] As used herein, the term "allogeneic cell" refers to any cell derived from a different subject of the same species.

[0402] As used herein, the term "autologous cell" refers to any cell derived from the same subject that is subsequently reintroduced into the subject.

[0403] As used herein, the term "xenogeneic cell" refers to any cell originating from a different subject of a different species.

[0404] In some embodiments, the CAR is engrafted on an immune effector cell (e.g., T cell, natural killer cell, macrophage, tumor infiltrating lymphocyte, etc.).

[0405] Polynucleotides and recombinant methods

[0406] The disclosure provides isolated polynucleotides, and the isolated polynucleotide encoding antibodies or their antigen-binding fragments and / or chimeric antigen receptors provided herein. As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise indicated, specific polynucleotide sequences also implicitly encompass variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences of their conservative modifications and sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acids Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0407] The DNA encoding the antibodies or antigen-binding fragments thereof provided herein is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of antibodies). Encoding DNA can also be obtained by synthetic methods.

[0408] Recombinant techniques known in the art can be used to encode the isolated polynucleotides of antibodies or their antigen-binding fragments and / or chimeric antigen receptors provided herein and inserted into vectors for further cloning (amplification of DNA) or expression. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0409] The disclosure provides a vector comprising a polynucleotide of separation provided herein. In certain embodiments, the polynucleotide encoding provided herein is an antibody or its antigen-binding fragment and / or chimeric antigen receptor provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably connected to a nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), λ phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0410] The carrier comprising the polynucleotide sequence of the antibody or its Fab and / or chimeric antigen receptor provided by this paper can be introduced into the host expression system (for example, host cell) for cloning or gene expression.In some embodiments, the host expression system provided by this paper is a microorganism, yeast or mammalian cell.In some embodiments, the microorganism is selected from the group consisting of Escherichia coli and Bacillus subtilis.In some embodiments, the yeast is a genus Saccharomyces.In some embodiments, the mammalian cell is selected from the group consisting of: COS, CHO-S, CHO-K1, HEK-293 and 3T3 cells.

[0411] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotic cells, yeast cells or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0412] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding anti-LILRB4 antibodies. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species and strains are commonly used and suitable for use herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0413] Suitable host cells for expressing the glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. A variety of baculovirus strains and variants and corresponding permissive insect host cells have been identified, and the permissive insect host cells are derived from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori. A variety of viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses herein according to the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.

[0414] However, most attention has been paid to vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical tumor cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CCL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); murine breast tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; mouse forestomach carcinoma cells (MFC); SNU620 cells; and human hepatoma line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293, MFC, SNU620, and derivatives thereof.

[0415] With the above-mentioned expression or cloning vector transformed host cell that is used for antibody production, and described host cell is cultivated in conventional nutrient medium, and described conventional nutrient medium is modified into the gene that is suitable for inducing promoter, selecting transformant or amplification coding desired sequence.In another embodiment, antibody can be produced by homologous recombination as known in the art.In certain embodiments, host cell can produce antibody or its Fab that this paper provides.

[0416] The disclosure also provides a method for expressing the antibody or its antigen binding fragment and / or chimeric antigen receptor provided herein, the method comprising culturing the host expression system provided herein under the condition of expressing the antibody or its antigen binding fragment and / or chimeric antigen receptor. The host expression system for producing the antibody or its antigen binding fragment and / or chimeric antigen receptor provided herein can be cultivated in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Methods in Enzymology 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. Reexamination Procedure 30,985 can be used as culture medium for the host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM Drugs), trace elements (defined as inorganic compounds whose final concentrations are usually in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art.

[0417] When using recombinant technology, antibodies can be produced in intracellular, periplasmic space, or directly secreted into culture medium. If antibodies are produced intracellularly, then as a first step, the particulate fragments of the fragments of host cells or dissolution can be removed, for example, by centrifugation or ultrafiltration. Carter et al., " Biology / Technology " 10: 163-167 (1992) describes a program for separating antibodies secreted into the periplasmic space of Escherichia coli. In short, the cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) in about 30 minutes. Cell debris can be removed by centrifugation. When the antibody is secreted into culture medium, usually first use commercially available protein concentration filter (for example, Amicon or Millipore Pellicon ultrafiltration unit) to concentrate the supernatant from such expression system. Protease inhibitors such as PMSF can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0418] Antibodies or antigen-binding fragments thereof and / or chimeric antigen receptors produced by the host expression system can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.

[0419] In certain embodiments, protein A fixed on a solid phase is used for immunoaffinity purification of antibodies and their antigen-binding fragments and / or chimeric antigen receptors. Whether protein A is suitable as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used for purifying antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., Journal of Immunological Methods 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., Journal of the European Molecular Biology Association (EMBO J.) 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Compared with the flow rate and processing time that can be achieved with agarose, a mechanically stable matrix (such as controlled pore glass or poly (styrene divinyl) benzene) can achieve faster flow rates and shorter processing times. Where the antibody comprises a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) can be used for purification. Other techniques for protein purification are available, depending on the antibody to be recovered, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0420] Following any preliminary purification steps, the mixture including the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5 and 4.5, preferably at low salt concentrations (e.g., about 0 to 0.25 M salt).

[0421] Pharmaceutical composition

[0422] The present disclosure further provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof and / or chimeric antigen receptors provided herein and one or more pharmaceutically acceptable carriers.

[0423] Pharmaceutically acceptable carriers for the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquids, gels or solid phase carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / partitioning agents, sequestering agents or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0424] Suitable components can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole (butylated hydroxanisol), butylated hydroxytoluene and / or propyl gallate. As disclosed herein, including one or more antioxidants such as methionine in the composition comprising the antibody or its Fab and conjugate provided herein reduces the oxidation of the antibody or its Fab. This oxidation reduction will prevent or reduce the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Therefore, in certain embodiments, a pharmaceutical composition is provided, which includes one or more antibodies or its Fab as disclosed herein and one or more antioxidants (such as methionine). Further provided are methods for preventing oxidation, extending shelf life, and / or improving therapeutic efficacy of the antibodies or antigen-binding fragments provided herein by mixing the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0425] To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multidose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0426] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0427] In some embodiments, pharmaceutical composition is formulated into injectable composition.Injectable pharmaceutical composition can be prepared in any conventional form, such as liquid solution, suspension, emulsion or solid form suitable for producing liquid solution, suspension or emulsion.Injection preparation can include sterile and / or pyrogen-free solution prepared for injection, sterile dry soluble product (such as lyophilized powder, including subcutaneous injection tablets) prepared for injection before use with solvent combination, sterile suspension prepared for injection, sterile dry insoluble product prepared for use with vehicle combination before use and sterile and / or pyrogen-free emulsion.Solution can be aqueous or non-aqueous.

[0428] In certain embodiments, the unit dose parenteral formulation is packaged in an ampoule, a vial, or a syringe with a needle. As is known and practiced in the art, all preparations for parenteral administration should be sterile and pyrogen-free.

[0429] In certain embodiments, a sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment thereof as disclosed herein in a suitable solvent. The solvent may also contain an excipient that can improve the stability of the powder or a reconstituted solution prepared from the powder or other pharmacological components. The excipients that can be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may contain a buffer such as citrate, sodium phosphate or potassium phosphate, or other such buffers known to those skilled in the art, and in one embodiment, the buffer is about neutral pH. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art, providing the desired formulation. In one embodiment, the resulting solution is dispensed into vials for freeze-drying. Each vial may contain a single dose or multiple doses of an antibody or antigen-binding fragment thereof or a composition thereof. It is acceptable for the vial to be overfilled beyond a small amount (e.g., about 10%) required for a single dose or a set of doses, so as to facilitate accurate sampling and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0430] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. In one embodiment, sterile and / or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the given selected therapy and can be determined empirically.

[0431] Reagent test kit

[0432] In certain embodiments, the disclosure provides a kit comprising an antibody or antigen binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein. In certain embodiments, the disclosure provides a kit comprising an antibody or antigen binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein and a second therapeutic agent. In certain embodiments, the second therapeutic agent is selected from the group consisting of: chemotherapeutic agents, anticancer drugs, radiotherapeutic agents, immunotherapeutic agents, antiangiogenic agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormone therapy agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators and cytokines.

[0433] As will be apparent to those skilled in the art, such kits may further include, if desired, one or more of the various conventional pharmaceutical kit components, such as a container with one or more pharmaceutically acceptable carriers, additional containers, etc. Instructions indicating the quantities of the components to be administered (either as an insert or as labels), directions for administration, and / or directions for mixing the components may also be included in the kit.

[0434] How to use

[0435] The present disclosure also provides a method for treating, preventing or alleviating a disease, disorder or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein. In certain embodiments, the disease, disorder or condition is a LILRB4-related disease, disorder or condition. In certain embodiments, the subject is a human.

[0436] In some embodiments, the LILRB4-associated disease, disorder or condition is characterized by expression or overexpression of LILRB4.

[0437] In certain embodiments, the disease, disorder or condition is an immune disease, an inflammatory disease, a cancer or a nervous system disease. In certain embodiments, the cancer is a LILRB4-expressing cancer. As used herein, a "LILRB4-expressing" cancer refers to a cancer characterized by expressing the LILRB4 protein in cancer cells (i.e., tumor-infiltrating immune cells) or expressing LILRB4 in cancer cells (i.e., tumor-infiltrating immune cells), and its expression level is significantly higher than the expected level of normal cells. Various methods can be used to determine the presence and / or amount of LILRB4 in a test biological sample of a subject. For example, a test biological sample can be exposed to an anti-LILRB4 antibody or an antigen-binding fragment thereof, which binds to the expressed LILRB4 protein and detects the protein. Alternatively, LILRB4 can also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, etc. In some embodiments, the test sample is derived from a cancer cell or tissue or a tumor-infiltrating immune cell. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from which the test sample is obtained. For example, the reference sample can be a non-diseased sample adjacent to or near the test sample (e.g., a tumor). In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the cancer is a LILRB4-expressing B-cell cancer.

[0438] In certain embodiments, the disease, disorder or condition is selected from the group consisting of Kawasaki disease, toxoplasmosis, multiple sclerosis, systemic lupus erythematosus, lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lung adenocarcinoma, lung squamous cell carcinoma, Lewis lung cancer or radiation therapy resistant Lewis lung cancer), peritoneal cancer, carcinoid, bone cancer, pancreatic cancer, primitive neuroectodermal tumor, skin cancer, gallbladder cancer, head and neck cancer, squamous cell carcinoma, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial carcinoma), anal region cancer (e.g., anal squamous cell carcinoma), stomach cancer (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma), liver cancer / hepatoma or liver cancer), bile duct cancer, sarcoma, colorectal cancer, fallopian tube cancer, salivary gland cancer, cervical cancer, endometrial cancer or uterine cancer, osteosarcoma, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, nasopharyngeal cancer, soft tissue sarcoma, polycythemia vera, urethral cancer, penile cancer, kidney or urethra cancer (e.g., renal rhabdoid tumor), cutaneous T-cell lymphoma, medulloblastoma, nephroblastoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, choroid plexus papilloma, renal cell carcinoma, renal pelvic cancer, central nervous system (CNS) neoplasms, soft tissue sarcomas (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi's sarcoma), spinal cord axis tumors, gliomas (e.g., ependymomas, astrocytomas, anaplastic astrocytomas, oligodendrogliomas), eye cancers (e.g., retinoblastomas), brain stem gliomas or mixed gliomas such as oligoastrocytomas), brain tumors (e.g., glioblastoma / glioblastoma multiforme (GBM), non-glioblastoma brain tumors, or meningiomas), melanomas (e.g., cutaneous or intraocular melanomas), thrombocytosis, mesothelioma, mycosis fungoides, Sezary syndrome, primary myelofibrosis, solitary plasmacytoma, vestibular schwannoma, Ewing's sarcoma, chondrosarcoma, MYH-associated polyposis, pituitary adenomas, pediatric cancers, such as pediatric sarcomas (e.g., neuroblastoma, rhabdomyosarcoma, and osteosarcoma), blood cancers, Lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia (e.g., lymphocytic / lymphoblastic leukemia), chronic or acute leukemia, mast cell leukemia, lymphocytic lymphoma, primary CNS lymphoma, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia (HCL), Burkitt's lymphoma (BL), multiple myeloma (e.g., relapsed or refractory multiple myeloma), T or B cell lymphoma, mantle cell lymphoma (MCL) (e.g.,In some embodiments, the disease, disorder or condition is acute myeloid leukemia. In some embodiments, the disease, disorder or condition is chronic myelomonocytic leukemia.

[0439] In some embodiments, the subject has been identified as having cancer cells or tumor-infiltrating immune cells that express LILRB4, optionally at levels significantly higher than those typically found on non-cancerous cells.

[0440] In another aspect, a method for treating, preventing or ameliorating a disease, disorder or condition in a subject that would benefit from modulating LILRB4 activity is provided, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein. In certain embodiments, the disease, disorder or condition is a LILRB4-related disease, disorder or condition as defined above.

[0441] The therapeutically effective amount of the antibody or antigen-binding fragment provided herein will depend on various factors known in the art, such as the subject's weight, age, past medical history, current medication, the subject's health and the potential for cross-reactions, allergies, sensitivities and adverse side effects, and the route of administration and the extent of disease development. Those skilled in the art (e.g., physicians or veterinarians) can proportionally reduce or increase the dosage according to these and other circumstances or required instructions.

[0442] In certain embodiments, the antibody or Fab provided herein and / or the chimeric antigen receptor provided herein can be administered in a therapeutically effective amount of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the administration dose can be changed during the course of treatment. For example, in certain embodiments, the initial administration dose can be higher than the subsequent administration dose. In certain embodiments, the administration dose can change according to the reaction of the subject during the course of treatment.

[0443] Dosage regimens may be adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0444] The antibodies or antigen-binding fragments thereof provided herein and / or the chimeric antigen receptors provided herein can be administered by any route known in the art, such as by parenteral routes (including subcutaneous, intraperitoneal, intravenous, intramuscular or intradermal injection) or non-parenteral routes (including transdermal, oral, intranasal, intraocular, sublingual, rectal or topical).

[0445] In some embodiments, the antibody or its antigen binding fragment provided herein and / or the chimeric antigen receptor provided herein can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibody or its antigen binding fragment disclosed herein and / or the chimeric antigen receptor provided herein can be administered in combination with a second therapeutic agent, and the second therapeutic agent is, for example, a chemotherapeutic agent, an anticancer drug, a radiotherapeutic agent, an immunotherapy agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, a cytokine, an active agent, an imaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a costimulatory molecule agonist, a co-inhibitory molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or its functional fragment, a detectable marker or a reporter gene, an antimicrobial agent, a gene editor, a beta agonist, a viral RNA inhibitor, a polymerase inhibitor, an interferon or a microRNA.

[0446] As used herein, the term "immunotherapy" refers to a type of therapy that stimulates the immune system to fight diseases such as cancer or strengthens the immune system in a general manner. Examples of immunotherapy include, but are not limited to, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0447] "Targeted therapy" is a type of therapy that acts on specific molecules associated with cancer, such as specific proteins that are present in cancer cells but not in normal cells or are more abundant in cancer cells, or target molecules in the cancer microenvironment that contribute to cancer growth and survival. Targeted therapy targets the therapeutic agent to the tumor, thereby sparing normal tissue from the effects of the therapeutic agent.

[0448] In some of these embodiments, the antibody or its antigen binding fragment provided herein and / or the chimeric antigen receptor provided herein and / or the pharmaceutical composition provided herein administered in combination with one or more additional therapeutic agents can be administered simultaneously with one or more additional therapeutic agents, and in some of these embodiments, the antibody or its antigen binding fragment provided herein and / or the pharmaceutical composition and the additional therapeutic agent can be administered as part of the same pharmaceutical composition. However, the antibody or its antigen binding fragment provided herein and / or the chimeric antigen receptor and / or the pharmaceutical composition provided herein administered in combination with another therapeutic agent need not be administered simultaneously with the agent or administered in the same composition as the agent. The antibody or its antigen binding fragment, or the chimeric antigen receptor or the pharmaceutical composition administered before or after another agent is considered to be administered in combination with the agent, as the phrase used herein, even if the antibody or antigen binding fragment, or the pharmaceutical composition or the chimeric antigen receptor and the second agent are administered by different routes. Where possible, the additional therapeutic agent administered in combination with an antibody or antigen-binding fragment thereof, chimeric antigen receptor, or pharmaceutical composition disclosed herein is administered according to the schedule listed in the product information sheet of the additional therapeutic agent or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457 57th Edition (November 2002)) or regimens well known in the art.

[0449] The present disclosure further provides a method for inactivating LILRB4-expressing cells in vivo or in vitro, the method comprising contacting the LILRB4-expressing cells with the antibody or antigen-binding fragment thereof provided herein and / or the chimeric antigen receptor provided herein and / or the pharmaceutical composition provided herein.

[0450] The present disclosure further provides a method for regulating LILRB4 activity in LILRB4-expressing cells, the method comprising exposing the LILRB4-expressing cells to an antibody or antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein. In some embodiments, the LILRB4-expressing cells are dendritic cells, monocytes, macrophages, B cells, Tregs, promast cells, endothelial cells or osteoclasts.

[0451] On the other hand, the disclosure provides a method for inducing phagocytosis of a target cell in vivo or in vitro, the method comprising exposing the target cell to an antibody or antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein. In some embodiments, the target cell is an antigen presenting cell, a cancer cell, or a cell infected by a pathogen.

[0452] In another aspect, the present disclosure provides a method of inducing TNF-α production, comprising exposing tolerogenic dendritic cells to an antibody or antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein.

[0453] In another aspect, the present disclosure provides a method for detecting the presence or amount of LILRB4 in a sample, the method comprising: contacting the sample with an antibody or antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein; and determining the presence or the amount of LILRB4 in the sample.

[0454] In another aspect, the present disclosure provides a method for diagnosing a LILRB4-associated disease, disorder or condition in a subject, the method comprising: a) obtaining a sample from the subject; b) contacting the sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein; c) determining the presence or amount of LILRB4 in the sample; and d) correlating the presence or amount of LILRB4 with the presence or status of the LILRB4-associated disease, disorder or condition in the subject.

[0455] In another aspect, the disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein, optionally conjugated to a detectable moiety, which can be used to detect LILRB4, optionally recombinant LILRB4, LILRB4 expressed on the cell surface, or LILRB4-expressing cells. The kit may further include instructions for use.

[0456] On the other hand, the present disclosure also provides the use of the antibodies or antigen-binding fragments thereof provided herein and / or the chimeric antigen receptors provided herein and / or the pharmaceutical compositions provided herein in the preparation of a medicament for treating, preventing or alleviating a LILRB4-related disease, disorder or condition in a subject, or in the preparation of a diagnostic reagent for diagnosing a LILRB4-related disease, disorder or condition.

[0457] On the other hand, the present disclosure also provides use of the antibodies or antigen-binding fragments thereof provided herein and / or the chimeric antigen receptors provided herein and / or the pharmaceutical compositions provided herein in the preparation of diagnostic reagents for diagnosing LILRB4-related diseases, disorders or conditions.

[0458] The following examples are provided to better illustrate the claimed invention and the examples should not be construed as limiting the scope of the present invention. All specific compositions, materials and methods described below fall within the scope of the present invention in whole or in part. These specific compositions, materials and methods are not intended to limit the present invention, but are only used to illustrate specific embodiments falling within the scope of the present invention. Those skilled in the art can develop equivalent compositions, materials and methods without using inventive abilities and without departing from the scope of the present invention. It should be understood that many changes can be made in the procedures described herein while still remaining within the limits of the present invention. It is the intention of the inventor of the present invention that such changes are all included within the scope of the present invention.

[0459] Example

[0460] Example 1. Antibody Generation

[0461] 1.1. Immunization

[0462] To generate antibodies targeting LILRB4, Balb / c and CD1 mice were immunized with human LILRB4 overexpressing cells (CHO-S-hLILRB4) and / or recombinant LILRB4 proteins in each group (as shown in Table 5 below). The immunogens were delivered to the mice by intrahepatic (IH) and / or intraperitoneal (IP) injection. The primary immunization was then boosted several times until the animals produced a satisfactory antiserum titer suitable for hybridoma development. Complete Freund's Adjuvant (CFA) was used for the primary immunization, and incomplete Freund's adjuvant was used for subsequent immunizations. Test blood sampling was performed and evaluated by FACS experiments using a CHO-S cell line (CHO-S-hLILRB4) that stably overexpresses human LILRB4 or by ELISA experiments on the extracellular domain of the recombinant human LILRB4 protein. The immunization schedule for each group of animals is shown in Tables 6, 7, 8, 9, 10 and 11 below.

[0463] Table 5: Animal groups

[0464]

[0465] Table 6: Immunization schedule for Group 1

[0466]

[0467] Table 7: Immunization schedule for Group 2

[0468]

[0469] Table 8: Immunization schedule for Group 3

[0470]

[0471]

[0472] Table 9: Immunization schedule for Group 4

[0473]

[0474] Table 10: Immunization schedule for Group 5

[0475]

[0476] Table 11: Immunization schedule for Group 6

[0477]

[0478] 1.2. Hybridoma Generation and Screening

[0479] Mice with the best immune response were subjected to spleen cell fusion, as identified by blood sampling for FACS and ELISA tests. FACS assays were performed using a CHO-S cell line stably overexpressing human LILRB4 (CHO-S-hLILRB4). In the ELISA assay, the extracellular domain of the recombinant human LILRB4 protein was coated as a ligand to test the antiserum concentration. Lymphocytes from the spleen and lymph nodes were fused with a mouse myeloma cell line (SP2 / 0) using an optimized electrofusion protocol. Multiple fusions were performed to ensure the success of the project.

[0480] The fusion was inoculated (2×10 4 Up to 10 5 Cells were added to a stack of 96-well plates. The growth of the cells in the plates was monitored and fed weekly. Wells with cell growth were screened by primary screening assays using FACS and / or other feasible assays (such as ELISA) within 10-14 days. Multiple fusions were performed and screened for each targeted antigen. Positive parent clones that showed positive binding to 293F-LILRB4 and positive ELISA signals from the primary screen were expanded into 24-well plates for secondary screening.

[0481] After primary screening, positive parental clones expanded into 24-well plates are screened again by FACS or ELISA assays as described below. Hybridomas of interest are selected to proceed to subcloning.

[0482] Parental hybridomas with the desired reactivity and isotype from the above screening funnel are then subcloned by multiple rounds of limiting dilution or single cell sorting until a single clone is obtained.

[0483] Subclone plates were screened by protein or cell-based ELISA, and subclones with good binding capacity were expanded to 24 wells for confirmation testing. The specificity and cross-reactivity of these subclones were confirmed by FACS analysis. In brief, parental 293F cells and 293F-hLILRB4 were incubated with antibodies produced by each subclone, respectively. Fluorescent dye-conjugated secondary antibodies were used to detect binding of primary antibodies to cells. Median fluorescence intensity (MFI) was measured by FACS analysis.

[0484] The desired subcloned cell lines were sequenced and further expanded into culture flasks for cryopreservation. Initially 0.5-13.0x10 6 Cells / vial were cryopreserved in 4-6 vials per cell line. A master cell bank and working cell bank were established for the most valuable cell lines selected.

[0485] As Fig.12 The results shown, a total of 28 antibodies with unique sequences were found, which showed positive binding to 293F cells stably overexpressing human LILRB4 protein (293F-hLILRB4), but did not show binding to parental 293F cells. Among these antibodies, 3 antibodies (i.e., 42-F5-B12-F2, 44-F10-B6-D4 and 42-A10-C1-E10) can also bind to cynomolgus / Macaca fascicularis LILRB4. The MFI of mouse antibodies staining 293F-hLILRB4, 293F-hLILRB1, 293F-hLILRB2, 293F-hLILRB3, 293F-hLILRB5, 293F-hLILRA1, 293F-hLILRA2, 293F-hLILRA3, 293F-hLILRA4, 293F-hLILRA5, CHO-S-mLILRB4, CHO-S-cynomolgus monkey LILRB4 (CHO-S-cynoLILRB4) detected by FACS are summarized in Tables 12 and 13 below.

[0486] Table 12: MFI of antibody binding to cell lines expressing different human LILRA family and human LILRB family proteins

[0487]

[0488]

[0489]

[0490] Table 13: FACS MFI of antibody binding to cynomolgus / Macaca fascicularis LILRB4 cell line

[0491]

[0492]

[0493] Notice:

[0494] " / " indicates no detectable binding.

[0495] Example 2. Antibody Characterization: Binding Affinity

[0496] Antibodies

[0497] Characterization of hybridoma antibody clones 2-G4-G8-E10, 36-F3-E7-H9, 26-H9-B9-B7, 27-D11-C2-A10, 32-F9-C9-B9, 27-F11-E10-G10, 27-G5-E9-B8, 4-E6-E4-B12, 25-G10-C5-G2, 42-F5-B12-F2, 7-B4-C1-C9, 36-F4-H1-D9, 10-H9-E3-G3, 44-F10-B6-D4, 25-E7-A10 -H6, 25-G9-G4-C12, 10-G3-B3-E7, 42-A10-C1-E10, 8-B3-F6-H8, 48-E11-H7-B5, 43-D12-F3-G11, 28-F10-B2-F6, 2-H1-D7-E5-D5, 2-H8-C9-F7-E2, 42-C8-A12-F3-D11, 26-C11-F8-B2-D3, 7-A7-F9-D8 and 41-E12-E10-D8, and their sequences were determined by the following method.

[0498] 2.2. Hybridoma Sequencing

[0499] Total RNA was isolated from hybridoma cells according to the technical manual of RNAiso Plus (TAKARA catalog number 9109). Total RNA was then reverse transcribed into cDNA using isoform-specific antisense primers or universal primers according to the technical manual of PrimeScript II 1st Strand cDNA Synthesis Kit (TAKARA catalog number 6210A). TM(Catalog No. R001A) amplified antibody fragments of VH and VL. The amplified antibody fragments were cloned individually into standard cloning vectors. Colony PCR was performed to screen clones with inserts of the correct size. No less than five colonies with inserts of the correct size for each fragment were sequenced. Sequences of different clones were compared and the consensus sequences of these clones were provided.

[0500] The variable region sequences of the hybridoma antibodies are provided in Table 3 above.

[0501] 2.3. Antibody binding affinity on 293F-hLILRB4 cell line

[0502] To evaluate the binding affinity of antibody candidates to hLILRB4 in a cell-based assay, sequences of 17 mouse antibodies from Table 12 were selected to generate and produce human IgG1 chimeric antibodies. Two human IgG1 chimeric antibody benchmark antibodies, IO-202 and NGM831, were also generated. In general, DNA encoding the variable regions of 17 mouse antibodies and the benchmark antibodies were synthesized and subcloned into expression vectors, in which human IgG constant genes were included in advance. The vectors were transfected into mammalian cells for recombinant protein expression, and the expressed antibodies were purified using a protein A affinity chromatography column. The resulting chimeric antibodies are referred to herein as ch27-F11-E10-G10, ch43-D12-F3-G11, ch36-F4-H1-D9, ch8-B3-F6-H8, ch44-F10-B6-D4, ch27-G5-E9-B8, ch48-E11-H7-B5, ch42-C8-A12-F3-D11, ch10-H9-E3-G 3, ch7-B4-C1-C9, ch25-E7-A10-H6, ch25-G9-G4-C12, ch2-H1-D7-E5-D5, ch27-D11-C2-A10, ch26-H9-B9-B7, ch4-E6-E4-B12 and ch2-H8-C9-F7-E2, wherein the prefix "ch" means "chimeric" and the following means a hybridoma antibody clone. For example, ch27-F11-E10-G10 means that it is a chimeric antibody derived from hybridoma antibody clone 27-F11-E10-G10. The two benchmark antibodies (which are produced as human IgG1 chimeric antibodies) are still referred to as IO-202 and NGM831, respectively.

[0503] The binding affinity of these chimeric antibodies and benchmark antibodies (i.e., IO-202 and NGM831) to 293F cells stably overexpressing human LILRB4 protein (293F-hLILRB4) was determined by FACS analysis. The binding affinity of these chimeric antibodies and benchmark antibodies to LILRB on different cell lines, including 293F-hLILRB4, 293F-hLILRB1, 293F-hLILRB2, 293F-hLILRB3, 293F-hLILRB5, 293F-hLILRA1, 293F-hLILRA2, 293F-hLILRA3, 293F-hLILRA4, 293F-hLILRA5, CHO-S-mLILRB4, CHO-S-cynomolgus monkey LILRB4 (CHO-S-cynoLILRB4), was determined by FACS. The results are summarized in Table 14 below.

[0504] The protocol for FACS analysis is described as follows:

[0505] (a) Cells were collected by centrifugation at 300 g for 3 min.

[0506] (b) Wash cells twice with FACS buffer by centrifugation at 300 g for 3 min and discard the supernatant.

[0507] (c) Resuspend the cells in 50 μl FACS buffer and plate them in assay plates (2×10 5 Cells / well) were added to the plate. 50 μL of a range of final concentrations (66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09, 0.03 nM) of primary antibody were added to the plate. Cells were incubated with antibodies for 1 hour at 4°C.

[0508] (d) Repeat step (b) and resuspend the cells with 100 μl of diluted secondary antibody and incubate at 4° C. in the dark for 1 hour.

[0509] (e) Repeat step (b) and resuspend the cells with 100 μl FACS buffer. Keep the cells in the dark for FACS analysis.

[0510] As shown in Table 14 and Figure 1 , 2 As shown in Tables 14 and 3, the binding affinity of the selected chimeric antibodies to the 293F-hLILRB4 cell line was higher, lower or comparable to that of the benchmark antibodies IO-202 and NGM831 (Tables 14 and Figure 1 ). The ch43-D12-F3-G11 and ch42-C8-A12-F3-D11 antibodies showed binding to human LILRB3 ( Figure 1 and Figure 2AB), and ch44-F10-B6-D4 showed significant cross-reactivity with cynomolgus monkey LILRB4 ( Figure 1 and Figure 3 AB) with good cross-reactivity.

[0511] Table 14: Binding affinity of chimeric antibodies to 293F-hLILRB4 cell line, 293F-hLILRB3 cell line, CHO-S-cynoLILRB4 cell line determined by FACS and binding affinity of chimeric antibodies to hLILRA6 protein determined by ELISA

[0512]

[0513]

[0514]

[0515] Notice:

[0516] " / " indicates no detectable binding.

[0517] 2.4 LILRB4 expression profile in human cancer cell lines

[0518] To compare LILRB4 expression in different human cancer cell lines, human LILRB4 APC-conjugated antibody (clone: ​​293623, catalog: FAB24251A-100) was used in FACS analysis. Figure 4 As shown, LILRB4 is expressed at high levels in some human cancer cell lines, such as THP-1 (acute myeloid leukemia, AML5) and MV-4-11 (acute myeloid leukemia, AML5).

[0519] Example 3. Antibody Characterization: Antibody-Dependent Cellular Cytotoxicity (ADCC)

[0520] 3.1. Determination of ADCC of selected antibodies

[0521] To measure the hLILRB4-specific ADCC effect, carboxyfluorescein succinimidyl ester (CFSE)-labeled THP-1 cells were used as target cells. 4 Cells / well were seeded in a 96-well flat-bottom sterile plate, and 2x10 5Freshly isolated human PBMCs were used as effector cells. Target cells, effector cells (FcR blocked before adding antibody to CFSE-labeled THP-1 cells) and antibodies with increasing concentrations were merged into 200 μL total volume in a U-shaped 96-well plate with X-VIVO culture medium containing 50 ng / mL of IL-2. Cells and antibodies were incubated for 20 hours at 37°C 5% CO2, followed by FACS analysis to measure the percentage of CFSE-labeled THP-1 cells. IO-202 and NGM831 were used as benchmark antibodies (which were produced as human IgG1 chimeric antibodies), and human IgG1 was used as a negative control.

[0522] The percentage of cytotoxicity was calculated according to the following equation:

[0523] (AT: percentage of CFSE-labeled THP-1 cells in the antibody-treated groups;

[0524] (NAT: Percentage of CFSE-labeled THP-1 cells in the negative control group)

[0525] Results

[0526] As shown in Table 15 and Figure 5 As shown, antibody ch2-H1-D7-E5-D5 showed a significantly potent ADCC effect and lower EC against THP-1 cells compared to benchmark antibodies IO-202 and NGM831. 50 .

[0527] Table 15: Cytotoxicity and EC of selected antibody ADCC effects on THP-1 cells 50

[0528] Clone number Maximum cytotoxicity (%) <![CDATA[EC 50 (nM) <!-- 69 -->]]> Isotype (hIgG1) 2.75 / IO-202 13.93 247.6 NGM831 -6.89 487.7 ch27-F11-E10-G10 18.7 / ch43-D12-F3-G11 2.39 4.145 ch36-F4-H1-D9 17.9 / ch8-B3-F6-H8 7.1 / ch44-F10-B6-D4 0.43 1.432 ch27-G5-E9-B8 16.4 / ch48-E11-H7-B5 / / ch42-C8-A12-F3-D11 14.52 / ch10-H9-E3-G3 16.61 / ch7-B4-C1-C9 13.91 / ch25-E7-A10-H6 11.73 / ch25-G9-G4-C12 11.03 / ch2-H1-D7-E5-D5 39.61 7.337 ch27-D11-C2-A10 20.2 / ch26-H9-B9-B7 15.17 / ch4-E6-E4-B12 14.56 / ch2-H8-C9-F7-E2 31.11 1.377

[0529] Notice:

[0530] " / " indicates that it cannot be detected.

[0531] Example 4. Antibody Characterization: Antibody-Dependent Cellular Phagocytosis (ADCP)

[0532] 4.1. Determination of ADCP of selected antibodies

[0533] To measure the hLILRB4-specific ADCP effect, CFSE-labeled MV-4-11 and Far-Red-labeled M2 macrophages differentiated from isolated human PBMC were added as effector cells. IO-202 and NGM831 were used as benchmark antibodies (which were produced as human IgG1 chimeric antibodies), while human IgG1 was used as a negative control. The ADCP assay was performed as follows:

[0534] (a) First, CD14 was isolated from PBMC using EasySep (STEMCELL Inc). + Monocytes.

[0535] (b) Monocytes were differentiated into M2 macrophages by culturing in X-VIVO 15 for 8 days with 100 ng / ml rhM-CSF on day 0 and day 3, and 100 ng / ml rhIL10 and rhTGF-β on day 5, respectively.

[0536] (c) 2×10 4 Far-Red labeled M2 macrophages were incubated with various concentrations of antibodies and 2 × 10 4 CFSE-labeled MV-4-11 were mixed, in which FcR was blocked before adding antibodies (effector:target=1:1).

[0537] (d) The mixture was incubated at 37°C in a CO2 incubator for 2 hours.

[0538] (e) Cells were digested with trypsin (1X) by centrifugation at 500 g for 3 min.

[0539] (f) Wash cells twice with FACS buffer by centrifugation at 500 g for 3 min.

[0540] (g) Phagocytosis was assessed by flow cytometry and the results are reported as the ratio of cells positive for both CFSE and Far-Red to the total macrophages in the sample.

[0541] Results

[0542] As shown in Table 16 and Figure 6 As shown, all chimeric antibodies showed effective ADCP effect on MV-4-11 cells. Compared with the benchmark antibody IO-202, the chimeric antibody ch2-H1-D7-E5-D5 showed a lower EC 50 (Table 16 and Figure 6 ).

[0543] Table 16: ADCP efficacy of 133.3 nM selected antibodies in macrophages and MV-4-11 cells

[0544]

[0545]

[0546] Example 5. Antibody Characterization: Reprogramming Tolerogenic Dendritic Cells by Blocking LILRB4-Fibronectin Interaction

[0547] 5.1. Anti-LILRB4 antibodies block LILRB4-fibronectin interaction and regulate tolerogenic dendritic cells Reprogramming

[0548] Dendritic cells (DCs), including monocyte-derived dendritic cells (moDCs), induced by immunosuppressive factors such as TGF-β and IL-10 in the tumor microenvironment are tolerant. LILRB4, which contains three cytoplasmic ITIMs (immunoreceptor tyrosine-based inhibitory motifs) in the intracellular domain, is significantly overexpressed on tolerant dendritic cells. FcγRI (CD64), which is a high-affinity IgG receptor, is significantly expressed on many myeloid cells such as dendritic cells and macrophages. IgG and FcγRI interact to trigger the intracellular ITAM (immunoreceptor tyrosine-based activation motif) signal of FcγRI to promote TNF-α production. It has been identified that there is a crosstalk between LILRB4 and FcγRI in the signaling pathway mediated by LILRB4 and FcγRI, in which LILRB4 regulates FcγRI-mediated ITAM activation to secrete TNF-α. Fibronectin is a functional ligand of LILRB4, promoting stimulation of unresponsive dendritic cells through FcγRI-IgG interaction. However, FcγRI signaling can be reversed by anti-LILRB4 antibody-mediated blockade of the fibronectin-LILRB4 interaction. Therefore, the inventors of the present invention evaluated the blocking ability of the claimed anti-LILRB4 antibodies in tolerogenic moDCs by this FcγR stimulation assay. IO-202 and NGM831 were used as benchmark antibodies, which were generated as human IgG1 chimeric antibodies. TNF-α secretion from tolerogenic dendritic cells was used as a readout for evaluating FcγR stimulation. The assay was performed as described below:

[0549] (a) Day 0: Monocytes were isolated from PBMCs and cultured at 5 × 10 6 The cells were resuspended in 3 ml of X-VIVO supplemented with 2 U / ml of dendritic cell culture factor, and then the cells were cultured in a 6-well plate.

[0550] (b) Day 2: Add 2 ml / well of fresh complete medium supplemented with 2 U / ml dendritic cell culture factors.

[0551] (c) Day 5: Immature moDCs were differentiated into tolDCs by adding 10 nmol / L dexamethasone and 100 nmol / L vitamin D3.

[0552] (d) Day 7:

[0553] (1) A 96-well plate (Corning) was co-coated with PBS containing 5 μg / mL fibronectin and 5 μg / mL human IgG1 at room temperature for 2 hours.

[0554] (2) The plate was washed twice with PBS and pre-incubated with RPMI1640 containing 10% FBS for 2 hours.

[0555] (3) Tolerogenic human dendritic cells (5-7 x 10 4 Cells / well) were plated on the coated wells and incubated with 5 μg / mL of the indicated antibodies for 5-20 minutes at room temperature.

[0556] (4) The cells in the coated wells were mixed thoroughly and incubated at 37°C in a CO2 incubator overnight.

[0557] (5) After incubation at 37°C overnight, the culture supernatant was collected and cytokine secretion was measured by ELISA assay using a human TNF-α kit according to the manufacturer's instructions.

[0558] Results

[0559] As shown in Table 17, all 17 chimeric LILRB4 antibodies significantly promoted TNF-α production. This suggests that these LILRB4 antibody candidates can effectively block the LILRB4-fibronectin interaction to reprogram tolerant dendritic cells into mature dendritic cells that stimulate T cell activation, proliferation and differentiation. Except for ch43-D12-F3-G11 and ch44-F10-B6-D4, 15 of the 17 antibody candidates showed more effective blocking or reprogramming effects compared to the benchmark antibodies IO-202 and NGM831.

[0560] Table 17: TNFα secretion in tolerogenic dendritic cells reprogrammed by antibody blockade of LILRB4-fibronectin interaction

[0561]

[0562]

[0563] Example 6. Antibody Characterization: Antigen Presentation and Co-stimulatory Capacity of Dendritic Cells

[0564] 6.1. LILRB4 antibody reprograms DCs for antigen presentation and co-stimulation

[0565] To test the ability of LILRB4 antibody candidates in modulating the expression of antigen presenting molecules and co-stimulatory molecules, assays were performed as follows: IO-202 and NGM831 were used as benchmark antibodies, which were generated as human IgG1 chimeric antibodies.

[0566] (a) Day 0: Monocytes were isolated from PBMCs and cultured at 5 × 106 The cells were resuspended in 3 ml of X-VIVO supplemented with 2 U / mL of dendritic cell culture factors, and then the cells were cultured in a 6-well plate.

[0567] (b) Day 2: Add 2 mL / well of fresh complete medium supplemented with 2 U / ml dendritic cell culture factors.

[0568] (c) Day 5: Immature DCs were differentiated into tolDCs by adding 10 nM dexamethasone and 100 nM vitamin D3.

[0569] (d) Day 7: Samples were examined by flow cytometry by staining for CD86-PE, HLA-DR-FITC, and HLA-ABC-APC.

[0570] Results

[0571] As shown in Table 18, all chimeric antibodies induced overexpression of antigen presenting molecules (HLA-DR and HLA-ABC) and co-stimulatory molecules (CD86) on DC. These results show that chimeric antibodies reprogram tolerant DC to activate T cells. Compared with benchmark antibodies IO-202 and NGM831, some of the antibodies (e.g., ch7-B4-C1-C9, ch25-E7-A10-H6, ch2-H8-C9-F7-E2, etc.) showed more effective expression-induction effects.

[0572] Table 18: MFI of antigen presenting molecules (HLA-DR and HLA-ABC) and co-stimulatory molecules (CD86) for antibody-induced reprogramming of dendritic cells

[0573] clone CD86 MFI HLA-DR MFI HLA-ABC MFI Isotype (hIgG1) 1052 2805 1601 IO-202 1650 3097 1560 NGM831 2087 3425 1603 ch27-F11-E10-G10 1578 3444 1714 ch43-D12-F3-G11 1792 3771 1935 ch36-F4-H1-D9 1777 3343 2027 ch8-B3-F6-H8 3148 4521 2056 ch44-F10-B6-D4 1165 3121 1910 ch27-G5-E9-B8 1977 3676 1641 ch48-E11-H7-B5 1564 3462 2028 ch42-C8-A12-F3-D11 1843 3629 1496 ch10-H9-E3-G3 1862 3340 1631 ch7-B4-C1-C9 2153 3654 2097 ch25-E7-A10-H6 2089 3375 1881 ch25-G9-G4-C12 1485 3789 1898 ch2-H1-D7-E5-D5 1927 4485 2279 ch27-D11-C2-A10 1661 3854 2097 ch26-H9-B9-B7 1512 3928 2139 ch4-E6-E4-B12 1623 3903 1983 ch2-H8-C9-F7-E2 2333 4250 1839

[0574] Example 7. Antibody Characterization: THP-1 and T Cell Co-culture Assays of Selected Chimeric Antibodies

[0575] The chimeric antibodies ch2-H1-D7-E5-D5 and ch8-B3-F6-H8 prepared in Example 2.3 were used for THP-1 and T cell co-culture assays. The protocol for the THP-1 and T cell co-culture assays is as follows:

[0576] (a) Human T cells (5x10 4 cells / well) were placed in the lower chamber of a 96-well transwell plate.

[0577] (b) Irradiated THP-1 cells (E:T ratio = 1:4) were cultured and treated with the indicated antibodies in the upper chamber of transwell inserts in U-bottom 96-well plates (pore size, 3 mm; Thermo Fisher Scientific).

[0578] (c) After culturing for 5 to 7 days in the lower chamber with anti-CD3 / CD28 coated beads (Thermo Fisher Scientific) and 50 U / mL rhIL2, representative T cells in the lower chamber were photographed using a microscope. Cells were stained with anti-CD3-PE or anti-CD8-APC / Fire750 and analyzed by flow cytometry.

[0579] like Fig.11 As shown in AD, chimeric antibodies ch2-H1-D7-E5-D5 and ch8-B3-F6-H8 reversed THP-1-mediated T cell suppression.

[0580] Example 8. Antibody Characterization: In vivo Efficacy Testing of Selected Chimeric Antibodies against Radiation-Resistant Lewis Lung Carcinoma (LLC1) and EL4-LILRB4 Lymphoma in Female hLILRB1 / hLILRB4 Transgenic Mice

[0581] The chimeric antibody ch8-B3-F6-H8 prepared in Example 2.3 was used in an in vivo efficacy test for the treatment of radiation therapy resistant Lewis lung carcinoma (LLC1) and EL4-LILRB4 lymphoma in female hLILRB1 / hLILRB4 transgenic mice. IO-202 was used as a positive control, and hIgG1 was used as a negative control.

[0582] Research design and methods

[0583] 8.1.1. Radiotherapy-resistant LLC1 model

[0584] The in vivo efficacy assay protocol for the radiotherapy-resistant LLC1 model is summarized in Table 19 below and described in detail below.

[0585] (a) LLC1 tumor cells were maintained in vitro in DMEM medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Cells in the exponential growth phase were harvested before tumor inoculation and quantified by cell counter.

[0586] (b) LLC1 tumor cells (3x10^6) in 0.1 ml PBS were subcutaneously inoculated in the right hind flank area of ​​each mouse for tumor development. The randomization date was denoted as day 0, and drug administration began from day 0.

[0587] (c) When the average tumor size reaches approximately 70-100 mm3 Randomization was started at 1:00 p.m. This study included 18 mice. All animals were randomly assigned to 3 study groups. Randomization was performed based on a randomized block design.

[0588] (d) Tumor volume was measured twice weekly in two dimensions using calipers and expressed in mm using the following formula: 3 To express volume in units:

[0589] V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L).

[0590] Dosing and tumor and body weight measurements were performed in a laminar flow cabinet.

[0591] Table 19: Study design for in vivo efficacy testing in the LLC1 model of radiotherapy resistance

[0592]

[0593]

[0594] 8.1.2.EL4-LILRB4 Lymphoma Model

[0595] The in vivo efficacy assay protocol for the EL4-LILRB4 lymphoma model is summarized in Table 20 below and described in detail below.

[0596] (a) EL4-LILRB4 lymphoma tumor cells were maintained in vitro in DMEM medium supplemented with 10% fetal bovine serum at 37° C. in an atmosphere of 5% CO 2 in air. Cells in the exponential growth phase were harvested before tumor inoculation and quantified by cell counter.

[0597] (b) Each mouse was intravenously inoculated with EL4-LILRB4 lymphoma tumor cells (0.2x10^6) in 0.2 ml PBS in the right hind flank area for tumor development. The randomization date is denoted as day 0, and drug administration started from day 0.

[0598] (c) When the total flux reaches about 10 6 photons / second. This study included 18 mice. All animals were randomly assigned to 3 study groups. Randomization was performed based on a randomized block design.

[0599] (d) Tumor flux was measured twice a week. Dosing and tumor and body weight measurements were performed in a laminar flow cabinet.

[0600] Table 20: Study design for in vivo efficacy testing in the EL4-LILRB4 lymphoma model

[0601]

[0602] Results

[0603] like Figure 7 As shown, ch8-B3-F6-H8 showed effective in vivo therapeutic effects. Figure 7 As shown in A, the combination of 10 mg / kg ch8-B3-F6-H8 and 10 Gy radiation therapy significantly inhibited LLC1 tumor growth in vivo, with a tumor growth inhibition (TGI) of 40%, while the benchmark antibody (i.e., IO-202) only exhibited a 24% TGI in the LLC1 murine syngeneic model. Figure 7 As shown in B, 3 mg / kg ch8-B3-F6-H8 significantly inhibited EL4-LILRB4 tumor growth in vivo with a TGI of 62%, while the benchmark antibody (i.e., IO-202) exhibited a 53% TGI in the EL4-LILRB4 syngeneic model. Figure 7 C) and EL4-LILRB4 models ( Figure 7 The body weight of mice in each group in D) did not change significantly.

[0604] Example 9. Humanized Antibodies of 2-H1-D7-E5-D5 and 8-B3-F6-H8: Generation and Antibody Characterization

[0605] 9.1. Humanized Antibody Production

[0606] Chimeric antibodies ch2-H1-D7-E5-D5 and ch8-B3-F6-H8 were selected to prepare humanized clones. The antibody sequences were aligned with human germline sequences to identify the best fit model. Based on the homology with the original mouse antibody sequence, the best matching human germline sequence was selected as the humanization template. Typically, antibody humanization is performed by comparing the IMGT (https: / / www.imgt.org) human antibody heavy and light chain variable strain gene database, heavy chain and light chain variable strain genes with high homology, wherein a murine antibody is selected as a template, and the CDR of the murine antibody is transplanted into the corresponding human template. The variable region sequence is formed in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As needed, the key amino acids in the framework sequence are restored and mutated to antibodies corresponding to murine antibodies to ensure the original affinity. The two humanized antibodies are designated as h2-H1-D7-E5-D5 and h8-B3-F6-H8, respectively, where the prefix "h" indicates "humanized" and the suffix "2-H1-D7-E5-D5" indicates, for example, the sequence number of the humanized antibody clone of ch2-H1-D7-E5-D5. The amino acid sequence information of h2-H1-D7-E5-D5 and h8-B3-F6-H8 is shown in Table 21 above. As can be seen in Table 21, the CDRs of h2-H1-D7-E5-D5 are the same as those of its corresponding mouse monoclonal antibody 2-H1-D7-E5-D5; the CDRs of h8-B3-F6-H8 are the same as those of its corresponding mouse monoclonal antibody 8-B3-F6-H8, except for HCDR2. Specifically, the amino acid sequence of HCDR2 of h8-B3-F6-H8 is as shown in SEQ ID NO: 158 (INP E The amino acid sequence of HCDR2 of the mouse monoclonal antibody 8-B3-F6-H8 is shown in SEQ ID NO: 42 (INP D While not wishing to be bound by any particular theory, it is believed that such HCDR2 changes favor the binding affinity to human LILRB4.

[0607] 9.2. Binding affinity testing of humanized antibodies

[0608] 9.2.1. Protein-based affinity testing by Biacore (Biointron)

[0609] The parameters of the protein-based affinity test performed by Biacore (Biacore Biotechnology) are as follows:

[0610] Analyte: LILRB4;

[0611] Running buffer: HBS-EP + ;

[0612] Flow rate: 30 μL / min;

[0613] Capture: Ab, 10 μl / min for 60 sec;

[0614] injection of serially diluted LILRB4;

[0615] Contact time: 180 seconds, dissociation time: 400 seconds;

[0616] Regeneration: pH 1.5Gly, 30 μl / min for 30 sec;

[0617] Methods: Multiple cycle kinetics / affinity using capture;

[0618] Machine model: Biacore 8K(GE)

[0619] Analysis temperature: 25℃

[0620] 9.2.2. Protein-based affinity testing by ELISA

[0621] The protocol for protein-based affinity testing by ELISA is as follows:

[0622] (a) 100 μl / well of diluted soluble protein (0.5 μg / mL) was added to a clear flat-bottom Immuno 96-well plate (Nunc, Cat. No. 442404) and incubated overnight at 4°C.

[0623] (b) The cells were washed three times with PBS containing 0.05% Tween-20 (PBST / BSA).

[0624] (c) Block the plate: add 200 μl of 2% BSA to each well and incubate at 37°C for 1 hour.

[0625] (d) Wash three times with PBS containing 0.05% Tween-20 (PBST / BSA).

[0626] (e) 100 μl of test antibody (primary antibody final concentration: adjust the starting concentration of antibody to 1000 nM or 120 nM and serially dilute the antibody at a 1:3 ratio) was added to each well and incubated at 37°C for 1 hour.

[0627] (f) Wash three times with PBS containing 0.05% Tween-20 (PBST / BSA).

[0628] (g) 100 μl of HRP-conjugated secondary antibody diluted 1:10,000 was added and incubated at 37°C for 1 hour.

[0629] (h) Wash three times with PBS containing 0.05% Tween-20 (PBST / BSA).

[0630] (i) 100 μl of substrate solution TMB (SURMODICS, catalog number TMBS-1000-01) was added to each well and incubated at room temperature in the dark for 30 minutes.

[0631] (j) 50 μl of stop solution was added to each well and read on the MD SpectraMax iD3 at 450 nm.

[0632] 9.2.3.293F-hLILRB4 Cell-Based Binding Affinity

[0633] The protocol for cell-based binding affinity on 293F-hLILRB4 cells was similar to that described in Example 2.3.

[0634] 9.2.4.293F-hLILRB4 Cell-Based Binding Affinity

[0635] The protocol for competitive cell-based binding affinity on 293F-hLILRB4 cells is as follows:

[0636] (a) Cells were harvested by centrifugation at 300 g for 3 min and the supernatant discarded.

[0637] (b) Wash cells twice with FACS buffer by centrifugation at 300 g for 3 min and discard the supernatant.

[0638] (c) Resuspend the cells in 50 μL FACS buffer and plate them in assay plates (2*10 5 50 μl of primary antibody was added to the plate at a range of final concentrations (working concentration of one competitor, e.g. 10 μg / ml for h2-H1-D7-E5-D5, and 50 μg / ml for the other 3 antibodies, including IO-202, NGM831, and h8-B3-F6-H8, from 3 serial dilutions of 50 μg / ml, 10 steps) and incubated at 4°C for 1 hour.

[0639] (d) Repeat step (b) and resuspend the cells with 100 μl of diluted secondary antibody and incubate at 4° C. in the dark for 1 hour.

[0640] (e) Repeat step (b) and resuspend the cells with 100 μl FACS buffer. Keep the cells in the dark for FACS analysis.

[0641] Results

[0642] like Figure 8As shown in AC, the binding affinity of humanized antibody h8-B3-F6-H8 on 293F-hLILRB4 cell line was high and comparable to benchmark antibodies (IO-202 and NGM831) in both ELISA and cell-based binding assays, and humanized antibody h2-H1-D7-E5-D5 also showed binding on 293F-hLILRB4 cell line in both ELISA and cell-based binding assays. Figure 8 As shown in DF, h8-B3-F6-H8 has a binding epitope similar to IO-202, while h2-H1-D7-E5-D5 has a specific binding epitope.

[0643] 9.3. Antibody Characterization: Mixed Lymphocyte Reaction (MLR) of Humanized Antibodies

[0644] The humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 prepared in Example 9.1 were used for MLR. IO-202 and NGM831 were used as positive controls, and hIgG1 was used as a negative control. The protocol for MLR of humanized antibodies is as follows:

[0645] (a) Day 0: Isolation of monocytes from PBMCs. 5x10 6 The cells were resuspended in 3 ml of X-VIVO supplemented with 2 U / ml of dendritic cell culture factors, and then the cells were cultured in a 6-well plate.

[0646] (b) Day 2: Add 2 ml / well of fresh complete medium supplemented with 2 U / ml dendritic cell culture factors.

[0647] (c) Day 5: Immature DCs were differentiated into tolDCs by adding 10 nmol / L dexamethasone and 100 nmol / L vitamin D3.

[0648] (d) Day 6: Order 50-100 million PBMCs for T cell isolation the next day.

[0649] (e) Day 7: After 48 hours of stimulation, immature DCs were differentiated into mature DCs and tolDCs, respectively.

[0650] (f) Day 7: T cells were isolated from PBMCs (about 15-30 million T cells were isolated from 50-100 million PBMCs). Half of the isolated T cells were labeled with CFSE, and the other half were not labeled.

[0651] (g) Day 7: 200,000 T cells / well and 25,000 tolDC cells / well were seeded in 96-well plates (tolDC are non-adherent cells).

[0652] (h) Day 7: Cells, mixtures of DCs and T cells were treated with PBS, hIgG1, IO-202, NGM831, h2-H1-D7-E5-D5 or h8-B3-F6-H8 (final concentration: 15 μg / ml), three replicates per group. In addition, two groups of T cells alone and tolDC alone were used as controls.

[0653] (i) Day 12: The supernatant was assayed using an IFN-γ ELISA kit.

[0654] like Fig. 9 As shown, humanized antibody h8-B3-F6-H8 re-enhanced tolDC to elicit T cell activation.

[0655] 9.4. Antibody Characterization: Macrophage and T Cell Co-culture Assays for Humanized Antibodies

[0656] The humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 prepared in Example 9.1 were used for macrophage and T cell co-culture assays. IO-202 and NGM831 were used as positive controls, and hIgG1 was used as a negative control. The protocol for the macrophage and T cell co-culture assay was as follows:

[0657] (a) Through EasySep TM Human CD14 Positive Selection Kit II (Stem Cell Company, 17858) was used to isolate monocytes.

[0658] (b) Monocytes were cultured in X-VIVO (55 μm β-ME) and 100 ng / mL rhM-CSF for 5 days. Half of the medium was replaced and cytokines were added every 2 to 3 days.

[0659] (c) 50 ng / mL TGF-β and rhIL-10 were added and cultured for another 2 days.

[0660] (d) Cells were digested with Accutase cell disruption medium for 10 min at 37°C, centrifuged and resuspended in X-VIVO (55 μM β-ME).

[0661] (e) Inoculation 3.3x10 4 , 1x10 4 / well macrophages, and antibodies (10 μg / mL) were added to 96-well plates and incubated at 37°C under 5% CO2 for 1 hour.

[0662] (f) Add 30 ng / mL soluble CD3 antibody to T cells. 1x10 5 T cells were seeded into 96-well plates at 100 μl / well and incubated at 37°C in 5% CO2 for 3 days.

[0663] (g) Thereafter, the supernatant was collected by IFN-γ centrifugation.

[0664] like Fig.10 As shown, humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 reversed macrophage-mediated T cell suppression.

[0665] 9.5. Antibody Characterization: Humanized Antibodies for Cytotoxic CD8 + T cell killing assay

[0666] The humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 prepared in Example 9.1 were used for cytotoxic CD8 + T cell killing assay. Cytotoxic CD8 + The protocol for the T cell killing assay is as follows:

[0667] (a) Isolation of CD3 from PBMC + T cells.

[0668] (b) 5x10 3 Target cells (THP-1 labeled with CFSE) were added to the wells of a 96-well U-bottom plate.

[0669] (c) 1x10 4 T cells were added to the wells (20:1 effector:target ratio).

[0670] (d) Target cells and T cells were mixed to a final concentration of 20 μg / ml.

[0671] (e) The cells were incubated for 20 hours and incubated at 37°C, 5% CO2 for 18 hours.

[0672] (f) Finally, the cells were washed and resuspended in 200 μL of staining buffer. The cells were acquired by FACS and the percentage of CFSE-labeled THP-1 cells was measured.

[0673] The percentage of cytotoxicity was calculated according to the following equation:

[0674]

[0675] (AT: percentage of CFSE-labeled THP-1 cells in the antibody-treated groups;

[0676] (NAT: Percentage of CFSE-labeled THP-1 cells in the negative control group)

[0677] like Fig.12 As shown, humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 enhanced the expression of CD8 + T cell-mediated cytotoxicity.

[0678] 9.6. Antibody Characterization: ADCC and ADCP Assays for Humanized Antibodies

[0679] The humanized antibody h2-H1-D7-E5-D5 prepared in Example 9.1 was used for ADCC assay and ADCP assay. The ADCC assay protocol was similar to that described in Example 3.1, and the ADCP assay protocol was similar to that described in Example 4.1, except that different test antibodies were used.

[0680] like Fig.13 As shown in AB, compared with the benchmark antibody IO-202, the humanized antibody h2-H1-D7-E5-D5 showed significantly effective ADCC and ADCC effects and lower EC against THP-1 cells 50 .

[0681] 9.7. Characterization of Antibodies for Treatment of EL4-LILRB4 Lymphoma in Female hLILRB1 / hLILRB4 Transgenic Mice In vivo efficacy testing of humanized antibodies

[0682] The humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 prepared in Example 9.1 were used in an in vivo efficacy test for treating EL4-LILRB4 lymphoma in female hLILRB1 / hLILRB4 transgenic mice. IO-202 was used as a positive control, and hIgG1 was used as a negative control.

[0683] The protocol for in vivo efficacy assays in the EL4-LILRB4 lymphoma model was similar to that described in Example 8.1.2, except that humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 were used to test antibodies.

[0684] like Fig.14 As shown in AB, compared with IO-202, humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 showed effective in vivo therapeutic effects, and h2-H1-D7-E5-D5 had the strongest in vivo therapeutic effect. Fig.14 As shown in A, 3 mg / kg h2-H1-D7-E5-D5 and 3 mg / kg h8-B3-F6-H8 significantly inhibited EL4-LILRB4 tumor growth in vivo, with TGIs of 77% and 60%, respectively, while the benchmark antibody IO-202 exhibited a 53% TGI in the EL4-LILRB4 syngeneic model. Fig.14 As shown in B, the body weight of mice in each group did not change significantly.

Claims

1. An antibody or antigen-binding fragment thereof that binds to LILRB4, the antibody or antigen-binding fragment thereof comprising: one, two or three heavy chain complementarity determining regions (HCDR1, HCDR2 and / or HCDR3), wherein the one, two or three heavy chain complementarity determining regions are contained within any one of the heavy chain variable (VH) region sequences selected from the group consisting of: SEQ ID NO: 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 154 and 156; and / or One, two or three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3), wherein the one, two or three light chain complementary determining regions are contained within any one of the light chain variable (VL) region sequences selected from the group consisting of: SEQ ID NO: 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 155 and 157.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment comprises at least one heavy chain or light chain complementary determining region (CDR), wherein the CDR comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 33, 34, 35, 36, 37, 38, 41, 42, 43, 44, 45, 46, 49, 50, 51, 52, 53, 54, 57, 58, 59, 60, 61, 62, 65, 66, 67, 68, 69, 70, 73, 74, 75, 76, 77, 78, 81, 82, 83, 84, 85, 86, 89, 90, 91, 92 , 93, 94, 97, 98, 99, 100, 101, 102, 105, 106, 107, 108, 109, 110, 113, 114, 115, 116, 117, 118, 121, 122, 123, 124, 125, 126, 129, 130, 131, 132, 133, 134, 137, 138, 139, 140, 141, 142, 145, 146, 147, 148, 149, 150 and 158.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising one or two or three of HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, 25, 26, 27, 33, 34, 35, 41, 42, 43, 49, 50, 51, 57, 58, 59, 65, 66, 67, 73, 74, 75, 81, 82, 83, 89, 90, 91, 97, 98, 99, 105, 106, 107, 113, 114, 115, 121, 122, 123, 129, 130, 131, 137, 138, 139, 145, 146, 147 and 158.

4. The antibody or antigen-binding fragment thereof of any of the preceding claims, comprising one or two or three of LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 21, 22, 28, 29, 30, 36, 37, 38, 44, 45, 46, 52, 53, 54, 60, 61, 62, 68, 69, 70, 76, 77, 78, 84, 85, 86, 92, 93, 94, 100, 101, 102, 108, 109, 110, 116, 117, 118, 124, 125, 126, 132, 133, 134, 140, 141, 142, 148, 149 and 150.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: i. HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137 and 145; ii. a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, 146 and 158; and iii. HCDR3, said HCDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139 and 147.

6. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: i. LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132, 140 and 148; ii. LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141 and 149; and iii. LCDR3, said LCDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134, 142 and 150.

7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: i. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 19; ii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 27; iii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 33, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 34, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 35; iv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:42, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43; v. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:158, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43; vi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:50, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:51; vii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 57, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 58, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 59; viii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 65, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 66, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 67; ix. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 73, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 74, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 75; x. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:81, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:82, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:83; xi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:89, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:90, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:91; xii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 97, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 98, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 99; xiii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 106, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 107; xiv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 114, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 115; xv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 122, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 123; xvi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 130, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 131; xvii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 138, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 139; or xviii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 145, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 146, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:

147.

8. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: i. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 20, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 22; ii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:28, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:29, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:30; iii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:36, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:37, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:38; iv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:44, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:45, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:46; v. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:52, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:53, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:54; vi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:60, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:61, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:62; vii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:68, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:69, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:70; viii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 76, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 77, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 78; ix. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:84, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:85, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:86; x. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:92, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:93, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:94; xi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 100, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 101, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 102; xii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 108, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 109, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 110; xiii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 116, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 118; xiv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 124, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 125, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 126; xv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 132, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 133, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 134; xvi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 140, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 141, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 142; or xvii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:148, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:149, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:

150.

9. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 18, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 19, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 20, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 21, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 22; ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:25, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:26, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:27, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:28, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:29, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:30; iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:33, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:34, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:35, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:36, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:37, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:38; iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:42, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:44, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:45, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:46; v. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:158, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:43, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:44, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:45, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:46; vi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:50, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:51, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:52, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:53, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:54; vii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:57, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:58, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:59, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:60, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:61, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:62; viii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:65, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:66, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:67, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:68, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:69, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:70; ix. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:73, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:74, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:75, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:76, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:77, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:78; x. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:81, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:82, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:83, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:84, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:85, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:86; xi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:89, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:90, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:91, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:92, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:93, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:94; xii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:97, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:98, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:99, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:100, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:101, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:102; xiii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 106, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 107, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 108, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 109, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 110; xiv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 114, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 115, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 116, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 117, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 118; xv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 122, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 123, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 124, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 125, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 126; xvi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 130, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 131, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 132, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 133, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 134; xvii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 138, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 139, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 140, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 141, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 142; or xviii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:145, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:146, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:147, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:148, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:149, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:

150.

10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising a VH region having an amino acid sequence as shown in SEQ ID NO: 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 154 or 156, or a homologous sequence thereof having at least 80% sequence identity thereto.

11. An antibody or antigen-binding fragment thereof according to any of the preceding claims, comprising a VL region having an amino acid sequence as shown in SEQ ID NO: 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 155 or 157, or a homologous sequence thereof having at least 80% sequence identity with SEQ ID NO: 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 155 or 157.

12. An antibody or antigen-binding fragment thereof according to any of the preceding claims, comprising a VH / VL amino acid sequence pair selected from the group consisting of: SEQ ID NO: 23 / 24, 31 / 32, 39 / 40, 47 / 48, 55 / 56, 63 / 64, 71 / 72, 79 / 80, 87 / 88, 95 / 96, 103 / 104, 111 / 112, 119 / 120, 127 / 128, 135 / 136, 143 / 144, 151 / 152, 154 / 155 and 156 / 157.

13. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, further comprising one or more amino acid residue substitutions or modifications, while retaining binding affinity to LILRB4.

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein at least one of the substitutions or modifications is located in one or more CDR sequences in the CDR sequences of the VH region or the VL region.

15. The antibody or antigen-binding fragment thereof according to claim 13, wherein at least one of the substitutions or modifications is located in one or more of the non-CDR sequences of the VH region or the VL region.

16. The antibody or antigen-binding fragment of the preceding claim, further comprising one or more non-natural amino acid (NNAA) substitutions.

17. The antibody or antigen-binding fragment of claim 16, wherein the NNAA is capable of being conjugated.

18. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which has one or more binding properties for LILRB4 selected from the group consisting of: i. capable of specifically binding to human LILRB4 as measured by FACS assay; ii. capable of specifically binding to human LILRB4 and human LILRB3 as measured by FACS assay; iii. capable of specifically binding to human LILRB4 and cynomolgus monkey LILRB4 as measured by FACS assay; iv. Has effective ADCP effect on human AML cell lines; v. Has effective ADCC effect on human AML cell lines; vi. being able to block LILRB4-fibronectin interaction to reprogram tolerogenic dendritic cells into mature dendritic cells that stimulate T cell activation as measured by FcγR stimulation assay; vii. Able to induce TNF-α production; viii. Ability to reprogram tolerant DCs to activate T cells; ix. Ability to reverse macrophage-mediated T cell suppression; x. Ability to reverse THP-1-mediated T cell suppression; and xi.Can enhance CD8 + T cell-mediated cytotoxicity against THP-1 cells.

19. An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof according to any one of the preceding claims for binding to LILRB4.

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

21. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a labeled antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein.

22. The antibody or antigen-binding fragment thereof according to any of the preceding claims, which is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), a scFv dimer (bivalent diabody), a camelized single domain antibody, a nanobody, a domain antibody or a bivalent domain antibody.

23. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, further comprising an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG.

24. The antibody or antigen-binding fragment thereof according to claim 23, wherein the Fc region is derived from human IgG1, IgG2, IgG3 or IgG4.

25. The antibody or antigen-binding fragment of claim 24, wherein the Fc region comprises the amino acid sequence shown in SEQ ID NO:

153.

26. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the light chain is a lambda light chain or a kappa light chain.

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

28. The antibody or antigen-binding fragment thereof according to claim 27, which is capable of binding to one or more additional antigens in addition to LILRB4, or specifically binds to a second epitope on LILRB4.

29. The antibody or antigen-binding fragment thereof of claim 28, wherein the one or more additional antigens other than LILRB4 are selected from the group consisting of CD3, CD16a, CD33, CD38, CD45, CD123, CD146, CD228, CLL-1, Flt3, TAF1, TgPRF, HVCN1, IL-6R, IL-11R, IL17A, IL-23R, IL-33, ILDR2, LAP, TSLP, TREM-1, ANGPT2, APOE, IFNAR, CypA, DOG-1, NKp30, CSF-1R, CCR2, LRRC15, Mesothelin, Dickkopf2, DLL3, HER-2, C10orf54, TrkA, MEKK1, KRAS, ERK, XPO1, mTORC1 / 2, PAK4, NAMPT, ATR, EGFR, FGFR, VEGF, c-MET, Her2, Her3, CTLA4 , GITA, CD112R, CD2, CD7, CD16, CD19, CD20, CD24, CD27, CD30, CD34, CD37, CD39, CD70, CD73, CD83, CD28, CD80(B7-1), CD86(B7-2), CD40, CD40L( CD154), CD47, SIRPα, CD122, CD137, CD137L, OX40(CD134), OX40L(CD252), BCMA (e.g., BCMA02), PSMA, CLDN18 (e.g., CLDN18.2), NKG2C, 4-1BB, LIG HT, PVRIG, SLAMF7, HVEM, BAFFR, ICAM-1, 2B4, LFA-1, GITR, ICOS(CD278), ICOSLG(CD275), LAG3(CD223), A2AR, B7-H3(CD276), B7-H4(VTCN1), B 7-H5, BTLA(CD272), BTLA, CD160, CTLA-4(CD152), GPRC5D, IDO1, IDO2, ILT3, TDO, KIR, LAIR-1, NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC -7(CD328), SIGLEC-9(CD329), SIGLEC-15, TIGIT, PVR(CD155), LILRB2, LILRB3, FLT3, FLT3L, TLR3, CLEC9A, DEC-205, STING, IL-12, IDO and TGFβ.

30. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, linked to one or more conjugate moieties.

31. The antibody or antigen-binding fragment thereof of claim 30, wherein the conjugate moiety comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a detectable label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a purification moiety or other anti-cancer drug.

32. The antibody or antigen-binding fragment thereof of claim 30 or 31, wherein the conjugate moiety is covalently linked directly or via a linker.

33. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of the preceding claims and one or more pharmaceutically acceptable carriers.

34. A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, a transmembrane region and an intracellular signaling region.

35. The chimeric antigen receptor of claim 34, wherein the transmembrane region comprises the transmembrane region of CD3, CD4, CD8 or CD28.

36. The chimeric antigen receptor of claim 34, wherein the intracellular signaling region is selected from the group consisting of: intracellular signaling region sequences of CD3, FcγRI, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR, or a combination thereof.

37. The chimeric antigen receptor of any one of claims 34 to 36, wherein the antigen binding fragment is a scFv.

38. The chimeric antigen receptor of any one of claims 34 to 37, wherein the chimeric antigen receptor is transplanted onto allogeneic cells, autologous cells, or xenogeneic cells.

39. The chimeric antigen receptor of any one of claims 34 to 38, wherein the chimeric antigen receptor is transplanted onto an immune effector cell.

40. The chimeric antigen receptor of any one of claims 34 to 39, wherein the chimeric antigen receptor is transplanted onto a T cell, a natural killer cell, a macrophage, or a tumor infiltrating lymphocyte.

41. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 29 and / or the chimeric antigen receptor according to any one of claims 34 to 40.

42. A vector comprising the isolated polynucleotide according to claim 41.

43. A host expression system comprising the vector according to claim 42 or having the polynucleotide according to claim 41 integrated into its genome.

44. The host expression system of claim 43, which is a microorganism, a yeast or a mammalian cell, optionally wherein the microorganism is selected from the group consisting of E. coli and B. subtilis, wherein the yeast is Saccharomyces, and wherein the mammalian cell is selected from the group consisting of COS, CHO-S, CHO-K1, HEK-293 and 3T3 cells.

45. A virus comprising the vector according to claim 42.

46. ​​A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or the pharmaceutical composition according to claim 33 and / or the chimeric antigen receptor according to any one of claims 34 to 40 and a second therapeutic agent.

47. A method for expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 29 or the chimeric antigen receptor according to any one of claims 34 to 40, the method comprising culturing the host expression system according to claim 43 under conditions where the antibody or antigen-binding fragment thereof according to any one of claims 1 to 29 or the chimeric antigen receptor according to any one of claims 34 to 40 is expressed.

48. A method for treating, preventing or ameliorating a disease, disorder or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or the pharmaceutical composition according to claim 33 and / or the chimeric antigen receptor according to any one of claims 34 to 40.

49. The method of claim 48, wherein the disease, disorder or condition is an immune disease, an inflammatory disease, cancer or a neurological disease.

50. The method of claim 49, wherein the cancer is a solid tumor or a hematological tumor.

51. The method of any one of claims 48 to 50, wherein the disease, disorder or condition is selected from the group consisting of Kawasaki disease, T. gondii, multiple sclerosis, systemic Lupus erythematosus, lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lung adenocarcinoma, lung squamous cell carcinoma, Lewis lung carcinoma or radiation therapy resistant Lewis lung carcinoma), peritoneal cancer, carcinoid, bone cancer, pancreatic cancer, primitive neuroectodermal tumor, skin cancer, gallbladder cancer, head and neck cancer, squamous cell carcinoma, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial carcinoma), anal region cancer (e.g., anal squamous cell carcinoma), gastric or stomach cancer. cancer (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatoma or hepatocellular carcinoma), bile duct cancer, sarcoma, colorectal cancer, fallopian tube cancer, salivary gland cancer, cervical cancer, endometrial cancer or uterine cancer, osteosarcoma, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, nasopharyngeal cancer, soft tissue sarcoma, polycythemia vera, urethral cancer, penile cancer, kidney or urethral cancer (e.g., renal rhabdoid tumor), cutaneous T-cell lymphoma, medulloblastoma, nephroblastoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, choroid plexus papilloma, renal cell carcinoma, renal pelvic cancer, central nervous system (CNS) neoplasms, soft tissue sarcomas (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi's sarcoma (Kaposi's sarcoma), spinal cord tumors, gliomas (e.g., ependymomas, astrocytomas, anaplastic astrocytomas, oligodendrogliomas), eye cancers (e.g., retinoblastomas), brain stem gliomas, or mixed gliomas such as oligoastrocytomas), brain tumors (e.g., glioblastoma / glioblastoma multiforme (GBM), non-glioblastoma brain tumors, or meningiomas), melanomas (e.g., cutaneous or intraocular melanomas), thrombocytosis, mesothelioma, mycosis fungoides, Sezary syndrome, primary myelofibrosis, solitary plasmacytoma, vestibular schwannoma, Ewing's sarcoma sarcoma), chondrosarcoma, MYH-associated polyposis, pituitary adenomas, pediatric cancers, such as pediatric sarcomas (e.g., neuroblastoma, rhabdomyosarcoma, and osteosarcoma), blood cancers, lymphomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemias (e.g.,lymphocytic / lymphoblastic leukemia), chronic or acute leukemia, mast cell leukemia, lymphocytic lymphoma, primary CNS lymphoma, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia (HCL), Burkitt's lymphoma (Burkitt's lymphoma, BL), multiple myeloma (e.g., relapsed or refractory multiple myeloma), T or B cell lymphoma, mantle cell lymphoma (MCL) (e.g., relapsed or refractory mantle cell lymphoma), malignant melanoma, diffuse large B cell lymphoma (DLBCL), DLBCL arising from follicular lymphoma, high-grade B cell lymphoma, primary mediastinal large B cell lymphoma, follicular lymphoma (FL), and primary mediastinal B cell lymphoma. , 52. The method of any one of claims 48 to 51, wherein the subject is a human.

53. The method of any one of claims 48 to 52, wherein the administration is by: a parenteral route, including subcutaneous, intraperitoneal, intravenous, intramuscular or intradermal injection; or a non-parenteral route, including transdermal, oral, intranasal, intraocular, sublingual, rectal or topical.

54. The method of any one of claims 48 to 53, wherein the method further comprises administering an additional therapeutic agent to a subject in need thereof.

55. The method of claim 54, wherein the additional therapeutic agent is selected from the group consisting of active agents, imaging agents, cytotoxic agents and angiogenesis inhibitors, kinase inhibitors, co-stimulatory molecule agonists, co-inhibitory molecule blockers, adhesion molecule blockers, anti-cytokine antibodies or functional fragments thereof, detectable markers or reporter genes, antimicrobial agents, gene editing agents, beta agonists, viral RNA inhibitors, polymerase inhibitors, interferons, and microRNAs.

56. The method of claim 54, wherein the additional therapeutic agent is administered to the subject in need thereof before, after, or concurrently with administration of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or the pharmaceutical composition according to claim 33 and / or the chimeric antigen receptor according to any one of claims 34 to 40.

57. A method for modulating LILRB4 activity in a LILRB4-expressing cell, the method comprising exposing the LILRB4-expressing cell to an antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or a pharmaceutical composition according to claim 33 and / or a chimeric antigen receptor according to any one of claims 34 to 40.

58. The method of claim 57, wherein the LILRB4-expressing cell is a dendritic cell, a monocyte, a macrophage, a B cell, a Treg, a promast cell, an endothelial cell, or an osteoclast.

59. A method of inducing phagocytosis of a target cell in vivo or in vitro, the method comprising exposing the target cell to an antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or a pharmaceutical composition according to claim 33 and / or a chimeric antigen receptor according to any one of claims 34 to 40.

60. The method of claim 59, wherein the target cell is an antigen presenting cell, a cancer cell, or a cell infected with a pathogen.

61. A method of inducing TNF-α production, the method comprising exposing tolerogenic dendritic cells to an antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or a pharmaceutical composition according to claim 33 and / or a chimeric antigen receptor according to any one of claims 34 to 40.

62. A method of reprogramming tolerant dendritic cells into mature dendritic cells, the method comprising exposing the tolerant dendritic cells to an antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or a pharmaceutical composition according to claim 33 and / or a chimeric antigen receptor according to any one of claims 34 to 40.

63. A method for detecting the presence or amount of LILRB4 in a sample, the method comprising: contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or the pharmaceutical composition according to claim 33 and / or the chimeric antigen receptor according to any one of claims 34 to 40; and determining the presence or the amount of LILRB4 in the sample.

64. A method of diagnosing a LILRB4-associated disease or condition in a subject, the method comprising: a) obtaining a sample from the subject; b) contacting the sample obtained from the subject with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or the pharmaceutical composition according to claim 33; c) determining the presence or amount of LILRB4 in the sample; and d) correlating the presence or the amount of LILRB4 with the presence or status of the LILRB4-associated disease or condition in the subject.

65. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or a pharmaceutical composition according to claim 33 and / or a chimeric antigen receptor according to any one of claims 34 to 40 in the preparation of a medicament for treating a LILRB4-related disease, disorder or condition in a subject.

66. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or a pharmaceutical composition according to claim 33 and / or a chimeric antigen receptor according to any one of claims 34 to 40 in the preparation of a diagnostic reagent for diagnosing a LILRB4-related disease, disorder or condition.

67. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 and / or the pharmaceutical composition according to claim 33 and / or the chimeric antigen receptor according to any one of claims 34 to 40, which can be used to detect LILRB4, optionally recombinant LILRB4, LILRB4 expressed on the cell surface or LILRB4-expressing cells.

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