Novel anti-LILRB4 antibody and its use

Abstract

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

Description

Technical Field

[0001] The present disclosure generally relates to novel anti-LILRB4 antibodies and antigen-binding fragments thereof and their use.

Background Art

[0002] Acute myeloid leukemia (AML) is one of the most common acute leukemias in adults. To effectively treat AML, new molecular targets and treatment approaches need to be identified. Leukocyte immunoglobulin-like receptors (LILRs) are a family of at least 13 receptors mainly expressed by lymphocytes and myelomonocytic cells (Rachel Thomas et al., Clin Rev Allergy Immunol., 2010 April; 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 in myeloid cells and other specific hematopoietic cells (Mori et al., J Immunol., 2008 Oct 1; 181(7):4742~51). Some members of the LILRB family are highly expressed in AML cells, and their expression shows a negative correlation with the overall survival of human AML patients. In addition, inhibition of the expression of some LILRBs individually inhibited different human leukemia cell lines in culture and blocked the development of leukemia in xenograft mice (see WO2013181438A2).

[0003] Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) is a protein encoded by the LILRB4 gene in humans. This encoded protein belongs to the subfamily B class of LILRs and contains two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LILRB4 is expressed in monocytic cells and transmits negative signals that inhibit the stimulation of immune responses. Additionally, LILRB4 functions in antigen capture and presentation. It is thought to help control inflammatory responses and cytotoxicity, focus immune responses, and limit autoreactivity. LILRB4 has also been proposed as a potential target for tumor immunotherapy. It has been shown to be expressed in tumor-associated macrophages and negatively regulate immune responses in tumors. Expression of LILRB4 in monocytic myeloid leukemia cells supports infiltration and inhibits T cell proliferation. IO-202, developed by Immune-Onc Therapeutics, is in a Phase I clinical trial for the treatment of AML and chronic myelomonocytic leukemia (CMML).

[0004] There is still a need for novel anti-LILRB4 antibodies.

Summary of the Invention

[0005] Throughout this disclosure, the articles "a," "an," and "the" refer to one or more than one (i.e., at least one) of the grammatical objects of the article in the specification. By way of 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 and comprises one or two or three heavy chain complementarity determining regions (HCDR1, HCDR2, and / or HCDR3) contained within any one of the heavy chain variable (VH) region sequences selected from the group consisting of SEQ ID NOs: 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 154, and 156; and / or one or two or three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable (VL) region sequences selected from the group consisting of SEQ ID NOs: 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 155, and 157 and provides an antibody or an antigen-binding fragment thereof comprising the same.

[0007] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises at least one heavy chain or light chain complementarity determining region (CDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises one or two or three of HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises one or two or three of LCDR1, LCDR2, and LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof provided herein is: i. HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, and 145; ii. HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, 146, and 158; and iii. HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139, and 147 and comprises.

[0011] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are: i. an LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132, 140, and 148; ii. an LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, and 149; and iii. an LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134, 142, and 150 and comprising.

[0012] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are: i. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19; ii. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; iii. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35; iv. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; v. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 158, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; vi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51; vii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 57, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59; viii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 67; ix. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75; x. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83; xi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91; xii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99; xiii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107; xiv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115; xv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123; xvi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 129, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 130, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 131; xvii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139; or xviii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 146, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 147 comprising.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is: i. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; iii. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; iv. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; v. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54; vi. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 60, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 61, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 62; vii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 68, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 69, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 70; viii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 76, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 77, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 78; ix. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 84, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 85, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 86; x. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 92, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 93, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 94; xi. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 100, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 101, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 102; xii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 108, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 109, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 110; xiii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 116, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 117, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 118; xiv. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 124, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 125, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 126; xv. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 132, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 133, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 134; xvi. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 140, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 141, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 142; or LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150 comprising.

[0014] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are: i. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; iii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; iv. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; v. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 158, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; vi. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54; vii. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 57, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 60, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 61, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 62; viii. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 67, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70; ix. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78; x. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 86; xi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 92, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 93, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94; xii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102; xiii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110; xiv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118; xv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 124, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 125, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 126; xvi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 129, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 130, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 131, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 132, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 133, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 134; xvii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 140, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 141, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 142; or xviii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 146, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 147, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150 is included.

[0015] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a VH region having the amino acid sequence set forth 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 with SEQ ID NO: 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 154 or 156.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a VL region having the amino acid sequence 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 with SEQ ID NO: 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 155, or 157.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises 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.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises one or more amino acid residue substitutions or modifications and still retains the binding affinity for LILRB4. In some embodiments, at least one of the substitutions or modifications is present in one or more of the CDR sequences of the VH or VL region. In some embodiments, at least one of the substitutions or modifications is present in one or more of the non-CDR sequences of the VH or VL region. In some embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises one or more non-natural amino acid (NNAA) substitutions. In some embodiments, it is possible to conjugate the NNAA.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof provided herein has one or more binding properties for LILRB4 selected from the group consisting of: i. When measured by FACS assay, it can specifically bind to human LILRB4; ii. When measured by FACS assay, it can specifically bind to human LILRB4 and human LILRB3; iii. When measured by FACS assay, it can specifically bind to human LILRB4 and cynomolgus monkey LILRB4; iv. It has a strong ADCP effect on human AML cell lines; v. It has a strong ADCC effect on human AML cell lines; vi. It can reprogram tolerogenic dendritic cells into mature dendritic cells that stimulate T cell activation when measured by FcγR stimulation assay by blocking the LILRB4 - fibronectin interaction; vii. It can induce the production of TNF - α; viii. It can reprogram tolerogenic DCs to activate T cells; ix. It can reverse macrophage - mediated T cell suppression; x. It can reverse THP - 1 - mediated T cell suppression; and xi. It can enhance CD8 + T cell - mediated cytotoxicity against THP - 1 cells.

[0020] In another aspect, the disclosure provides an antibody or an antigen - binding fragment thereof that competes with the above - described antibody or its antigen - binding fragment with respect to binding to LILRB4.

[0021] In some embodiments, the antibody or its antigen - binding fragment provided herein is a chimeric, humanized, or human antibody or its antigen - binding fragment.

[0022] In some embodiments, the antibody or its antigen - binding fragment provided herein is a labeled antibody, a bivalent antibody, an anti - idiotype antibody, or a fusion protein.

[0023] 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 (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), camelized single-domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0024] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise an Fc region. In some embodiments, the Fc region is the Fc region of a human immunoglobulin (Ig). In some embodiments, the Fc region is the Fc region of human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 153.

[0025] In some embodiments, the light chain of the antibodies or antigen-binding fragments thereof provided herein is a λ light chain or a κ light chain.

[0026] 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, one or more additional antigens other than LILRB4 are selected from the group consisting of CD3, CD16a, LILRB43, LILRB48, 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, LIGHT, PVRIG, 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.

[0027] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are linked to one or more conjugate moieties. In some embodiments, the conjugate moieties include clearance modifiers, chemotherapeutic agents, toxins, radioisotopes, lanthanides, detectable labels, DNA alkylating agents, topoisomerase inhibitors, tubulin binders, purification moieties, or other anti-cancer agents. In some embodiments, the conjugate moieties are covalently attached either directly or via a linker.

[0028] In another aspect, the disclosure provides a chimeric antigen receptor comprising an antibody or antigen-binding fragment thereof provided herein, a transmembrane region, and an intracellular signaling region. In some embodiments, the transmembrane region comprises the transmembrane region of CD3, CD4, CD8, or CD28. In some embodiments, the intracellular signaling region is selected from the group consisting of the intracellular signaling region sequences of CD3, FcγRI, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR, or combinations thereof. In some embodiments, the antigen-binding fragment of the chimeric antigen receptor is a scFv. In some embodiments, the chimeric antigen receptor is grafted onto allogeneic cells, autologous cells, or heterologous cells. In some embodiments, the chimeric antigen receptor is grafted onto immune effector cells. In some embodiments, the chimeric antigen receptor is grafted onto T cells, natural killer cells, macrophage cells, or tumor-infiltrating lymphocytes.

[0029] In another aspect, the disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the disclosure, and / or a chimeric antigen receptor, and one or more pharmaceutically acceptable carriers.

[0030] In another aspect, the disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof of the disclosure, and / or a chimeric antigen receptor.

[0031] In another aspect, the disclosure provides a vector comprising the isolated polynucleotide of the disclosure.

[0032] In another aspect, the present disclosure provides a host expression system that includes the vector of the present disclosure or in which the polynucleotide of the present disclosure is integrated into its genome. In some embodiments, the host expression system of the present disclosure is a microorganism, yeast, or 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.

[0033] In another aspect, the present disclosure provides a virus that includes the vector of the present disclosure.

[0034] In another aspect, the present disclosure provides a kit that includes the antibody of the present disclosure or an antigen-binding fragment thereof, and / or the chimeric antigen receptor of the present disclosure, and / or the pharmaceutical composition of the present disclosure, and a second therapeutic agent.

[0035] In another aspect, the present disclosure provides a method for expressing the antibody of the present disclosure or an antigen-binding fragment thereof, and / or the chimeric antigen receptor of the present disclosure, the method including culturing the host expression system of the present disclosure under conditions in which the antibody of the present disclosure or an antigen-binding fragment thereof or the chimeric antigen receptor of the present disclosure is expressed.

[0036] In another aspect, the present disclosure provides a method for treating, preventing, or alleviating a disease, disorder, or condition in a subject, the method including administering to the subject a therapeutically effective amount of the antibody of the present disclosure or an antigen-binding fragment thereof, and / or the chimeric antigen receptor, and / or the pharmaceutical composition.

[0037] In another aspect, the present disclosure provides the use of the antibody of the present disclosure or an antigen-binding fragment thereof, and / or the chimeric antigen receptor, and / or the pharmaceutical composition in the manufacture of a medicament for treating an LILRB4-related disease, disorder, or condition in a subject.

[0038] In another aspect, the present disclosure provides for the use of an antibody or antigen-binding fragment thereof, and / or a chimeric antigen receptor, and / or a pharmaceutical composition of the present disclosure in the manufacture of a diagnostic reagent for diagnosing LILRB4-related diseases, disorders, or conditions.

[0039] In some embodiments, the disease, disorder, or condition is an immune disease, an inflammatory disease, 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 an LILRB4-expressing B cell cancer. In some embodiments, the disease, disorder, or condition is 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 cancer, or radiation-resistant Lewis lung cancer), abdominal cancer, carcinoid cancer, bone cancer, pancreatic cancer, undifferentiated neuroectodermal tumor, skin cancer, gallbladder cancer, head or neck cancer, squamous cell carcinoma, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial cancer), anal region cancer (e.g., anal squamous cell carcinoma), gastric cancer [gastric cancer] or gastric cancer [stomach cancer] (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatic tumor, or liver cancer), cholangiocarcinoma, 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 ureteral cancer (e.g., renal oncocytoma), cutaneous T cell lymphoma, medulloblastoma, nephroblastoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, choroid plexus papilloma, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) neoplasm, soft tissue sarcoma (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi sarcoma), spinal cord axis tumor, glioma (e.g., ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, eye cancer (e.g., retinoblastoma), brainstem glioma, or mixed glioma, e.g., oligoastrocytoma), brain tumor (e.g., glioblastoma / multiform glioblastoma (GBM), non-glioblastoma brain tumor, or meningioma), melanoma (e.g., skin or intraocular melanoma), thrombocytosis, mesothelioma, fungating polyposis, Sézary syndrome, idiopathic myelofibrosis, solitary plasmacytoma, vestibular schwannoma, Ewing sarcoma, chondrosarcoma, MYH-related polyposis, pituitary adenoma, pediatric cancer, e.g., pediatric sarcoma (e.g., neuroblastoma, rhabdomyosarcoma, and osteosarcoma), hematological cancer, lymphoma, Hodgkin lymphoma, non-Hodgkin 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 myelogenous leukemia (CML), acute myelogenous leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic lymphoblastic leukemia, acute lymphoblastic leukemia, hairy cell leukemia (HCL), Burkitt 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, and is selected from the group consisting of. In some embodiments, the disease, disorder, or condition is acute myelogenous leukemia. In some embodiments, the disease, disorder, or condition is chronic myelomonocytic leukemia.,

[0040] In some embodiments, the subject is human.

[0041] In some embodiments, administration is via a parenteral route including subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection; or via a non-parenteral route including transdermal, oral, intranasal, intraocular, sublingual, rectal, or topical surface.

[0042] In some embodiments, a method of treating, preventing, or alleviating a disease, disorder, or condition in 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 an active agent, a contrast agent, a cytotoxic agent, and an angiogenesis inhibitor, a kinase inhibitor, a costimulatory molecule agonist, a coinhibitory molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or a functional fragment thereof, a detectable label or reporter, an antibacterial agent, a gene editing agent, a beta agonist, a viral RNA inhibitor, a polymerase inhibitor, an interferon, and a microRNA. In some embodiments, the additional therapeutic agent is administered to a subject in need thereof before, after, or simultaneously with the antibody or an antigen-binding fragment thereof, and / or a pharmaceutical composition, and / or a chimeric antigen receptor of the present disclosure.

[0043] In another aspect, the present disclosure provides a method of modulating LILRB4 activity in LILRB4-expressing cells, the method comprising exposing the LILRB4-expressing cells to the antibody or an antigen-binding fragment thereof, and / or a pharmaceutical composition, and / or a chimeric antigen receptor of the present disclosure.

[0044] In some embodiments, the LILRB4-expressing cells are dendritic cells, monocytes, macrophages, B cells, Tregs, progenitor mast cells, endothelial cells, or osteoclasts.

[0045] In another aspect, the present disclosure provides a method of inducing phagocytosis of target cells in vivo or in vitro, the method comprising exposing the target cells to the antibody or an antigen-binding fragment thereof, and / or a pharmaceutical composition, and / or a chimeric antigen receptor of the present disclosure. In some embodiments, the target cells are antigen-presenting cells, cancer cells, or cells infected with a pathogen.

[0046] In another aspect, the present disclosure provides a method of inducing the production of TNF-α, the method comprising exposing tolerogenic dendritic cells to the antibody or an antigen-binding fragment thereof, and / or a pharmaceutical composition, and / or a chimeric antigen receptor of the present disclosure.

[0047] 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 an antibody of the present disclosure or an antigen-binding fragment thereof, and / or a pharmaceutical composition, and / or a chimeric antigen receptor.

[0048] In another aspect, the present disclosure provides a method of detecting the presence or amount of LILRB4 in a sample, the method comprising contacting the sample with an antibody of the present disclosure or an antigen-binding fragment thereof, and / or a pharmaceutical composition, and / or a chimeric antigen receptor, and determining the presence or amount of LILRB4 in the sample.

[0049] In another aspect, the present disclosure provides a method of diagnosing an LILRB4-related disease or disorder in a subject, the method comprising: a) obtaining a sample from the subject; b) contacting the sample obtained from the subject with an antibody of the present disclosure or an antigen-binding fragment thereof, and / or a pharmaceutical composition, and / or a chimeric antigen receptor; c) determining the presence or amount of LILRB4 in the sample; and d) correlating the presence or amount of LILRB4 with the presence or condition of an LILRB4-related disease or condition in the subject.

[0050] In another aspect, the present disclosure provides a kit comprising an antibody of the present disclosure or an antigen-binding fragment thereof, and / or a pharmaceutical composition, and / or a chimeric antigen receptor, which is useful in the detection of LILRB4, optionally recombinant LILRB4, LILRB4 expressed on the cell surface, or LILRB4-expressing cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0051]

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Figure 14

[0052] The following description of the present disclosure is intended merely to illustrate various embodiments of the present disclosure. Therefore, the specific modifications considered should not be construed as limitations on 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 is understood that embodiments of such equivalents are included herein. All references cited herein, including publications, patents, and patent applications, are hereby incorporated by reference in their entirety.

[0053] Definitions As used herein, the term "antibody" includes any immunoglobulin that binds specifically to an antigen, monoclonal antibodies, polyclonal antibodies, multivalent antibodies, bivalent antibodies, monovalent antibodies, multispecific antibodies, or bispecific antibodies. A native intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, and each heavy chain comprises a variable region (VH), as well as first, second, third, and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are classified as lambda or kappa, and each light chain comprises a variable region (VL) and a constant region. An antibody has a "Y" shape, and the stem of the Y comprises the second and third constant regions of two heavy chains joined together via disulfide bonds. Each arm of the Y comprises the variable region of a single heavy chain and the first constant region bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are involved in antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, and heavy chain CDRs including HCDR1, HCDR2, HCDR3). The three CDRs are interposed between adjacent stretches known as framework regions (FRs) (light chain FRs including LFR1, LFR2, LFR3, and LFR4, and heavy chain FRs including HFR1, HFR2, HFR3, and HFR4) that are more highly conserved than the CDRs and form a scaffold structure that holds the highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are further classified into subclasses such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain).

[0054] In certain embodiments, the antibodies provided herein include any antigen-binding fragment thereof. As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody that contains 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 the intact 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 diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bispecific antibodies, multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies. An antigen-binding fragment is capable of binding to the same antigen or epitope to which the parent antibody binds.

[0055] By "Fab" with respect to an antibody is meant that portion of the antibody consisting of a single light chain (both variable and constant regions) linked by a disulfide bond to a single heavy chain variable region and the first constant region.

[0056] "Fab’" refers to a Fab fragment that includes a portion of the hinge region.

[0057] "F(ab’)2" refers to a dimer of Fab’.

[0058] By "Fc" with respect to an antibody (e.g., an antibody of the IgG, IgA, or IgD isotype) is meant that portion of the antibody consisting of the second and third constant domains of the first heavy chain linked via a disulfide bond to the second and third constant domains of the second heavy chain. For antibodies of the IgM and IgE isotypes, the Fc further includes a fourth constant domain. The Fc portion of an antibody is involved in various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0059] "Fv" with respect to an antibody refers to the smallest fragment of an antibody that has a complete antigen-binding site. The Fv fragment consists of a variable region of a single heavy chain bound to the variable region of a single light chain.

[0060] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light-chain variable region and a heavy-chain variable region connected via a linker (e.g., a peptide sequence) or directly to each other (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0061] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv connected to the Fc region of an antibody.

[0062] "Camelized single-domain antibody", "heavy-chain antibody", or "HCAb" refers to an antibody that contains two VH domains and no light chain (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; US Patent No. 6,005,079). Heavy-chain antibodies are originally derived from camelids (camels, dromedaries, and llamas). Although lacking a light chain, camelized antibodies have a genuine antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. et al., Immunogenetics. Apr;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 generated by the adaptive immune system (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0063] "Nanobody" refers to an antibody fragment consisting of a VHH domain from a heavy-chain antibody, as well as two constant domains, CH2 and CH3.

[0064] "Diabody" or "dAb" includes small antibody fragments having two antigen-binding sites, the fragments including a VH domain connected to a VL domain within the same polypeptide chain (VH-VL or VL-VH) (see, for example, Holliger P. et al., Proc Natl Acad Sci USA. Jul 15;90(14):6444-8(1993); European Patent No. 404097; WO93 / 11161). By using a linker that is too short for the two domains within the same chain to pair with each other, the domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0065] "Domain antibody" refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. In certain examples, two or more VH domains are covalently linked by a peptide linker to create a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.

[0066] As used herein, the term "valence" refers to the presence of a specified number of antigen-binding sites on a given molecule. The term "monovalent" refers to an antibody or antigen-binding fragment having only a single antigen-binding site; the term "multivalent" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites. Thus, the terms "bivalent", "tetravalent", and "hexavalent" indicate the presence of two, four, and six antigen-binding sites, respectively, on an antigen-binding molecule. In some embodiments, an antibody or its antigen-binding fragment is bivalent.

[0067] As used herein, a "bispecific" antibody refers to an engineered antibody having fragments derived from two different monoclonal antibodies and 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.

[0068] As used herein, a "multispecific" antibody refers to an antibody that specifically binds to at least two distinct antigens or at least two distinct epitopes within the same antigen. A multispecific antibody can bind, for example, to 2, 3, 4, 5, or more distinct antigens or distinct epitopes within the same antigen.

[0069] In certain embodiments, a "scFv dimer" is a bivalent diabody or bispecific scFv (BsFv) comprising a VH-VL (linked by a peptide linker) that forms a dimer with another VH-VL such that the VH of one moiety cooperates with the VL of the other moiety to form two binding sites capable of targeting the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, a "scFv dimer" is a bispecific diabody comprising VH1-VL2 (linked by a peptide linker) associated with VL1-VH2 (linked by a peptide linker) such that VH1 and VL1 cooperate and VH2 and VL2 cooperate and each cooperating pair has a different antigen specificity.

[0070] "dsFv" refers to a disulfide-stabilized Fv fragment in which the bond 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')", comprises three peptide chains: two VH moieties are linked by a peptide linker (e.g., a long flexible linker) and each is bound to two VL moieties via a disulfide bridge. In some embodiments, dsFv-dsFv' is bispecific in that the heavy and light chains paired by each disulfide have different antigen specificities.

[0071] As used herein, the term "chimeric" means an antibody or antigen-binding fragment having portions of a heavy and / or light chain from one species and the remaining heavy and / or light chain from a different species. In an exemplary example, a chimeric antibody can 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.

[0072] As used herein, the term "humanized" means that an antibody or antigen-binding fragment includes CDRs derived from a non-human animal, FR regions derived from a human, and optionally a constant region derived from a human. The CDRs of the humanized antibodies provided in the present disclosure may contain mutations (s) as compared to the CDRs of their parent antibodies.

[0073] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or an antigen-binding fragment thereof and an antigen.

[0074] An antibody or antigen-binding fragment thereof that "specifically binds" or "is specifically bound" to a target (e.g., an epitope) is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to "specifically bind" if it reacts or associates more frequently, more rapidly, for a longer period of time, and / or with greater affinity with a particular cell or substance than with alternative cells or substances. An antibody "specifically binds" to a target if it binds with greater affinity, avidity, more readily, and / or for a longer period of time 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, avidity, more readily, and / or for a longer period of time than it binds to other LILRB4 epitopes or non-LILRB4 epitopes. By reading this definition, it is also understood 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, "specifically bound" or "specifically binds" does not necessarily (although it may) require exclusive binding. Generally, although not necessarily, the reference to binding means specific binding.

[0075] The ability to "compete with respect to binding to LILRB4", as used herein, refers to the ability of a first antibody or antigen-binding fragment to inhibit the binding interaction between LILRB4 and a second anti-LILRB4 antibody to any detectable extent. In certain embodiments, an antibody or antigen-binding fragment that competes with respect to 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 can be greater than 95%, or greater than 99%.

[0076] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies can bind to the same or closely related epitopes within an antigen if they exhibit competitive binding with respect to the antigen. Epitopes can be linear or conformational (i.e., including spatially separated amino acid residues). For example, an antibody or antigen-binding fragment can be considered to bind to the same / closely related epitope as a reference antibody if it blocks the binding of the reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%.

[0077] As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group, along with a side chain specific to each amino acid. The names of amino acids are represented in the standard one-letter or three-letter notations in this disclosure, which are summarized below.

[0078]

Table A

[0079] "Conservative substitution" when referring to an amino acid sequence refers to the replacement of an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made among amino acid residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), amino acid residues having neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), amino acid residues having acidic side chains (e.g., Asp, Glu), amino acid residues having basic side chains (e.g., His, Lys, and Arg), or amino acid residues having aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not cause significant changes in the three-dimensional structure of a protein and can thus retain the biological activity of the protein.

[0080] As used herein, the term "identical" 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 with another sequence when optimally aligned.

[0081] "Percent (%) 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 percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) that are identical when compared to the reference sequence to which it is being compared, by the total number of amino acid residues (or bases) in the shorter of the candidate sequence or the reference sequence. Conservative substitutions of amino acid residues may or may not be considered as identical residues. Alignments for the purpose of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the U.S. National Center for Biotechnology Information (NCBI); see also Altschul S.F. 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 D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art may use the default parameters provided by the tools, or may generate, upon request, parameters that are appropriate for the alignment, for example, by selecting a suitable algorithm.

[0082] "Effector function", as used herein, refers to the biological activity resulting from 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 an antibody with C1q on the C1 complex; antibody-dependent cellular cytotoxicity (ADCC) mediated by the binding of the Fc region of an 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.

[0083] "Antibody-dependent cellular cytotoxicity" or "ADCC", as used herein, refers to a cell-mediated reaction in which effector cells expressing Fc receptors (FcR) recognize bound antibodies or antigen-binding fragments on target cells and subsequently cause lysis of the target cells. "ADCC activity" or "ADCC effect" refers to the ability of bound antibodies or antigen-binding fragments on target cells to induce an ADCC reaction as described above.

[0084] "Complement-dependent cytotoxicity" or "CDC", as used herein, refers to a mechanism by which an antibody can mediate specific target cell lysis through the activation of the complement system of an organism. In CDC, C1q binds to the antibody, this binding induces the complement cascade, resulting in the formation of a membrane attack complex (MAC) (C5b to C9) on the surface of the target cell as a result of classical pathway complement activation. "CDC activity" or "CDC effect" refers to the ability of bound antibodies or antigen-binding fragments on target cells to induce a CDC reaction as described above.

[0085] "Antibody-dependent cellular phagocytosis" or "ADCP", as used herein, refers to an immunological mechanism of elimination that targets tumor cells with a monoclonal antibody and promotes clearance from the body by phagocytic immune cells. The ADCP effect of an antibody can be measured by methods well known in the art, such as those described in Example 4 of the present disclosure.

[0086] As used herein, a "target cell" refers to a cell to which an antibody containing an Fc region specifically binds via a protein moiety that is generally C-terminal to the Fc region. An "effector cell" is a leukocyte that expresses one or more Fc receptors and performs an effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. Effector cells can be isolated from natural sources, such as blood or PBMCs, as is known in the art.

[0087] An "isolated" substance has been changed by human hand from its natural state. When an "isolated" composition or substance occurs naturally, it has been changed or removed from, or both, its original environment. For example, a polynucleotide or polypeptide that occurs naturally in a living animal is not "isolated," but is "isolated" if it is sufficiently separated from coexisting materials in its natural state to exist in a substantially pure state. 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 having a purity of 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% as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis, capillary electrophoresis) or chromatography methods (e.g., ion exchange chromatography, or reverse phase HPLC).

[0088] As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to effect expression of that protein. A vector can be used to transform, transduce, or transfect a host cell so as to effect expression of genetic elements carried within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. The category of animal viruses used as vectors includes retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and parvoviruses (e.g., SV40). A vector can contain various elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, selectable elements, and a reporter gene. In addition, a vector can contain an origin of replication. A vector can also include materials that assist its entry into cells, including but not limited to viral particles, liposomes, or protein coatings. A vector can be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence provided herein encoding an antibody or an antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, parvoviruses (e.g., SV40), lambda phage, and M13 phage, plasmid 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, p15TV-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.

[0089] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide and / or vector can be introduced or has been introduced.

[0090] As used herein, the term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and worms. Unless otherwise noted, the terms "patient", "subject", or "individual" are used interchangeably herein.

[0091] The term "anti-tumor activity" means a reduction in tumor cell proliferation, survival rate, or metastatic activity. For example, anti-tumor activity can be demonstrated by a decrease in the abnormal cell growth rate that occurs during treatment, or by the stability or reduction of tumor size, or by a longer survival due to treatment compared to a control that does not receive treatment. Such activity can be evaluated using accepted 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 for examining anti-tumor activity.

[0092] "Treating" or "treatment" of a disease, disorder, or condition, as used herein, includes preventing or alleviating the disease, disorder, or condition, slowing the rate of onset or occurrence of the disease, disorder, or condition, reducing the risk that the disease, disorder, or condition will occur, preventing or delaying the occurrence of symptoms associated with the disease, disorder, or condition, reducing or eliminating symptoms associated with the disease, disorder, or condition, producing a complete or partial regression of the disease, disorder, or condition, curing the disease, disorder, or condition, or any combination thereof.

[0093] The terms "diagnosing," "diagnosis," or "diagnosed" refer to the identification of a pathological condition, disease, or condition, such as the identification of a LILRB4-related disease, or the identification of a subject having a LILRB4-related disease from which benefit can be obtained from a particular treatment regimen. In some embodiments, the diagnosis includes the identification of an abnormal amount or activity of LILRB4. In some embodiments, the diagnosis refers to the identification of cancer in a subject.

[0094] As used herein, the terms "biological sample" or "sample" refer to a biological composition obtained from or derived from a subject of interest that contains cell entities and / or other molecular entities that are characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. 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 of skill 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 mucus and sputum), peritoneal fluid, pleural fluid, chest fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, abdominal fluid, ascites, or pericardial fluid. In some embodiments, the biological sample is tissue or cells obtained from the subject's stomach, heart, liver, spleen, lung, kidney, skin, or blood vessels.

[0095] As used herein, the term "LILRB4" refers to leukocyte immunoglobulin-like receptor subfamily B member 4, and includes any variant, conformation, isoform, and species homolog of LILRB4 that is naturally expressed by cells or expressed by cells transfected with the LILRB4 gene. For example, LILRB4 as described herein can refer to a leukocyte immunoglobulin-like receptor subfamily B member 4 protein derived from any vertebrate origin, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). Exemplary sequences of the human LILRB4 protein are as described, for example, in UniProtKB entry number: Q8NHJ6, or GenBank accession number AAB68665.1. As used herein, the term "LILRB4" includes any form of LILRB4, such as 1) an unprocessed, natural LILRB4 molecule, a "full-length" LILRB4 chain, or a naturally occurring variant of LILRB4, such as a splice variant or allelic variant; 2) any form of LILRB4 resulting from processing in cells; or 3) fragments (e.g., truncated, extracellular / membrane-spanning domains) or modified forms (e.g., mutant, glycosylated / PEGylated, His-tag / immunofluorescent fusion) of full-length, recombinantly produced LILRB4 subunits.

[0096] As used herein, the term "anti-LILRB4 antibody" refers to an antibody that specifically binds to LILRB4 (e.g., human LILRB4). The terms "anti-human LILRB4 antibody" or "anti-hLILRB4 antibody" refer to an antibody that specifically binds to human LILRB4.

[0097] "Associated with LILRB4" or "LILRB4 - related" diseases, disorders, or conditions, as used herein, refer to any disease, disorder, or condition caused by, exacerbated by, or otherwise related to an increase or decrease in the expression or activity of LILRB4. In some embodiments, LILRB4 - related diseases, disorders, or conditions are disorders related to excessive cell proliferation, such as cancer. In certain embodiments, an LILRB4 - related disease or condition is characterized by expressing or overexpressing LILRB4 and / or an LILRB4 - related gene.

[0098] The term "pharmaceutically acceptable" indicates that the designated carrier, medium, diluent, excipient(s), and / or salt is generally chemically and / or physically compatible with the other components that make up the formulation and is physiologically compatible with the subject to which it is administered.

[0099] The term "LILRB4 - expressing cell", as used herein, refers to a cell that expresses LILRB4 on its surface.

[0100] Anti - LILRB4 antibody 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 (e.g., specifically binding) to LILRB4 (e.g., human LILRB4).

[0101] The binding affinity of an antibody or its antigen - binding fragment provided herein is represented by the ratio (k off / k on ) of the dissociation rate to the association rate when the binding between the antigen and the antigen - binding molecule has reached equilibrium, which can be represented by the K D value. The antigen - binding affinity (e.g., K D) can be appropriately determined using suitable methods known in the art, including, for example, flow cytometry assays. In some embodiments, the binding of different concentrations of an antibody or its antigen-binding fragment to 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 the K D value can be calculated by fitting to a one-site saturation equation: Y = B max * X / (K D +X) using Prism version 5 (GraphPad Software, San Diego, CA), where B max refers to the maximum specific binding of the test antibody to the antigen.

[0102] The binding of the antibodies or their antigen-binding fragments provided herein to LILRB4 can also be represented by the "half maximal effective concentration" (EC 50 ) value, which refers to the antibody concentration at which 50% of its maximum binding is observed. The EC 50 value 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 assay, and other binding assays.

[0103] In certain embodiments, the antibodies or their antigen-binding fragments provided herein are capable of specifically binding to human LILRB4 when measured by FACS assay. For example, the antibodies or their antigen-binding fragments provided herein bind to human LILRB4 with an EC 50 of 5 nM or less (e.g., 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less) when measured by FACS assay.

[0104] 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 FACS assay. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have an EC 50 of 15 nM or less (e.g., 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less) when binding to human LILRB3 as measured by FACS assay. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have an EC 50 of 15 nM or less (e.g., 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less) when binding to human LILRB4 as measured by FACS assay.

[0105] 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 have cross-reactivity with human and cynomolgus monkey LILRB4 as measured by FACS assay.

[0106] As used herein, the term "cross-reactivity" refers to the ability of a binding protein to bind to targets other than the target used in the generation of the binding protein. Generally, a binding protein binds to the target tissue / antigen with an appropriate high affinity, but shows a moderately low affinity for non-target normal tissues / antigens. Individual binding proteins are generally selected to meet two criteria: (1) antibody binding to tissues compatible with the known expression of the antibody target, visualized using staining methods known in the art, and (2) similar staining patterns between tissues from the same organs of humans and toxicology assessment species (e.g., mice and cynomolgus monkeys). These methods and other methods for assessing cross-reactivity are known to those skilled in the art (e.g., US20090311253A1).

[0107] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein exhibit a strong ADCP effect against human AML cell lines (e.g., as measured by a FACS assay). In certain embodiments, the ADCP effect is measured by the method described in Example 4 of the present disclosure.

[0108] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein exhibit a strong ADCC effect against human AML cell lines (e.g., as measured by a FACS assay). In certain embodiments, the ADCC effect is measured by the method described in Example 3 of the present disclosure.

[0109] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of blocking the LILRB4-fibronectin interaction and reprogramming tolerogenic dendritic cells into mature dendritic cells that stimulate T cell activation as measured by the FcγR stimulation assay. In certain embodiments, the LILRB4-fibronectin interaction is measured by the method described in Example 5 of the present disclosure. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of inducing the production of TNF-α. Secretion of TNF-α from tolerogenic dendritic cells was used as a readout to evaluate FcγR stimulation.

[0110] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of reprogramming tolerogenic dendritic cells (DCs) to activate T cells. DCs are the most specialized antigen-presenting cells and play an important role in regulating the immune response between innate and adaptive immunity. Mature DCs exhibit strong co-stimulatory and T cell activation capabilities. Mature DCs have high expression levels of antigen-presenting molecules (HLA-DR and HLA-ABC) and co-stimulatory molecules (CD80 and CD86), and activate T cells for anti-tumor purposes. The interaction between HLA and the T cell receptor induces primary signal transduction, and the interaction between CD28 and CD80 / CD86 on naive T cells causes co-stimulatory signal transduction. Both interactions lead to the activation and proliferation of T cells for the defense against tumor cells. However, the expression of antigen-presenting molecules and co-stimulatory molecules in tolerogenic DCs is attenuated, while the expression of LILRB4 in tolerogenic DCs is upregulated. However, treatment of tolerogenic DCs with anti-LILRB4 antibodies (e.g., the 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 anti-LILRB4 antibodies to reprogram tolerogenic DCs to activate T cells is measured by the method described in Example 6 of the present disclosure.

[0111] 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 an anti-LILRB4 antibody to reverse macrophage-mediated T cell suppression is measured by the method described in Example 9.4 of the present disclosure.

[0112] 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 an anti-LILRB4 antibody to reverse THP-1-mediated T cell suppression is measured by the method described in Example 7 of the present disclosure.

[0113] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein enhance CD8 + T cell-mediated cytotoxicity of THP-1 cells. In certain embodiments, the ability of an anti-LILRB4 antibody to enhance CD8 + T cell-mediated cytotoxicity of THP-1 cells is measured by the method described in Example 9.5 of the present disclosure.

[0114] Exemplary anti-LILRB4 antibodies In certain embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to LILRB4, one, two, or three heavy chain complementarity determining regions (HCDR1, HCDR2, and / or HCDR3) contained within any one of the heavy chain variable (VH) region sequences selected from the group consisting of SEQ ID NOs: 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 complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable (VL) region sequences selected from the group consisting of SEQ ID NOs: 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 155, and 157 An antibody or an antigen-binding fragment thereof comprising the same is provided.

[0115] Those skilled in the art can define or identify the CDR boundaries of the VH or VL regions by methods well known in the art as long as the amino acid sequences of the VH or VL regions are known. For example, the CDR boundaries of an antibody or its antigen-binding fragment can be defined or identified according to the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., 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, A.M., J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83(1989); Kabat E.A. et al., Sequences of Proteins of immunological Interest, 5th Ed. Public Health 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 their antigen-binding fragments provided herein are identified according to the Kabat convention. In some embodiments, the CDR boundaries of the antibodies or their antigen-binding fragments provided herein are identified according to the IMGT convention. In some embodiments, the CDR boundaries of the antibodies or their antigen-binding fragments provided herein are identified according to the Chothia convention.In some embodiments, the CDR boundaries of the antibodies or antigen-binding fragments thereof provided herein are identified according to the Al-Lazikani convention.

[0116] In certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to LILRB4 and comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of 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-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.

[0117] The antibody "27-F11-E10-G10", as used herein, 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.

[0118] The antibody "43-D12-F3-G11", as used herein, 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.

[0119] The antibody "36-F4-H1-D9", as used herein, 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.

[0120] The antibody "8-B3-F6-H8", as used herein, 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.

[0121] The antibody "44-F10-B6-D4", as used herein, 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.

[0122] The antibody "27-G5-E9-B8", as used herein, 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.

[0123] The antibody "48-E11-H7-B5", as used herein, 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.

[0124] The antibody "42-C8-A12-F3-D11", as used herein, 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.

[0125] The antibody "10-H9-E3-G3", as used herein, 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.

[0126] The antibody "7-B4-C1-C9", as used herein, 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.

[0127] The antibody "25-E7-A10-H6", as used herein, 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.

[0128] The antibody "25-G9-G4-C12", as used herein, 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.

[0129] The antibody "2-H1-D7-E5-D5", as used herein, 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.

[0130] The antibody "27-D11-C2-A10", as used herein, 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.

[0131] The antibody "26-H9-B9-B7", as used herein, 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.

[0132] The antibody "4-E6-E4-B12", as used herein, 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.

[0133] The antibody "2-H8-C9-F7-E2", as used herein, 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.

[0134] The specific amino acid sequences of the heavy chain variable regions and light chain variable regions of each of the above exemplary antibodies are shown in Tables 3 and 21 below.

[0135] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 23, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 24.

[0136] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 31, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 32.

[0137] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 39, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 40.

[0138] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 47, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 48.

[0139] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 55, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 56.

[0140] 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 set forth in SEQ ID NO: 63, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 64.

[0141] 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 set forth in SEQ ID NO: 71, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 72.

[0142] 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 set forth in SEQ ID NO: 79, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 80.

[0143] 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 set forth in SEQ ID NO: 87, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 88.

[0144] 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 set forth in SEQ ID NO: 95, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 96.

[0145] 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 set forth in SEQ ID NO: 103, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 104.

[0146] 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 set forth in SEQ ID NO: 111, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 112.

[0147] 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 set forth in SEQ ID NO: 119, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 120.

[0148] 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 set forth in SEQ ID NO: 127, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 128.

[0149] 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 set forth in SEQ ID NO: 135, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 136.

[0150] 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 set forth in SEQ ID NO: 143, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 144.

[0151] 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 set forth in SEQ ID NO: 151, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 152.

[0152] 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 set forth in SEQ ID NO: 154, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 155.

[0153] 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 set forth in SEQ ID NO: 156, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 157.

[0154] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise 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 NOs: 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.

[0155] In certain 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 NOs: 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.

[0156] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise one or two or three of LCDR1, LCDR2, and LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.

[0157] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise an HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137 and 145; an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, 146 and 158; and an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139 and 147.

[0158] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise an LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132, 140 and 148; an LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141 and 149; and an LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134, 142 and 150.

[0159] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are i. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19; ii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; iii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35; iv. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; v. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 158, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; vi. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51; vii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 57, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59; viii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 67; ix. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75; x. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83; xi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91; xii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99; xiii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107; xiv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115; xv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123; xvi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 129, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 130, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 131; xvii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139; or xviii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 146, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 147 is included.

[0160] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are i. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 20, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 22; ii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 28, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 30; iii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 36, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 38; iv. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 44, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 45, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 46; v. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 52, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 53, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 54; vi. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 60, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 61, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 62; vii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 68, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 69, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 70; viii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 76, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 77, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 78; ix. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 84, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 85, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 86; x. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 92, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 93, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 94; xi. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102; xii. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110; xiii. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118; xiv. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 124, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 125, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 126; xv. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 132, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 133, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 134; xvi. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 140, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 141, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 142; or xvii. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150 comprising.

[0161] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are i. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; iii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; iv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; v. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 158, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; vi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54; vii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 57, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 60, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 61, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 62; viii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 67, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70; ix. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78; x. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 85, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 86; xi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 92, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 93, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94; xii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102; xiii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110; xiv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118; xv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 124, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 125, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 126; xvi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 129, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 130, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 131, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 132, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 133, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 134; xvii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 140, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 141, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 142; or xviii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 146, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 147, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150 comprising.

[0162] The sequence numbers of the heavy chain (designated as "H") variable region, light chain (designated as "L") variable region, HCDR, and LCDR of each of the above 17 monoclonal antibodies are shown in Table 1 below. The amino acid sequences of each CDR of the 17 exemplary monoclonal antibodies are shown in Table 2 below. Unless otherwise specified, the CDR boundaries described in Table 2 below were defined or identified according to the Kabat convention. The amino acid sequences of each VH and VL of the 17 exemplary monoclonal antibodies are shown in Table 3 below.

[0163]

Table 1

[0164]

Table 2-1

Table 2-2

Table 2-3

[0165]

Table 3-1

Table 3-2

Table 3-3

[0166] Assuming that each of the 17 exemplary monoclonal antibodies binds to LILRB4 and that the antigen-binding specificity is provided primarily by the CDR1, CDR2, and CDR3 regions, the HCDR1, HCDR2, and HCDR3 sequences as well as the LCDR1, LCDR2, and LCDR3 sequences 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 as well as LCDR1, LCDR2, and LCDR3) to generate an anti-LILRB4 antibody or an antigen-binding fragment 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 mixing and matching VH CDR sequences, the HCDR1, HCDR2, and / or HCDR3 sequences from a particular VH sequence are replaced with structurally similar CDR sequence(s). Similarly, when mixing and matching VL CDR sequences, the LCDR1, LCDR2, and / or LCDR3 sequences from a particular VL sequence are preferably replaced with structurally similar CDR sequence(s). For example, the HCDR1s of antibodies 25-G9-G4-C12 and 2-H1-D7-E5-D5 share some structural similarity and are thus amenable to mixing and matching. It will be readily apparent to those 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 with respect to the 17 exemplary monoclonal antibodies.

[0167] It is known that CDRs are involved in antigen binding. However, it has been found that not all six CDRs are necessarily essential or non-exchangeable. In other words, it is possible to exchange, vary, or modify one or more CDRs of each of the 17 exemplary monoclonal antibodies and still retain specific binding affinity for LILRB4.

[0168] In certain embodiments, the anti-LILRB4 antibodies and antigen-binding fragments provided herein comprise a heavy chain CDR3 sequence of one of 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-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. In certain 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 NOs: 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 thus is thought to contact the antigen most extensively and provide the most free energy to the affinity of the antibody for the antigen. Similarly, the heavy chain CDR3 is thought to be the most diverse CDR of the antigen-binding site with respect to length, amino acid composition, and conformation by a plurality of diversification mechanisms (Tonegawa S. Nature. 302:575-81). The diversity of the heavy chain CDR3 is sufficient to generate most antibody specificities (Xu JL, Davis MM. Immunity. 13:37-45) as well as the desired antigen-binding affinity (Schier R, et al., J Mol Biol. 263:551-67).

[0169] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a VH region having the amino acid sequence set forth 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%, or 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.

[0170] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a VL region having the amino acid sequence 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%, or 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.

[0171] In certain 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.

[0172] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein include suitable framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof are capable of binding to LILRB4. The CDR sequences provided in Table 2 above are obtained from murine antibodies, but they can be grafted onto 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 techniques.

[0173] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are humanized. Humanized antibodies or antigen-binding fragments thereof are desirable due to their reduced immunogenicity in humans. A humanized antibody is chimeric in its variable region because the non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of an antibody or antigen-binding fragment can be essentially carried out by substituting the non-human (e.g., murine) CDR genes with the corresponding human CDR genes in the human immunoglobulin genes (see, for example, Jones et al., (1986) Nature 321:522-525; Riechmann et al., (1988) Nature 332:323-327; Verhoeyen et al., (1988) Science 239:1534-1536).

[0174] Suitable human heavy and light chain variable domains can be selected to achieve this purpose using methods known in the art. In an exemplary example, a "best fit" approach can be used, where non-human (e.g., rodent) antibody variable domain sequences are screened or BLASTed against a database of known human variable domain sequences to identify the human sequence closest to the non-human query sequence and used as a human scaffold structure for grafting the non-human CDR sequences (see, e.g., Sims et al., (1993) J. Immunol. 151:2296; Chothia et al., (1987) J. Mol. Biol. 196:901). Alternatively, a framework derived from the consensus sequence of all human antibodies may be used for grafting non-human CDRs (see, e.g., Carter et al., (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al., (1993) J. Immunol., 151:2623).

[0175] In some embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein are humanized. In certain embodiments, the humanized antibodies or antigen-binding fragments thereof provided herein are composed of substantially all human sequences except for the CDR sequences that are non-human. In some embodiments, the variable region, FR, and, if present, the constant region are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences may be derived from different human immunoglobulin genes. For example, the FR region may be derived from one human antibody and the constant region may be derived from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises human heavy chain HFR1, HFR2, HFR3 and HFR4, and / or light chain LFR1, LFR2, LFR3 and LFR4.

[0176] In some embodiments, the present disclosure provides a humanized antibody of clone 2-H1-D7-E5-D5 (also referred to herein as "humanized 2-H1-D7-E5-D5" or "h2-H1-D7-E5-D5") or an antigen-binding fragment thereof. In some embodiments, the humanized 2-H1-D7-E5-D5 or an antigen-binding fragment thereof provided herein comprises one, two, or three HCDRs (HCDR1, HCDR2, and / or HCDR3) contained within the heavy-chain variable region sequence of SEQ ID NO: 154; and / or one, two, or three LCDRs (LCDR1, LCDR2, and / or LCDR3) contained within the light-chain variable region sequence of SEQ ID NO: 155. In some embodiments, the humanized 2-H1-D7-E5-D5 or an antigen-binding fragment thereof provided herein comprises HCDR1, HCDR2, and HCDR3 contained within the heavy-chain variable region sequence of SEQ ID NO: 154; and comprises LCDR1, LCDR2, and LCDR3 contained within the light-chain variable region sequence of SEQ ID NO: 155. In some embodiments, the humanized 2-H1-D7-E5-D5 or an antigen-binding fragment thereof provided herein comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and LCDR3 comprising the amino acid sequence set forth 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.

[0177] In some embodiments, the present disclosure provides a humanized antibody of clone 8-B3-F6-H8 (also referred to herein as "humanized 8-B3-F6-H8" or "h8-B3-F6-H8") or an antigen-binding fragment thereof. In some embodiments, the humanized 8-B3-F6-H8 or antigen-binding fragment thereof provided herein comprises one, two, or three HCDRs (HCDR1, HCDR2, and / or HCDR3) contained within the heavy chain variable region sequence of SEQ ID NO: 156; and / or one, two, or three LCDRs (LCDR1, LCDR2, and / or LCDR3) contained within 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, and HCDR3 contained within the heavy chain variable region sequence of SEQ ID NO: 156; and comprises LCDR1, LCDR2, and LCDR3 contained within 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 comprising the amino acid sequence set forth in SEQ ID NO: 41, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the humanized 8-B3-F6-H8 or antigen-binding fragment thereof provided herein comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 158, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and LCDR3 comprising the amino acid sequence set forth 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.

[0178]

Table 21

[0179] In some embodiments, the FR region derived from a human 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 the human FR are replaced with the corresponding residues from the parental non-human antibody. This may be desirable in certain embodiments to closely approximate the humanized antibody or fragment thereof to the non-human parental antibody structure and optimize the binding characteristics (e.g., increase the binding affinity). In certain embodiments, the humanized antibodies or antigen-binding fragments thereof provided herein include 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer than 1 amino acid residue substitutions in each of the human FR sequences, or 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer than 1 amino acid residue substitutions in all of the FR sequences of the heavy or light chain variable domains. In some embodiments, such changes in amino acid residues can be present in the heavy chain FR region only, the light chain FR region only, or both chains. In certain embodiments, one or more amino acids of the human FR sequence are randomly mutated to increase the binding affinity. In certain embodiments, one or more amino acids of the human FR sequence are reverted to the corresponding amino acid(s) of the parental non-human antibody to increase the binding affinity.

[0180] In some embodiments, the anti-LILRB4 antibodies and antigen-binding fragments thereof provided herein include all or part of the heavy chain variable domain and / or all or part of the light chain variable domain. In one embodiment, the anti-LILRB4 antibody or antigen-binding fragment thereof provided herein is a single domain antibody consisting of all or part of the heavy chain variable domain provided herein. More information regarding such single domain antibodies is available in the art (see, e.g., U.S. Patent No. 6,248,516).

[0181] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein further comprise an Fc region. In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein further comprise the Fc region of human immunoglobulin (Ig). In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein further comprise a constant region optionally further comprising heavy and / or light chain constant regions. In certain embodiments, the heavy chain constant region comprises CH1, hinge, and / or CH2-CH3 regions (or optionally CH2-CH3-CH4 regions). In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein comprise the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgM. In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein comprise a lambda (λ) or kappa (κ) light chain. The constant region of the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein may be identical to the wild-type constant region sequence or may differ by one or more mutations.

[0182] In certain embodiments, the heavy chain constant region comprises an Fc region. The Fc region is known to mediate effector functions such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of antibodies. Fc regions of different Ig isotypes have different abilities to induce effector functions. For example, it is recognized that the Fc regions of IgG1 and IgG3 induce both ADCC and CDC more effectively than the Fc regions of IgG2 and IgG4. In certain embodiments, the anti-LILRB4 antibodies and antigen-binding fragments thereof provided herein comprise the Fc region of the IgG1 or IgG3 isotype capable of inducing ADCC or CDC; or alternatively, the constant region of the IgG4 or IgG2 isotype that may reduce or abrogate effector function. In some embodiments, the Fc region is derived from human IgG1 with enhanced effector function. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 153. APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 153)

[0183] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have specific binding affinity for human LILRB4 sufficient to provide for diagnostic and / or therapeutic use.

[0184] The antibodies or antigen-binding fragments thereof provided herein can be monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, multispecific antibodies, labeled antibodies, bivalent antibodies, anti-idiotypic antibodies, or fusion proteins. Recombinant antibodies are antibodies prepared in vitro using recombinant methods rather than in animals.

[0185] In certain embodiments, the present disclosure provides an anti-LILRB4 antibody or an antigen-binding fragment thereof that competes with the antibodies or antigen-binding fragments thereof provided herein with respect to binding to LILRB4. In certain embodiments, the present disclosure provides an anti-LILRB4 antibody or an antigen-binding fragment thereof that competes with any one of antibody 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-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 with respect to binding to human LILRB4. In some embodiments, the present disclosure provides an anti-LILRB4 antibody or an antigen-binding fragment thereof that competes with the antibodies or antigen-binding fragments thereof provided herein with respect to the same epitope.

[0186] The ability to "block binding" or "compete with respect to the same epitope", as used herein, refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human LILRB4 and an 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 may be greater than 85% or greater than 90%.

[0187] One skilled in the art will recognize that when a human monoclonal antibody binds to the same epitope as the antibodies of the present disclosure (e.g., mouse monoclonal 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-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), it is possible to determine without undue experimentation by checking whether the former prevents the latter from binding to the LILRB4 antigen polypeptide. As shown by the decrease in binding to the LILRB4 antigen polypeptide by the antibodies of the present disclosure, if a test antibody competes with the antibodies of the present disclosure, the two antibodies bind to the same or closely related epitopes. Or if the binding of the test antibody to the LILRB4 antigen polypeptide is inhibited by the antibodies of the present disclosure, the two antibodies bind to the same or closely related epitopes.

[0188] In certain embodiments, the present disclosure provides anti-LILRB4 antibodies or antigen-binding fragments thereof that compete with respect to binding of antibody IO-202 to LILRB4. In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments that compete with respect to binding to LILRB4 with the antibodies or antigen-binding fragments thereof provided herein are not IO-202. Information on IO-202 is described, for example, in WO2016144728A2, WO2018022881A2, Mi Deng et al., Nature. 2018 Oct;562(7728):605~609, Xun Gui et al., Cancer Immunol Res 2019;7:1244~57.

[0189] In certain embodiments, the present disclosure provides an anti-LILRB4 antibody or an antigen-binding fragment thereof that competes with respect to binding of antibody NGM831 to LILRB4. In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment that competes with respect to binding to LILRB4 with the antibody or antigen-binding fragment thereof provided herein is not NGM831. Information on NGM831 is described, for example, in US20210221887A1, Kevin J Paavola et al., Cancer Immunol Res. 2021 Nov;9(11):1283-1297.

[0190] "IO-202", as used herein, refers to an antibody or an antigen-binding fragment thereof comprising one or two or three heavy chain CDRs contained within the heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and one or two or three light chain CDRs contained within the light chain variable region having the amino acid sequence of SEQ ID NO: 8. In certain embodiments, IO-202 comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the 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 are identified according to the IMGT convention.

[0191] As used herein, "NGM831" refers to an antibody or an antigen-binding fragment thereof that includes one or two or three heavy-chain CDRs contained within a heavy-chain variable region having the amino acid sequence of SEQ ID NO: 15 and one or two or three light-chain CDRs contained within a light-chain variable region having the amino acid sequence of SEQ ID NO: 16. In certain embodiments, NGM831 includes HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and LCDR3 comprising the 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 are identified according to the IMGT convention.

[0192]

Table 4

[0193] Antibody variant The antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

[0194] In certain embodiments, the antibody variant includes one or more amino acid residue substitutions or modifications, yet still retains binding affinity for LILRB4. In certain embodiments, at least one of the substitutions or modifications is present in one or more of the CDR sequences of the VH region or VL region. In certain embodiments, at least one of the substitutions or modifications is present in one or more of the non-CDR sequences of the VH region or VL region. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein further include one or more non-natural amino acid (NNAA) substitutions. In certain embodiments, the NNAA can be conjugated.

[0195] For example, an antibody variant comprises one or more of the CDR sequences provided in Table 2 or Table 21 above, one or more non-CDR sequences of the heavy or light chain variable regions provided in Table 3 or Table 21 above, and / or one or more amino acid residue substitutions or modifications in the constant region (e.g., the Fc region). Such variants retain the binding specificity for LILRB4 of their parental antibody, but have one or more desired properties conferred by the modification(s) or substitution(s). For example, antibody variants can have improved antigen binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, enhanced effector function(s), improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine residues), among others.

[0196] The parental antibody sequence can be screened using methods known in the art, such as "alanine scan mutagenesis", to identify residues suitable or preferred for modification or substitution (see, for example, Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and exchanged for neutral or negatively charged amino acids (e.g., alanine or polyalanine), the modified antibody produced, and screened for the desired property. If a substitution at a particular amino acid position demonstrates the desired functional change, that position can be identified as a potential residue for modification or substitution. The potential residue can be further evaluated by substituting it with different types of residues (e.g., cysteine residues, positively charged residues, etc.).

[0197] Affinity variant Antibody affinity variants may contain modifications or substitutions in one or more of the CDR sequences, one or more of the FR sequences provided in Table 2 or Table 21 above, or the heavy or light chain variable region sequences provided in Table 3 or Table 21 above. Since it is well known in the art that CDR regions are adjacent to two FR regions in the variable region, FR sequences can be readily identified by those skilled in the art based on the CDR sequences in Table 2 or Table 21 above and the variable region sequences in Table 3 or Table 21 above. The affinity variant retains specific binding affinity for LILRB4 of the parental antibody or has improved LILRB4-specific binding affinity compared to the parental antibody. In certain embodiments, at least one (or all) of the substitution(s) in the CDR sequence, FR sequence, or variable region sequence comprises a conservative substitution.

[0198] One of ordinary skill in the art may substitute one or more amino acid residues 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 and still have the resulting antibody or antigen-binding fragment retain binding affinity or ability for LILRB4 or even retain improved binding affinity or ability. Various methods known in the art can be used to achieve this purpose. For example, a library of antibody variants (e.g., Fab or scFv variants) can be generated, expressed by phage display technology, and then screened for binding affinity to human LILRB4. In another example, computer software can be used to virtually simulate the binding of an antibody to human LILRB4 and identify the amino acid residues on the antibody that form the binding interface. Such residues may be avoided in substitutions to prevent reduction of binding affinity or may be targeted for substitution to provide stronger binding.

[0199] In certain embodiments, the humanized antibodies or antigen-binding fragments thereof provided herein include one or more amino acid residue substitutions in one or more of the CDR sequences and / or one or more of the FR sequences. In certain embodiments, the affinity variant includes a total of 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer substitutions in the CDR sequences and / or FR sequences.

[0200] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequence(s) set forth in Table 2 or Table 21 above, and still retain a specific binding affinity for LILRB4 that is similar to or even at a higher level than that of its parental antibody, and include one, two, or three CDR sequences.

[0201] In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequence(s) set forth in Table 3 or Table 21 above, and still retains a specific binding affinity for LILRB4 that is similar to or at a higher level than that of its parental antibody, and includes one or more variable region sequences. In some embodiments, 1 to 10 amino acids in total are substituted, inserted, or deleted in the variable region sequences set forth in Table 3 or Table 21 above. In some embodiments, the substitution, insertion, or deletion is present in a region outside the CDR (e.g., FR).

[0202] Glycosylation variants The anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein also include glycosylation variants that can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment thereof.

[0203] 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) can be attached. Glycosylation of an antibody is typically either N-linked or O-linked. N-linkage refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine. Removal of a native glycosylation site can be readily achieved, for example, by altering the amino acid sequence such that one of the above tripeptide sequences (in the case of an N-linked glycosylation site) or serine or threonine residue (in the case of an O-linked glycosylation site) present in the sequence is substituted. A novel glycosylation site can be similarly created by introducing such a tripeptide sequence or serine or threonine residue.

[0204] Cysteine engineered variants The anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein also include cysteine engineered variants that contain one or more introduced free cysteine amino acid residues.

[0205] A free cysteine residue is a residue that is not part of a disulfide bridge. Cysteine engineered variants are useful for conjugating, through engineered cysteine sites, for example, maleimide or haloacetyl, cytotoxic compounds and / or contrast compounds, labels, or radioisoptypes. Methods for engineering an antibody or antigen-binding fragment thereof to introduce free cysteine residues are known in the art; see, for example, WO2006 / 034488.

[0206] Fc variant The anti-LILRB4 antibodies and antigen-binding fragments provided herein also include Fc variants that contain one or more amino acid residue modifications or substitutions in their Fc region and / or hinge region.

[0207] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments provided herein contain one or more amino acid substitutions (s) that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants may have an extended pharmacokinetic half-life since it binds to FcRn at acidic pH allowing it to escape degradation in the lysosome and then traffic and be released from the cell. Methods for engineering antibodies or antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art and are described, for example, in Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 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., J. Immunology, 176:346-356 (2006).

[0208] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein include one or more amino acid substitutions (s) that alter ADCC. Substituting certain amino acid residues in the CH domains of the Fc region can provide enhanced ADCC activity. Alternatively or in addition, the carbohydrate structure of the antibody can be altered to enhance ADCC activity. Methods for altering ADCC activity by engineering antibodies are 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., PNAS, 2006,103(11):4005-4010; Ryan MC.et al., Mol.Cancer Ther., 2007,6:3009-3018; Richards JO,.et al., Mol Cancer Ther.2008,7(8):2517-27; Shields R.L.et al., J.Biol.Chem, 2002,277:26733-26740; Shinkawa T.et al., J.Biol.Chem, 2003,278:3466-3473.

[0209] In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions (s) that alter CDC, e.g., by enhancing or impairing C1q binding and / or CDC (see, e.g., WO 99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent 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 different amino acid residues to alter Clq binding and / or enhance CDC (see U.S. Patent No. 6,194,551 by Idusogie et al.). One or more amino acid substitutions (s) can also be introduced to alter the complement fixation ability of the antibody (see PCT application WO 94 / 29351 by Bodmer et al.).

[0210] In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof provided herein comprises one or more amino acid substitutions (s) at positions 234 and / or 235 of a human immunoglobulin (according to EU numbering). In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof provided herein comprises two amino acid substitutions at positions 234 and 235 of a human immunoglobulin (e.g., IgG1) (according to EU numbering). In certain embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof provided herein comprises the L234A and L235A (according to EU numbering) amino acid substitutions.

[0211] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein include one or more amino acid substitutions (s) at the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include the introduction of a protrusion into a first Fc polypeptide and the introduction of a cavity into a second Fc polypeptide, and the protrusion may be positioned in the cavity to facilitate the interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies having these modifications are known in the art, such as those described in U.S. Patent No. 5,731,168.

[0212] Antigen-binding fragment Also provided herein are anti-LILRB4 antigen-binding fragments. For example, various types of antigen-binding fragments are known in the art, including exemplary antibodies whose CDRs are shown in Table 2 or Table 21 above and whose variable sequences are shown in Table 3 or Table 21 above, and different variants thereof (e.g., affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants, etc.), and they can be developed based on the anti-LILRB4 antibodies provided herein.

[0213] In certain embodiments, the anti-LILRB4 antigen-binding fragments 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 (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), camelized single-domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0214] To produce such antigen-binding fragments, a variety of techniques can be used. Exemplary methods include enzymatic digestion of intact antibodies (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 E. coli (e.g., in the case of Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., in the case of ScFv), and formation of F(ab’)2 fragments by chemical coupling of two Fab’-SH fragments (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0215] In certain embodiments, the antigen-binding fragment is a scFv. Generation of scFvs is described, for example, in WO93 / 16185; U.S. Patent Nos. 5,571,894; and 5,587,458. The ScFv may be fused to an effector protein at either the amino or carboxyl terminus to provide a fusion protein (see, e.g., Antibody Engineering, ed. Borrebaeck).

[0216] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. Any molecule having more than bivalent is considered multivalent and includes, for example, trivalent, tetravalent, hexavalent, etc.

[0217] A bivalent molecule can be monospecific if both of its binding sites are specific with respect to binding to the same antigen or the same epitope. In certain embodiments, this provides a stronger binding to the antigen or epitope than the monovalent counterpart. Similarly, a multivalent molecule can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valence of the binding site and the second valence of the binding site are either structurally identical (i.e., having the same sequence) or structurally different (i.e., having different sequences despite having the same specificity).

[0218] 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 if two of its binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0219] Bispecific or multispecific antibodies 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 moiety having a binding specificity different from that of the anti-LILRB4 antibody or antigen-binding fragment 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.

[0220] In certain embodiments, the second specificity is for a tumor-associated antigen or an epitope thereof. The term "tumor-associated antigen" refers to antigens that are present or can be present on the surface of tumor cells and are located on or within tumor cells. In some embodiments, only tumor cells can present tumor-associated antigens, and normal cells, i.e., non-tumor cells, cannot. In some other embodiments, the tumor-associated antigen can be expressed exclusively on tumor cells or can exhibit tumor-specific mutations compared to non-tumor cells. In some other embodiments, the tumor-associated antigen can be found in both tumor cells and non-tumor cells, but is overexpressed in tumor cells compared to non-tumor cells or is accessible for antibody binding in tumor cells because the tumor tissue architecture is less dense compared to non-tumor tissue. In some embodiments, the tumor-associated antigen is located in the vasculature of the tumor.

[0221] 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 other than LILRB4 or a second epitope on LILRB4.In certain embodiments, 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, LIGHT, PVRIG, 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.

[0222] 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.

[0223] Conjugate In some embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof provided herein further comprise one or more conjugate moieties. The conjugate moieties can be linked to the antibody or antigen-binding fragment thereof. A conjugate moiety is a moiety that can bind to the antibody or antigen-binding fragment thereof. It is contemplated that a variety of conjugate moieties can be linked to the antibodies or antigen-binding fragments thereof provided herein (see, e.g., “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties can be linked to the antibody or antigen-binding fragment thereof by, among other methods, covalent bonding (e.g., disulfide bonding), affinity binding, intercalation, cooperative binding, complex formation, association, admixture, or addition. In some embodiments, the antibody or antigen-binding fragment thereof can be linked to one or more conjugates via a linker or crosslinker. The linker or crosslinker comprises reactive chemical groups that can react with the anti-LILRB4 antibody or fragment thereof. The reactive chemical groups can be N-succinimidyl esters and N-sulfosuccinimidyl esters. In addition, the linker can comprise reactive chemical groups that can be dithiopyridyl groups that can react with a 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., Biochem. J., 173:723-737 (1978)), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Patent No. 4,563,304), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfosuccinimidyl 4-(2-pyridyldithio)butanoate) (see, e.g., U.S. Patent Application Publication No. 20090274713), N-succinimidyl 4-(2-pyridyldithio)pentaenoate (SPP) (see, e.g., CAS Registry No. 341498-08-6), 2-iminothiolane, or acetylsuccinic anhydride. For example, an antibody or cell-binding agent can be modified with a cross-linking reagent, and the antibody or cell-binding agent containing a free or protected thiol group thus derived is reacted with a disulfide or thiol-containing maytansinoid 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.

[0224] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can be engineered to contain specific sites other than the epitope-binding portion that can be utilized to bind 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 bonding with the conjugate moiety.

[0225] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein may be indirectly linked to a conjugate moiety or linked through another conjugate moiety. For example, the antibodies or antigen-binding fragments thereof provided herein may be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin. In some embodiments, the conjugate moiety includes a clearance modifier (e.g., a polymer such as PEG that extends the half-life), 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 binder, a purification moiety or other anti-cancer agent (e.g., an agonist of toll-like receptor 7 (TLR-7), TLR-8 and / or TLR-9, siRNA, an antibody or antigen-binding fragment thereof, a peptide (e.g., a small peptide), etc.).

[0226] 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, taxol, taxoids, CC-1065 and CC-1065 analogs, duocarmycin and duocarmycin analogs, enediynes such as calicheamicin, dolastatin and dolastatin analogs including auristatin, tomatamycin derivatives, leptomycin derivatives, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil and morpholinodoxorubicin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, DM4, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin 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., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin and anthramycin (AMC)), mitotic inhibitors (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin binders.

[0227] Examples of detectable labels 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), radioisotopes (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 Bi, and 32 P, other lanthanides), luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules or gold for detection may be mentioned.

[0228] In certain embodiments, the conjugate moiety can be a clearance modifier that helps to increase the half-life of the antibody. Exemplary examples include water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol / propylene glycol and the like. The polymer can be a polymer of any molecular weight and can be branched or unbranched. The number of polymers binding to the antibody can vary and if more than one polymer is bound, they can be the same or different molecules.

[0229] In certain embodiments, the conjugate moiety can be a purification moiety such as magnetic beads.

[0230] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are used as a base for conjugates.

[0231] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are conjugated to a signal peptide. A signal peptide (sometimes called a signal sequence, leader sequence, or leader peptide) can be used to facilitate the secretion and isolation of the antibodies or antigen-binding fragments thereof provided herein. Signal peptides are typically characterized by a core of hydrophobic amino acids that are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow for cleavage of the signal sequence from the mature protein as they pass through the secretory pathway. Thus, the present invention relates to the polypeptides described having a signal sequence, as well as polypeptides in which the signal sequence has been proteolytically cleaved (i.e., cleavage products). In one embodiment, a nucleic acid sequence encoding the signal sequence can be operably linked in an expression vector to a protein of interest, such as a protein that is not normally secreted or is otherwise difficult to isolate. The signal sequence directs, for example, the secretion of the protein from a eukaryotic cell host into which the expression vector is transformed, and the signal sequence is cleaved thereafter or simultaneously. The protein can then be readily purified from the extracellular medium by methods recognized in the art. Alternatively, the signal sequence can be linked to the protein of interest using a sequence that facilitates purification, such as a GST domain.

[0232] Chimeric antigen receptor In certain embodiments, the present disclosure provides chimeric antigen receptors comprising the antibodies or antigen-binding fragments thereof provided herein, a transmembrane region, and an intracellular signaling region.

[0233] The terms "chimeric antigen receptor", "CAR", or "CARs", as used herein, refer to an engineered receptor that grafts antigen specificity onto a cell (e.g., a T cell, such as a naive T cell, central memory T cell, effector memory T cell, regulatory T cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors. In some embodiments, a CAR comprises an antigen-specific targeting region (e.g., an antigen-binding fragment of an anti-LILRB4 antibody provided herein), an extracellular region, a transmembrane region, one or more co-stimulatory regions, and an intracellular signaling region.

[0234] In some embodiments, the antigen-specific targeting region is a scFv. In some embodiments, the transmembrane region comprises the transmembrane region of CD3, CD4, CD8, or CD28. In some embodiments, the co-stimulatory region comprises the co-stimulatory domains of CD28, ICOS, CD27, 4-1BB, OX40, and CD40L. In some embodiments, the intracellular signaling region is selected from the group consisting of the intracellular signaling region sequences of CD3, FcγRI, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR, or a combination thereof.

[0235] A CAR can be grafted onto various cells, such as allogeneic cells, autologous cells, or heterologous cells.

[0236] The term "allogeneic cell", as used herein, refers to any cell derived from a different subject of the same species.

[0237] The term "autologous cell", as used herein, refers to any cell derived from the same subject that is later reintroduced.

[0238] The term "heterologous cell", as used herein, refers to any cell derived from a different subject of a different species.

[0239] In some embodiments, the CAR is grafted into immune effector cells, such as T cells, natural killer cells, macrophage cells, tumor infiltrating lymphocytes, and the like.

[0240] Polynucleotides and Recombinant Methods The present disclosure provides isolated polynucleotides encoding the antibodies or antigen-binding fragments thereof, and / or chimeric antigen receptors provided herein. The term "nucleic acid" or "polynucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and polymers thereof in single-stranded or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the explicitly shown sequences. 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 bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acid 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)).

[0241] The DNA encoding the antibodies or antigen-binding fragments thereof provided herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody). The encoding DNA can also be obtained by synthetic methods.

[0242] The isolated polynucleotides encoding the antibodies or antigen-binding fragments thereof and / or chimeric antigen receptors provided herein can be inserted into vectors using recombinant techniques known in the art 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: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoters (e.g., SV40, CMV, EF-1α), and transcription termination sequences.

[0243] The present disclosure provides vectors comprising the isolated polynucleotides provided herein. In certain embodiments, the polynucleotides provided herein encode an antibody or antigen-binding fragment thereof and / or chimeric antigen receptor provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, and M13 phage, plasmid 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, p15TV-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.

[0244] A vector comprising a polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof and / or a chimeric antigen receptor provided herein can be introduced into a host expression system (e.g., a host cell) for cloning or gene expression. In certain embodiments, the host expression systems provided herein are microorganisms, yeast, or mammalian cells. In certain embodiments, the microorganisms are selected from the group consisting of Escherichia coli and Bacillus subtilis. In certain embodiments, the yeast is Saccharomyces. In certain embodiments, the mammalian cells are selected from the group consisting of COS, CHO-S, CHO-K1, HEK-293, and 3T3 cells.

[0245] Suitable host cells for cloning or expressing DNA in the vectors herein are the prokaryotic cells, yeast, or higher eukaryotic cells described above. Suitable prokaryotic cells for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella as well as Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0246] In addition to prokaryotic cells, eukaryotic cell microorganisms, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for anti-LILRB4 antibody-encoding vectors. Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic cell host microorganism. However, many other genera, species, and strains, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as 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 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Trichopodium, and Aspergillus hosts, such as A. nidulans and A. niger are commonly available and useful herein.

[0247] Host cells suitable for expressing the glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrates include plant and insect cells. A number of baculovirus strains and variants, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm) have been identified. A variety of virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 variant of Bombyx mori NPV, have been published, and such viruses can be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as host cells.

[0248] However, the most interest lies in vertebrate cells, and growing vertebrate cells (tissue culture) has become an everyday procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human fetal kidney cell 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 carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; mouse pre-gastric cancer cells (MFC), SNU620 cells, and human hepatoma cell line (HepG2). In some embodiments, the host cells are mammalian cultured cell lines such as CHO, BHK, NS0, 293, MFC, SNU620 and their derivatives.

[0249] Transform a host cell with the above expression or cloning vector for antibody production and culture it in a conventional nutrient medium appropriately modified for inducing a promoter, selecting transformants, or amplifying a gene encoding a desired sequence. In another embodiment, the antibody may be produced by homologous recombination known in the art. In certain embodiments, the host cell is capable of producing the antibodies or antigen-binding fragments thereof provided herein.

[0250] The present disclosure also provides a method of expressing an antibody or antigen-binding fragment thereof and / or a chimeric antigen receptor provided herein, the method comprising culturing a host expression system provided herein under conditions in which the antibody or antigen-binding fragment thereof and / or the chimeric antigen receptor is expressed. The host expression systems used to produce the antibodies or antigen-binding fragments thereof and / or chimeric antigen receptors provided herein can be cultured in a variety of media. Commercially available media such as Ham’s F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco’s Modified Eagle Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90 / 03430; WO87 / 00195; or U.S. Patent No. 30,985 may be used as a culture medium for host cells. Any of these media may be supplemented with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™ drug), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source, as needed. Other optional supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions such as temperature, pH, and the like are those previously used for the host cells selected for expression and will be apparent to those of skill in the art.

[0251] Using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the medium. When antibodies are produced intracellularly, as a first step, particulate debris, which is either host cells or lysed fragments, is removed, for example, by centrifugation or ultracentrifugation. Carter et al., Bio / Technology 10:163-167 (1992) describe procedures for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed over about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When antibodies are secreted into the medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. To inhibit proteolysis, protease inhibitors, such as PMSF, may be included in any of the foregoing steps, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0252] Antibodies or antigen-binding fragments thereof and / or chimeric antigen receptors prepared from a host expression system can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0253] In certain embodiments, Protein A immobilized on a solid phase is used for the immunoaffinity purification of antibodies and their antigen-binding fragments and / or chimeric antigen receptors. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains can be purified using Protein A (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is bound is most often agarose, although other matrices are available. Mechanically stable matrices such as porous glass or poly(styrene divinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABXTM resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, heparin chromatography, SEPHAROSE™ chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0254] After any optional preliminary purification step(s), the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer between about pH 2.5 and 4.5, preferably at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0255] Pharmaceutical composition The present disclosure further provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof and / or a chimeric antigen receptor provided herein, and one or more pharmaceutically acceptable carriers.

[0256] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquids, gels, or solid carriers, aqueous media, non-aqueous media, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0257] Suitable components can include, for example, antioxidants, bulking agents, binders, disintegrants, buffers, preservatives, lubricants, flavorants, 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 hydroxytoluene, and / or propyl gallate. As disclosed herein, including one or more antioxidants such as methionine in a composition comprising an antibody or an antigen-binding fragment thereof and a conjugate provided herein reduces the oxidation of the antibody or an antigen-binding fragment thereof. This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving the stability of the antibody and maximizing its shelf life. Accordingly, in certain embodiments, provided is a pharmaceutical composition comprising one or more antibodies or antigen-binding fragments thereof disclosed herein and one or more antioxidants such as methionine. Further provided is a method of preventing oxidation of an antibody or antigen-binding fragment provided herein, extending its shelf life, and / or improving its efficacy by mixing the antibody or antigen-binding fragment with one or more antioxidants such as methionine.

[0258] To further illustrate, pharmaceutically acceptable carriers can include, for example, sodium chloride injection solution, Ringer's injection solution, isotonic dextrose injection solution, sterile water injection solution, or dextrose and lactate Ringer's injection solution, non-aqueous media such as fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffering agents such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, or polyvinyl pyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), metal ion sequestering agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antibacterial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers containing phenol or cresol, mercury compounds, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubilizing enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0259] 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.

[0260] In certain embodiments, the pharmaceutical composition is formulated into an injectable preparation. The injectable pharmaceutical composition can be prepared in any convenient form, such as a liquid solution, suspension, emulsion, or a solid form suitable for generating a liquid solution, suspension, or emulsion. The preparation of the injection can include a sterile and / or pyrogen-free solution ready for injection, a sterile dry soluble product, such as an implant tablet, a lyophilized powder ready to be mixed with a solvent immediately before use, a sterile suspension ready for injection, a sterile dry insoluble product ready to be mixed with a vehicle immediately before use, and a sterile and / or pyrogen-free emulsion. The solution can be either aqueous or non-aqueous.

[0261] In certain embodiments, the unit-dose parenteral preparation is packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration are known in the art and must be sterile and pyrogen-free, as practiced.

[0262] In certain embodiments, the sterile lyophilized powder is prepared by dissolving the antibodies or antigen-binding fragments disclosed herein in a suitable solvent. The solvent may contain excipients that improve stability, or other pharmacological components of the powder or the reconstitution solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain, for example in one embodiment, a buffer at a substantially neutral pH, such as citric acid, sodium or potassium phosphate, or other such buffers known to those of skill in the art. The desired formulation is provided by lyophilization under standard conditions known to those of skill in the art after subsequent filter sterilization of the solution. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dosage or multiple dosages of the antibody or its antigen-binding fragment or composition thereof. Overfilling the vial with a small amount (e.g., about 10%) in excess of the amount required for the dosage or dosage set is acceptable to facilitate accurate aspiration of the sample and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as from about 4°C to room temperature.

[0263] Reconstituting the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, sterile water and / or pyrogen-free water, or other suitable carriers for liquids, are added to the lyophilized powder for reconstitution. The exact amount depends on the selected treatment being given and can be determined empirically.

[0264] Kit In certain embodiments, the present 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 present 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 a chemotherapeutic agent, an anti-cancer agent, a radiation therapy agent, an immunotherapy agent, an anti-angiogenesis 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 chelating agent, and a cytokine.

[0265] Such kits may further include, if desired, one or more pharmaceutically acceptable carriers, one or more additional containers, etc., such as various conventional pharmaceutical kit components that will be readily apparent to those skilled in the art. Instructions for use, either as an insert or label indicating the amount of the components to be administered, guidelines for administration, and / or guidelines for mixing the components, can also be included in the kit.

[0266] Method of Use The present disclosure also provides a method of 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 human.

[0267] In some embodiments, the LILRB4-related disease, disorder, or condition is characterized by the expression or overexpression of LILRB4.

[0268] In certain embodiments, the disease, disorder, or condition is an immune disease, an inflammatory disease, cancer, or a neurological disease. In certain embodiments, the cancer is a LILRB4-expressing cancer. A "LILRB4-expressing" cancer, as used herein, refers to a cancer characterized by expressing the LILRB4 protein in cancer cells, tumor-infiltrating immune cells, or expressing LILRB4 at a level significantly higher than expected for normal cells in cancer cells, tumor-infiltrating immune cells. Various methods can be used to determine the presence and / or amount of LILRB4 in a test biological sample from a subject. For example, the test biological sample can be exposed to an anti-LILRB4 antibody or an antigen-binding fragment thereof that binds to and detects the expressed LILRB4 protein. Alternatively, LILRB4 can also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, and the like. In some embodiments, the test sample is derived from cancer cells or tissue, or tumor-infiltrating immune cells. 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 whom 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., tumor). In certain embodiments, the cancer is a solid tumor or a hematological tumor. In certain embodiments, the cancer is a LILRB4-expressing B cell cancer.

[0269] In certain embodiments, the disease, disorder, or condition is 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, squamous cell lung cancer, Lewis lung cancer, or radiation-resistant Lewis lung cancer), abdominal cancer, carcinoid cancer, bone cancer, pancreatic cancer, undifferentiated neuroectodermal tumor, skin cancer, gallbladder cancer, head or neck cancer, squamous cell carcinoma, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial cancer), anal region cancer (e.g., anal squamous cell carcinoma), gastric cancer or stomach cancer (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatoma, or liver carcinoma), cholangiocarcinoma, 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 cancer or ureteral cancer (e.g., renal oncocytoma), cutaneous T cell lymphoma, medulloblastoma, nephroblastoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, choroid plexus papilloma, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) neoplasm, soft tissue sarcoma (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi sarcoma), spinal cord tumors, glioma (e.g., ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, eye cancer (e.g., retinoblastoma), brainstem glioma, or mixed glioma, e.g., oligoastrocytoma), brain tumor (e.g., glioblastoma / multiform glioblastoma (GBM), non-glioblastoma brain tumor, or meningioma), melanoma (e.g., skin or intraocular melanoma), thrombocytosis, mesothelioma, fungating polyposis, Sézary syndrome, idiopathic myelofibrosis, solitary plasmacytoma, vestibular schwannoma, Ewing sarcoma, chondrosarcoma, MYH-related polyposis, pituitary adenoma, pediatric cancer, e.g., pediatric sarcoma (e.g., neuroblastoma, rhabdomyosarcoma, and osteosarcoma), blood cancer, lymphoma, Hodgkin lymphoma, non-Hodgkin 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 myelogenous leukemia (CML), acute myelogenous leukemia (AML), chronic myelomonocytic leukemia (CMML),Selected from the group consisting of chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia (HCL), Burkitt 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.,

[0270] In some embodiments, the subject is identified as having cancer cells or tumor-infiltrating immune cells that express LILRB4 at levels significantly higher than levels normally found in non-cancerous cells, optionally.

[0271] In another aspect, provided is a method of treating, preventing, or alleviating a disease, disorder, or condition in a subject that would benefit from modulation of LILRB4 activity, 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 an LILRB4-related disease, disorder, or condition as defined above.

[0272] The therapeutically effective amount of the antibody or antigen-binding fragment provided herein depends on various factors known in the art, such as, for example, the subject's body weight, age, past medical history, current medications, health status, and potential for cross-reactivity, allergies, hypersensitivities, and adverse side effects, as well as the route of administration and the degree of disease onset. One of ordinary skill in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage as indicated by these and other circumstances or requirements.

[0273] In certain embodiments, the antibodies or antigen-binding fragments provided herein and / or the chimeric antigen receptors provided herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the administered dose can vary over the course of the treatment. For example, in certain embodiments, the initial administered dose may be higher than subsequent administered doses. In certain embodiments, the administered dose may vary during the course of the treatment in response to the subject's response.

[0274] The dosing regimen may be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0275] 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, for example, administration can be via a parenteral route including subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection; or via a non-parenteral route including transdermal, oral, intranasal, intraocular, sublingual, rectal, or topical surface.

[0276] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein and / or the chimeric antigen receptors provided herein can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibodies or antigen-binding fragments thereof disclosed herein and / or the chimeric antigen receptors provided herein can be combined with a second therapeutic agent, such as a chemotherapeutic agent, an anti-cancer agent, a radiation therapy 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 activator, a contrast agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a co-stimulatory molecule agonist, a co-inhibitory molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or a functional fragment thereof, a detectable label or reporter, an antibacterial agent, a gene editing agent, a beta agonist, a viral RNA inhibitor, a polymerase inhibitor, an interferon, or a microRNA.

[0277] As used herein, the term "immunotherapy" refers to a type of treatment that stimulates the immune system to fight diseases such as cancer or enhances the immune system in a general way. Examples of immunotherapy include, but are not limited to, checkpoint regulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0278] "Targeted therapy" is a type of treatment 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 therapeutic agents to tumors, thereby preventing the effects of the therapeutic agents from reaching normal tissues.

[0279] In certain of these embodiments, the antibodies or antigen-binding fragments thereof provided herein, and / or the chimeric antigen receptors provided herein, and / or the pharmaceutical compositions provided herein, which are administered in combination with one or more additional therapeutic agents, may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the antibodies or antigen-binding fragments thereof and / or pharmaceutical compositions provided herein, and the additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. However, the antibodies or antigen-binding fragments thereof and / or chimeric antigen receptors and / or pharmaceutical compositions provided herein that are administered "in combination with" another therapeutic agent need not be administered simultaneously with or in the same composition as the agent. Antibodies or antigen-binding fragments thereof, or chimeric antigen receptors or pharmaceutical compositions that are administered before or after another agent are considered to be administered "in combination with" the agent as the term is used herein even if the antibody or antigen-binding fragment, or pharmaceutical composition or chimeric antigen receptor and the second agent are administered by different routes, if possible. The additional therapeutic agent administered in combination with the antibody or antigen-binding fragment thereof, chimeric antigen receptor, or pharmaceutical composition disclosed herein is administered according to the schedule set forth in the product information sheet for the additional therapeutic agent or according to Physicians’ Desk Reference 2003 (Physicians’ Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)) or according to protocols well known in the art.

[0280] The present disclosure further provides a method of inactivating LILRB4-expressing cells in vivo or in vitro, the method comprising contacting the LILRB4-expressing cells 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.

[0281] The present disclosure further provides a method of modulating LILRB4 activity in LILRB4-expressing cells, the method comprising exposing the LILRB4-expressing cells to an antibody or an 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, progenitor mast cells, endothelial cells, or osteoclasts.

[0282] In another aspect, the present disclosure provides a method of inducing phagocytosis of target cells in vivo or in vitro, the method comprising exposing the target cells to an antibody or an 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 cells are antigen-presenting cells, cancer cells, or cells infected with a pathogen.

[0283] In another aspect, the present disclosure provides a method of inducing the production of TNF-α, the method comprising exposing tolerogenic dendritic cells to an antibody or an antigen-binding fragment thereof provided herein, and / or a chimeric antigen receptor provided herein, and / or a pharmaceutical composition provided herein.

[0284] In another aspect, the present disclosure provides a method of detecting the presence or amount of LILRB4 in a sample, the method comprising contacting the sample with an antibody or an 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 amount of LILRB4 in the sample.

[0285] In another aspect, the present disclosure provides a method for diagnosing an LILRB4-related 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 an 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 an LILRB4-related disease, disorder, or condition in the subject.

[0286] In another aspect, the present disclosure provides a kit comprising an antibody or an 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 useful for detecting LILRB4, optionally recombinant LILRB4, LILRB4 expressed on the cell surface, or LILRB4-expressing cells. The kit may further comprise instructions for use.

[0287] In another aspect, the present disclosure also provides the use of an antibody or an antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein in the manufacture of a medicament for treating, preventing, or alleviating an LILRB4-related disease, disorder, or condition in a subject and in the manufacture of a diagnostic agent for diagnosing an LILRB4-related disease, disorder, or condition in a subject.

[0288] In another aspect, the present disclosure also provides the use of an antibody or an antigen-binding fragment thereof provided herein, and / or a chimeric antigen receptor provided herein, and / or a pharmaceutical composition provided herein in the manufacture of a diagnostic reagent for diagnosing an LILRB4-related disease, disorder, or condition.

[0289] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials, and methods described below fall, in whole or in part, within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention but are merely intended to illustrate specific embodiments that fall within the scope of the invention. One of ordinary skill in the art may develop equivalent compositions, materials, and methods without practicing the capabilities of the invention and without departing from the scope of the invention. It is understood that many variations can be made to the procedures described herein and still be within the scope of the invention. It is the intention of the inventors that such variations be included within the scope of the invention.

Example

[0290] [Example 1] Antibody Generation 1.1. Immunization To generate antibodies targeting LILRB4, in each group, Balb / c mice and CD1 mice were immunized with human LILRB4-overexpressing cells (CHO-S-hLILRB4) and / or recombinant LILRB4 protein (as shown in Table 5 below). The immunogens were delivered to the mice by intrahepatic injection (I.H.) and / or intraperitoneal injection (I.P.). After the primary immunization, several booster immunizations were performed until the animals developed a satisfactory antiserum titer suitable for hybridoma development. Complete Freund's adjuvant (CFA) was used for the primary immunization, and incomplete Freund's adjuvant (IFA) was used for subsequent immunizations. Test bleedings were performed and evaluated using FACS against a CHO-S cell line stably overexpressing human LILRB4 (CHO-S-hLILRB4) or ELISA using the extracellular domain of recombinant human LILRB4 protein. The immunization protocols for the animals in each group are shown in Tables 6, 7, 8, 9, 10, and 11 below.

[0291]

Table 5

[0292]

Table 6

[0293]

Table 7

[0294]

Table 8

[0295]

Table 9

[0296]

Table 10

[0297]

Table 11

[0298] 1.2. Generation and Screening of Hybridomas Spleen cell fusion was performed on the mice that responded best to immunization as determined by test bleeding FACS and ELISA. The FACS assay was performed on a CHO-S cell line (CHO-S-hLILRB4) that stably overexpresses human LILRB4. In the ELISA assay, the extracellular domain of the recombinant human LILRB4 protein was coated as a ligand and the antiserum concentration was measured. Lymphocytes from the spleen and lymph nodes were fused to a mouse myeloma cell line (SP2 / 0) using an optimized electrofusion protocol. Multiple fusions were performed to ensure the success of the project.

[0299] The fusions were seeded into multiple 96-well plates (2 × 10 4 ~105 Cells). The plates were monitored for growth and the medium was changed weekly. Wells in which the cells grew were screened on days 10 - 14 in a primary screening assay by FACS and / or other feasible assays such as ELISA. Multiple fusions of each targeted antigen were performed and screened. From the primary screening, positive parental clones showing binding positivity and positive ELISA signals against 293F-LILRB4 were expanded into 24-well plates for secondary screening.

[0300] After the primary screening, the positive parental clones expanded into 24-well plates were screened again by the assays described in the following FACS or ELISA assays. The desired hybridomas were selected and subcloning was advanced.

[0301] Parental hybridomas with the desired reactivity and isotype from the above screening funnel were subcloned by multiple rounds of limiting dilution or single cell sorting until monoclonal clones were obtained.

[0302] The subcloning plates were screened by protein or cell-based ELISA, and subclones with good binding ability were expanded into 24 wells for confirmation tests. The specificity and cross-reactivity of these subclones were confirmed by FACS analysis. Briefly, parental 293F cells and 293F-hLILRB4 were incubated with the antibodies produced by each subclone, respectively. A fluorescent dye-conjugated secondary antibody was used to detect the binding of the primary antibody to the cells. The mean fluorescence intensity (MFI) was measured by FACS analysis.

[0303] The desired subclone cell lines were sequenced and further expanded into culture flasks for cryopreservation. Cells 0.5 - 13.0×10 6Cells were initially cryopreserved at 4 to 6 vials per cell line per vial. Master cell banks and working cell banks were established for most of the selected valuable cell lines.

[0304] As shown in the results in Table 12, a total of 28 antibodies with unique sequences showing positive binding to 293F cells (293F-hLILRB4) that stably overexpress human LILRB4 protein were discovered, but they did not show binding to parental 293F cells. Among these, three antibodies (i.e., 42-F5-B12-F2, 44-F10-B6-D4, and 42-A10-C1-E10) were also able to bind to LILRB4 of cynomolgus monkey (Macaca fascicularis). The MFIs 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-Macaca fascicularis LILRB4 (CHO-S-cynoLILRB4) detected by FACS are summarized in Tables 12 and 13 below.

[0305] [Table 12-1] [Table 12-2]

[0306] [Table 13]

[0307] [Example 2] Characterization of Antibodies: Binding Affinity 2.1. Antibodies The 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 were characterized, and their nucleotide sequences were identified by the following method.

[0308] 2.2. Sequencing of Hybridomas Total RNA was isolated from hybridoma cells according to the technical manual of RNAiso Plus (TAKARA catalog number 9109). Next, the total RNA was reverse transcribed into cDNA using an isotype-specific antisense primer or a universal primer according to the technical manual of PrimeScript II 1st Strand cDNA Synthesis Kit (TAKARA catalog number 6210A). The VH and VL antibody fragments were amplified according to TaKaRa Taq (trademark) (catalog number R001A). The amplified antibody fragments were individually cloned into a standard cloning vector. Colony PCR was performed to screen for clones having inserts of the correct size. Five or more colonies having inserts of the correct size were sequenced for each fragment. The sequences of different clones were aligned to provide the consensus sequence of these clones.

[0309] The variable region sequences of the hybridoma antibodies are provided in Table 3 above in this specification.

[0310] 2.3.293 Binding affinity of antibodies for F-hLILRB4 cell line To evaluate the binding affinity of antibody candidates for hLILRB4 in a cell-based assay, the sequences of 17 mouse antibodies were selected from Table 12, and human IgG1 chimeric antibodies were prepared and produced. Also, two reference antibodies, IO-202 and NGM831, were prepared as human IgG1 chimeric antibodies. Briefly, the DNAs encoding the variable regions of the 17 mouse antibodies and the reference antibodies were synthesized and subcloned into an expression vector pre-containing the human IgG constant gene. The vector was 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-G3, 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, where the prefix "ch" indicates "chimeric" and the following indicates the hybridoma antibody clone. For example, ch27-F11-E10-G10 indicates that it is a chimeric antibody derived from the hybridoma antibody clone 27-F11-E10-G10. The two reference antibodies prepared as human IgG1 chimeric antibodies are also named IO-202 and NGM831, respectively.

[0311] The binding affinities of these chimeric antibodies and reference antibodies (i.e., IO-202 and NGM831) to 293F cells (293F-hLILRB4) that stably overexpress human LILRB4 protein were determined by FACS analysis. The binding affinities of these chimeric antibodies and reference antibodies to LILRB were measured by FACS in 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, and CHO-S-Macaca fascicularis LILRB4 (CHO-S-cynoLILRB4). The results are summarized in Table 14 below.

[0312] The protocol for FACS analysis is described below: (a) Cells were harvested by centrifugation at 300 g for 3 minutes. (b) The cells were washed twice with FACS buffer by centrifuging at 300 g for 3 minutes and discarding the supernatant. (c) The cells were resuspended and seeded into assay plates in 50 μl of FACS buffer (2×10 5 cells / well). 50 μL of primary antibody was added to the plates at a series of final concentrations (66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09, 0.03 nM). The cells were incubated with the antibody at 4°C for 1 hour. (d) Step (b) was repeated, and the cells were resuspended with 100 μl of diluted secondary antibody and incubated at 4°C in the dark for 1 hour. (e) Step (b) was repeated, and the cells were resuspended in 100 μl of FACS buffer. The cells were stored in the dark for FACS analysis.

[0313] As shown in Table 14 and Figures 1, 2, and 3, the binding affinity of the selected chimeric antibodies for the 293F-hLILRB4 cell line was higher than, lower than, or equivalent to that of the reference antibodies IO-202 and NGM831 (Table 14 and Figure 1). The ch43-D12-F3-G11 and ch42-C8-A12-F3-D11 antibodies showed significant cross-reactivity with human LILRB3 (Figures 1 and 2A–B), and the ch44-F10-B6-D4 antibody showed good cross-reactivity with cynomolgus monkey LILRB4 (Figures 1 and 3A–B).

[0314]

Table 14-1

Table 14-2

Table 14-3

[0315] 2.4 Expression pattern of LILRB4 in human cancer cell lines To compare the expression of LILRB4 in different human cancer cell lines, a human LILRB4 APC-conjugated antibody (clone: 293623, catalog: FAB24251A-100) was used for FACS analysis. As shown in Figure 4, the expression level of LILRB4 was high in some human cancer cell lines such as THP-1 (acute myeloid leukemia, AML5) and MV-4-11 (acute myeloid leukemia, AML5).

[0316] [Example 3] Characterization of antibodies: Antibody-dependent cell cytotoxicity (ADCC) 3.1. Determination of ADCC of the selected antibodies To measure the ADCC effect specific for hLILRB4, carboxyfluorescein succinimidyl ester (CFSE)-labeled THP-1 cells were seeded at 5×10 4 cells / well in a 96-well flat-bottom sterile plate and used as target cells, and 2×10 5Individual fresh isolated human PBMCs were added as effector cells. Target cells, effector cells (those with FcR blocked prior to addition of antibody to CFSE-labeled THP-1 cells), and increasing concentrations of antibody were combined in a total volume of 200 μL in a U-bottom 96-well plate with X-VIVO medium containing 50 ng / mL of IL-2. After incubating the cells and antibody for 20 hours at 37 °C and 5% CO2, the percentage of CFSE-labeled THP-1 cells was measured by FACS analysis. IO-202 and NGM831 (prepared as human IgG1 chimeric antibodies) were used as reference antibodies, and human IgG1 was used as a negative control.

[0317] The percentage of cytotoxicity was calculated according to the following formula:

[0318]

Number

[0319] 3.2. Results As shown in Table 15 and Figure 5, the ch2-H1-D7-E5-D5 antibody showed a significantly stronger ADCC effect against THP-1 cells and a lower EC 50 compared to the reference antibodies IO-202 and NGM831.

[0320]

Table 15

[0321] [Example 4] Characterization of Antibodies: Antibody-Dependent Cellular Phagocytosis (ADCP) 4.1. Determination of ADCP of Selected Antibodies To measure the ADCP effect specific to hLILRB4, CFSE-labeled MV-4-11 and Far-Red-labeled M2 macrophages differentiated from isolated human PBMCs were added as effector cells. IO-202 and NGM831 were used as reference antibodies (prepared as human IgG1 chimeric antibodies), and human IgG1 was used as a negative control. The ADCP assay was performed as described below: (a) First, CD14 + monocytes were isolated from PBMCs by EasySep (STEMCELL Inc.). (b) Monocytes were cultured in X-VIVO15 for 8 days with 100 ng / ml rhM-CSF on days 0 and 3, and with 100 ng / ml rhIL10 and rhTGF-β on day 5 to differentiate into M2 macrophages. (c) 2×10 4 Far-Red-labeled M2 macrophages were mixed with various concentrations of antibodies and 2×10 4 CFSE-labeled MV-4-11 cells, and FcR was blocked before adding the antibodies (effector:target = 1:1). (d) The mixture was incubated in a 37 °C CO2 incubator for 2 hours. (e) Cells were digested with trypsin (1×) and centrifuged at 500 g for 3 minutes. (f) Cells were washed twice with FACS buffer by centrifugation at 500 g for 3 minutes. (g) Phagocytosis ability was evaluated by flow cytometry, and the results were recorded as the ratio of CFSE and Far-Red positive cells to all macrophages in the sample.

[0322] 4.2. Results As shown in Table 16 and Figure 6, all chimeric antibodies showed a strong ADCP effect on MV-4-11 cells. The chimeric antibody ch2-H1-D7-E5-D5 showed a lower EC 50 compared to the reference antibody IO-202 (Table 16 and Figure 6).

[0323]

Table 16

[0324] [Example 5] Characterization of antibodies: Reprogramming of tolerogenic dendritic cells by blocking the LILRB4-fibronectin interaction 5.1 Anti-LILRB4 antibodies block the LILRB4-fibronectin interaction and reprogram tolerogenic dendritic cells 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 tolerogenic. LILRB4, which contains three cytoplasmic ITIMs (immunoreceptor tyrosine-based inhibitory motifs) in its intracellular domain, is significantly overexpressed in tolerogenic dendritic cells. FcγRI (CD64), a high-affinity IgG receptor, is significantly expressed in many myeloid cells such as dendritic cells and macrophages. The interaction between IgG and FcγRI induces the intracellular ITAM (immunoreceptor tyrosine-based activation motif) signal of FcγRI and promotes the production of TNF-α. It has been revealed that there is crosstalk between the signal transduction pathways via LILRB4 and FcγRI, and LILRB4 regulates FcγRI-mediated ITAM activation to secrete TNF-α. Fibronectin is a functional ligand of LILRB4 and contributes to dendritic cells that do not respond to stimulation via the FcγRI-IgG interaction. However, FcγRI signaling can be reversed by blocking the fibronectin-LILRB4 interaction via anti-LILRB4 antibodies. Therefore, the inventors evaluated the inhibitory ability of the anti-LILRB4 antibodies described in the claims via this FcγR stimulation assay in tolerogenic moDCs. As reference antibodies, IO-202 and NGM831 prepared as human IgG1 chimeric antibodies were used. The secretion of TNF-α from tolerogenic dendritic cells was used as a readout for evaluating FcγR stimulation. The assay was performed as described below: (a) Day 0: Isolate monocytes from PBMCs and resuspend 5×10 6 cells in 3 ml of X-VIVO supplemented with 2 U / ml of dendritic cell growth factor, and then culture the cells in a 6-well plate. (b) Day 2: Add 2 ml of fresh complete medium supplemented with 2 U / ml of dendritic cell growth factor per well. (c) Day 5: Add 10 nmol / L of dexamethasone and 100 nmol / L of vitamin D3 to differentiate immature moDCs into tolDCs. (d) Day 7: (1) Co - coat a 96-well plate (Corning) with 5 μg / mL of fibronectin and 5 μg / mL of human IgG1 in PBS at room temperature for 2 hours. (2) Wash the plate twice with PBS and pre - incubate it with RPMI1640 containing 10% FBS for 2 hours. (3) Plate tolerogenic human dendritic cells (5 - 7×10 4 cells / well) in X - VIVO15 medium into the coated wells and incubate with 5 μg / mL of the designated antibody at room temperature for 5 - 20 minutes. (4) Mix the cells in the coated wells well and incubate overnight in a 37°C CO2 incubator. (5) After overnight incubation at 37°C, collect the culture supernatant and measure cytokine secretion according to the manufacturer's instructions using an ELISA assay with a human TNF - α kit.

[0325] 5.2. Results As shown in Table 17, all 17 chimeric LILRB4 antibodies significantly promoted the production of TNF - α. This suggests that these LILRB4 antibody candidates strongly block the LILRB4 - fibronectin interaction, reprogram tolerogenic dendritic cells into mature dendritic cells, and stimulate T cell activation, proliferation, and differentiation. Among the 17 antibody candidates, 15 except ch43 - D12 - F3 - G11 and ch44 - F10 - B6 - D4 showed a stronger blocking effect or reprogramming effect compared to the reference antibodies IO - 202 and NGM831.

[0326]

Table 17

[0327] [Example 6] Characterization of Antibodies: Antigen-Presenting Ability and Co-Stimulatory Ability of Dendritic Cells 6.1. LILRB4 Antibodies Reprogram Antigen Presentation and Co-Stimulation of DCs To test the ability of LILRB4 antibody candidates to regulate the expression of antigen-presenting molecules and co-stimulatory molecules, the assays described below were performed. As reference antibodies, IO-202 and NGM831 prepared as human IgG1 chimeric antibodies were used. (a) Day 0: Monocytes were isolated from PBMCs and resuspended in 3 mL of X-VIVO supplemented with 2 U / mL of dendritic cell growth factor at 5×10 6 cells, and then the cells were cultured in a 6-well plate. (b) Day 2: 2 mL of fresh complete medium supplemented with 2 U / ml of dendritic cell growth factor was added per well. (c) Day 5: 10 nM of dexamethasone and 100 nM of vitamin D3 were added to differentiate immature DCs into tolDCs. (d) Day 7: Samples were detected by flow cytometry with CD86-PE, HLA-DR-FITC, and HLA-ABC-APC staining.

[0328] 6.2. Results As shown in Table 18, all chimeric antibodies induced overexpression of antigen-presenting molecules (HLA-DR and HLA-ABC) and co-stimulatory molecules (CD86) in DCs. From these results, it was shown that chimeric antibodies reprogram tolerogenic DCs to activate T cells. Some of the antibodies (e.g., ch7-B4-C1-C9, ch25-E7-A10-H6, ch2-H8-C9-F7-E2, etc.) showed a stronger expression induction effect compared to the reference antibodies IO-202 and NGM831.

[0329]

Table 18

[0330] [Example 7] Characterization of antibodies: Co - culture assay of selected chimeric antibodies with THP - 1 and T cells The chimeric antibodies ch2 - H1 - D7 - E5 - D5 and ch8 - B3 - F6 - H8 prepared in Example 2.3 were used in the co - culture assay of THP - 1 and T cells. The protocol for the co - culture assay of THP - 1 and T cells is as follows: (a) Human T cells (5×10 4 cells per well) isolated from the peripheral blood of healthy donors were placed in the lower chamber of a 96 - well transwell plate. (b) Irradiated THP - 1 cells (E:T ratio = 1:4) were cultured in the upper chamber of a transwell insert (pore size 3 mm; Thermo Fisher) in a U - bottom 96 - well plate and treated with the indicated antibodies. (c) After culturing in the lower chamber with anti - CD3 / CD28 - coated beads (Thermo Fisher) and 50 U / mL of rhIL2 for 5 - 7 days, representative T cells in the lower chamber were photographed using a microscope. T cells were stained with anti - CD3 - PE or anti - CD8 - APC / Fire750 and analyzed by flow cytometry.

[0331] As shown in Figures 11A - D, the chimeric antibodies ch2 - H1 - D7 - E5 - D5 and ch8 - B3 - F6 - H8 reversed THP - 1 - mediated T cell suppression.

[0332] [Example 8] Characterization of antibodies: In vivo efficacy test of selected chimeric antibodies in the treatment of radiation - resistant Lewis lung cancer (LLC1) and EL4 - LILRB4 lymphoma in female hLILRB1 / hLILRB4 transgenic mice 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-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.

[0333] 8.1. Test Design and Methods 8.1.1. Radiation-Resistant LLC1 Model The protocol for the in vivo efficacy assay in the radiation-resistant LLC1 model is summarized in Table 19 below and detailed as follows. (a) LLC1 tumor cells were maintained in vitro at 37°C in a 5% CO2 atmosphere in air using DMEM medium supplemented with 10% fetal bovine serum. Prior to tumor inoculation, cells in the exponential growth phase were harvested and quantified using a cell counter. (b) Each mouse was subcutaneously inoculated with 0.1 ml of LLC1 tumor cells (3 × 10^6) in PBS in the right posterior flank to develop tumors. The randomization day was designated as day 0, and administration was initiated on day 0. (c) Randomization was initiated when the average tumor size reached approximately 70 - 100 mm 3 . Eighteen mice were enrolled in this study. All animals were randomly assigned to three test groups. Randomization was performed based on the block method. (d) Tumor volume was measured two-dimensionally twice a week using calipers, and the volume was expressed in mm 3 using the following formula: V = (L × W × W) / 2, where V is the tumor volume, L is the tumor length (the longest dimension of the tumor), and W is the tumor width (the longest dimension of the tumor perpendicular to L). Administration, as well as tumor and body weight measurements, were performed within a laminar flow cabinet.

[0334] [Table 19]

[0335] 8.1.2. EL4-LILRB4 Lymphoma Model The protocol for the in vivo efficacy assay in the EL4-LILRB4 lymphoma model is summarized in Table 20 below and is detailed as follows. (a) EL4-LILRB4 lymphoma tumor cells were maintained in vitro at 37 °C in a 5% CO2 atmosphere in air using DMEM medium supplemented with 10% fetal bovine serum. Before tumor inoculation, cells in the exponential growth phase were harvested and quantified using a cell counter. (b) Each mouse was inoculated intravenously with 0.2 ml of EL4-LILRB4 lymphoma tumor cells (0.2 × 10^6) in PBS into the right hind flank to generate tumors. The randomization day was designated as day 0, and dosing was started on day 0. (c) Randomization was started when the total flux reached approximately 10 6 p / s. Eighteen mice were enrolled in this study. All animals were randomly assigned to three test groups. Randomization was performed based on the block method. (d) Tumor flux was measured twice a week. Dosing, as well as tumor and body weight measurements, were performed in a laminar flow cabinet.

[0336]

Table 20

[0337] 8.2. Results As shown in Figure 7, ch8-B3-F6-H8 showed strong in vivo efficacy. For example, as shown in Figure 7A, 10 mg / kg of ch8-B3-F6-H8 in combination with 10 Gy of radiotherapy significantly inhibited LLC1 tumor growth in vivo with a 40% tumor growth inhibition rate (TGI), while the reference antibody (i.e., IO-202) showed only 24% TGI in the LLC1 syngeneic model. As shown in Figure 7B, 3 mg / kg of ch8-B3-F6-H8 significantly inhibited EL4-LILRB4 tumor growth in vivo with a 62% TGI, while the reference antibody (i.e., IO-202) showed 53% TGI in the EL4-LILRB4 syngeneic model. In addition, in the LLC1 model (Figure 7C) and the EL4-LILRB4 model (Figure 7D), the body weights of the mice in each group did not change significantly.

[0338] [Example 9] Humanized antibodies of 2-H1-D7-E5-D5 and 8-B3-F6-H8: Generation and antibody characterization 9.1 Generation of humanized antibodies The chimeric antibodies ch2-H1-D7-E5-D5 and ch8-B3-F6-H8 were selected as clones for humanization. The antibody sequences were aligned with human germline sequences to identify the best-fit model. The human germline sequence with the best match was selected as a template for humanization based on its homology to the original mouse antibody sequence. Generally, humanization of antibodies is performed by comparing the IMGT (https: / / www.imgt.org) human antibody heavy and light chain variable germline gene databases, selecting the heavy and light chain variable germline genes with high homology to the murine-derived antibody as templates, and transplanting the CDRs of the murine-derived antibody into the corresponding human templates. A variable region sequence with the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 was formed. If necessary, important amino acids in the framework sequence were reverted to ensure the original affinity and mutated to the amino acids corresponding to the murine antibody. The two humanized antibodies were named 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 serial 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 from Table 21, the CDRs of h2-H1-D7-E5-D5 are identical to those of the corresponding mouse monoclonal antibody 2-H1-D7-E5-D5; the CDRs of h8-B3-F6-H8 are identical to those of the corresponding mouse monoclonal antibody 8-B3-F6-H8 except for HCDR2. In particular, the amino acid sequence of HCDR2 of h8-B3-F6-H8 is as described in SEQ ID NO: 158 (INP E SSAI), while the amino acid sequence of HCDR2 of the corresponding mouse monoclonal antibody 8-B3-F6-H8 is as described in SEQ ID NO: 42 (INP D SSAI). Without wishing to be bound by any particular theory, such a change in HCDR2 is considered favorable for the binding affinity to human LILRB4.

[0339] 9.2. Binding Affinity Test of Humanized Antibodies 9.2.1. Protein-based Affinity Test by Biacore (Biointron) The parameters of the protein-based affinity test by Biacore (Biointron) were as follows: Analyte: LILRB4; Running buffer: HBS-EP + ; Flow rate: 30 μL / min; Capture: Abs, 10 μL / min, for 60 seconds; Injection of serially diluted LILRB4; Contact time: 180 seconds, dissociation time: 400 seconds; Regeneration: pH 1.5 Gly, 30 μL / min, for 30 seconds; Method: Affinity using multi-cycle kinetics / capture; Machine model: Biacore 8K (GE) Analysis temperature: 25 °C

[0340] 9.2.2. Protein-based Affinity Test by ELISA The protocol of the protein-based affinity test by ELISA is as follows: (a) Diluted soluble protein (0.5 μg / mL) was added to a Clear Flat-Bottom Immuno 96-well plate (Nunc, catalog number 442404) at 100 μl / well and incubated overnight at 4 °C. (b) Cells were washed three times with PBS containing 0.05% Tween-20 (PBST / BSA). (c) Block the plate: 200 μl of 2% BSA was added to each well and incubated at 37 °C for 1 hour. (d) Wash three times with PBS containing 0.05% Tween-20 (PBST / BSA). (e) 100 μl of the test antibody (final concentration of primary antibody: adjust the starting concentration of the antibody to 1000 nM or 120 nM and serially dilute the antibody at a ratio of 1:3) was added to each well and incubated at 37 °C for 1 hour. (f) Wash three times with PBS containing 0.05% Tween-20 (PBST / BSA). (g) 100 μl of HRP-conjugated secondary antibody was added at a dilution of 1:10,000 and incubated at 37 °C for 1 hour. (h) It was washed three times with PBS (PBST / BSA) containing 0.05% Tween-20. (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. (j) 50 μl of Stop Solution was added to each well and read at 450 nm using an MD SpectraMax iD3.

[0341] 9.2.3. Binding Affinity of Cell-Based to 293F-hLILRB4 Cells The protocol for the cell-based binding affinity to 293F-hLILRB4 cells is the same as the protocol described in Example 2.3.

[0342] 9.2.4. Competitive Cell-Based Binding Affinity to 293F-hLILRB4 Cells The protocol for the competitive cell-based binding affinity to 293F-hLILRB4 cells is as follows: (a) The cells were harvested by centrifugation at 300 g for 3 minutes, and the supernatant was discarded. (b) The cells were washed twice with FACS buffer by centrifuging at 300 g for 3 minutes and discarding the supernatant. (c) The cells were resuspended and seeded onto an assay plate in 50 μl of FACS buffer (2 × 10 5 cells / well). 50 μl of primary antibody was added to the plate at a series of final concentrations (the working concentration of one competing antibody, e.g., h2-H1-D7-E5-D5, is 10 μg / ml, and the working concentrations of the other three antibodies including IO-202, NGM831, and h8-B3-F6-H8 are 50 μg / ml, with three consecutive dilutions in a 10-fold gradient from 50 μg / ml), and incubated at 4 °C for 1 hour. (d) Repeat step (b), resuspend the cells with 100 μl of diluted secondary antibody, and incubate at 4 °C in the dark for 1 hour. (e) Repeat step (b), resuspend the cells with 100 μl of FACS buffer. The cells were stored in the dark for FACS analysis.

[0343] 9.2.5. Results As shown in FIGS. 8A - C, the binding affinity of the humanized antibody h8 - B3 - F6 - H8 to the 293F - hLILRB4 cell line was high, comparable to the reference antibodies (IO - 202 and NGM831) in ELISA and cell - based binding assays. Also, the humanized antibody h2 - H1 - D7 - E5 - D5 showed binding to the 293F - hLILRB4 cell line in ELISA and cell - based binding assays. As shown in FIGS. 8D - F, h8 - B3 - F6 - H8 had a binding epitope similar to IO - 202, while h2 - H1 - D7 - E5 - D5 had a specific binding epitope.

[0344] 9.3. Characterization of Antibodies: Mixed Lymphocyte Reaction (MLR) of Humanized Antibodies The humanized antibodies h2 - H1 - D7 - E5 - D5 and h8 - B3 - F6 - H8 prepared in Example 9.1 were used in the MLR. IO - 202 and NGM831 were used as positive controls, and hIgG1 was used as a negative control. The protocol for the MLR of humanized antibodies is as follows: (a) Day 0: Monocytes were isolated from PBMC. 5×10 6 cells were resuspended in 3 ml of X - VIVO supplemented with 2 U / ml of dendritic cell growth factor and cultured in a 6 - well plate. (b) Day 2: 2 ml of fresh complete medium supplemented with 2 U / ml of dendritic cell growth factor was added per well. (c) Day 5: Immature DCs were differentiated into tolDCs by adding 10 nmol / L of dexamethasone and 100 nmol / L of vitamin D3. (d) Day 6: The next day, 50 - 100 million PBMCs were ordered for T - cell isolation. (e) Day 7: After 48 hours of stimulation, immature DCs differentiated into mature dendritic cells and tolDCs, respectively. (f) Day 7: T cells were isolated from PBMCs (approximately 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 was not labeled. (g) Day 7: 200,000 cells of T cells per well and 25,000 cells of tolDCs per well were seeded in 96 - well plates (tolDCs are non - adherent cells). (h) Day 7: The mixed cells 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), and repeated 3 times for each group. Also, two groups of T cells alone and tolDCs alone were used as controls. (i) Day 12: The supernatant was assayed by IFN - γ ELISA kit.

[0345] As shown in Figure 9, the humanized antibody h8 - B3 - F6 - H8 reactivated tolDCs and primed the activation of T cells.

[0346] 9.4. Characterization of antibodies: Co - culture assay of humanized antibodies with macrophages and T cells The humanized antibodies h2 - H1 - D7 - E5 - D5 and h8 - B3 - F6 - H8 prepared in Example 9.1 were used in the co - culture assay of macrophages and T cells. IO - 202 and NGM831 were used as positive controls, and hIgG1 was used as a negative control. The protocol for the co - culture assay of macrophages and T cells is as follows: (a) Monocytes were separated by EasySep™ Human CD14 Positive Selection Kit II (Stemcell, 17858). (b) Monocytes were cultured in X - VIVO (55 μm β - ME) and 100 ng / mL of rhM - CSF for 5 days. Half of the medium was replaced, and cytokines were added every 2 - 3 days. (c) TGF-β at 50 ng / mL and rhIL-10 were added, and the cells were cultured for an additional 2 days. (d) The cells were digested with Accutase enzyme cell detachment medium at 37 °C for 10 minutes, centrifuged, and resuspended in X-VIVO (55 μm β-ME). (e) 3.3×10 4 cells and 1×10 4 macrophages per well were seeded, antibody (10 μg / mL) was added to the 96-well plate, and the plate was incubated at 37 °C, 5% CO2 for 1 hour. (f) Soluble CD3 antibody at 30 ng / mL was added to the T cells. 1×10 5 T cells per well were seeded in the 96-well plate and cultured at 37 °C, 5% CO2 for 3 days. (g) Subsequently, the supernatant was collected by centrifugation for IFN-γ.

[0347] As shown in Figure 10, the humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 reversed macrophage-mediated T cell suppression.

[0348] 9.5. Characterization of Antibodies: Cytotoxic CD8 + T Cell Killing Assay The humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 prepared in Example 9.1 were used in the cytotoxic CD8 + T cell killing assay. The protocol for the cytotoxic CD8 + T cell killing assay is as follows: (a) CD3 + T cells were isolated from PBMC. (b) 5×10 3 target cells (THP-1 with labeled CFSE) were added to the wells of a 96-well U-bottom plate. (c) 1×10 4 T cells were added to the wells (effector:target ratio of 20:1). (d) The target cells and T cells were mixed at a final concentration of 20 μg / ml. (e) The cells were incubated at 37 °C for 20 hours and at 5% CO2 for 18 hours. (f) Finally, the cells were washed and resuspended in 200 μL of staining buffer. The cells were acquired by FACS, and the proportion of CFSE-labeled THP-1 cells was measured.

[0349] The proportion of cytotoxicity was calculated according to the following formula:

[0350]

Number

[0351] As shown in Figure 12, the humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 enhanced CD8 + T cell-mediated cytotoxicity against THP-1 cells.

[0352] 9.6. Characterization of antibodies: ADCC assay and ADCP assay of humanized antibodies The humanized antibody h2-H1-D7-E5-D5 prepared in Example 9.1 was used in the ADCC assay and the ADCP assay. The protocol of the ADCC assay was the same as the protocol described in Example 3.1, and the protocol of the ADCP assay was the same as the protocol described in Example 4.1 except that different test antibodies were used.

[0353] As shown in Figures 13A - B, the humanized antibody h2-H1-D7-E5-D5 showed significantly stronger ADCC and ADCP effects against THP-1 cells and a lower EC 50 compared with the reference antibody IO-202.

[0354] 9.7. Characterization of antibodies: In vivo efficacy test of humanized antibodies in the treatment of EL4-LILRB4 lymphoma in female hLILRB1 / hLILRB4 transgenic mice 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 in the treatment of 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.

[0355] The protocol for the in vivo efficacy assay in the EL4-LILRB4 lymphoma model was the same as the protocol described in Example 8.1.2, except that the humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 were used as test antibodies.

[0356] As shown in FIGS. 14A-B, the humanized antibodies h2-H1-D7-E5-D5 and h8-B3-F6-H8 showed potent in vivo efficacy compared to IO-202, and h2-H1-D7-E5-D5 had the strongest in vivo efficacy. In particular, as shown in FIG. 14A, 3 mg / kg of h2-H1-D7-E5-D5 and 3 mg / kg of h8-B3-F6-H8 significantly inhibited EL4-LILRB4 tumor growth in vivo with 77% and 60% TGI, respectively, while the reference antibody IO-202 showed 53% TGI in the EL4-LILRB4 syngeneic model. In addition, as shown in FIG. 14B, the body weights of the mice in each group did not change significantly.

Claims

1. An antibody that binds to LILRB4 or an antigen-binding fragment thereof, containing one, two, or three heavy chain complementarity-determining regions (HCDR1, HCDR2, and / or HCDR3) within any one heavy chain variable (VH) region sequence selected from the group consisting of SEQ ID NOs: 154, 156, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, and 151; and / or One, two, or three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within one of the light chain variable (VL) region sequences selected from the group consisting of SEQ ID NOs: 155, 157, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, and 152. An antibody containing an antigen-binding fragment thereof.

2. Sequence numbers 113, 114, 115, 116, 117, 118, 41, 158, 43, 44, 45, 46, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 33, 34, 35, 36, 37, 38, 42, 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 The antibody or antigen-binding fragment according to claim 1, comprising at least one heavy chain or light chain complementarity determining region (CDR) containing an amino acid sequence selected from the group consisting of 98, 99, 100, 101, 102, 105, 106, 107, 108, 109, 110, 121, 122, 123, 124, 125, 126, 129, 130, 131, 132, 133, 134, 137, 138, 139, 140, 141, 142, 145, 146, 147, 148, 149, and 150.

3. a) One, two, or three HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 113, 114, 115, 41, 158, 43, 17, 18, 19, 25, 26, 27, 33, 34, 35, 42, 49, 50, 51, 57, 58, 59, 65, 66, 67, 73, 74, 75, 81, 82, 83, 89, 90, 91, 97, 98, 99, 105, 106, 107, 121, 122, 123, 129, 130, 131, 137, 138, 139, 145, 146, and 147, and / or b) One, two, or three LCDR1, LCDR2, and LCDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 116, 117, 118, 44, 45, 46, 20, 21, 22, 28, 29, 30, 36, 37, 38, 52, 53, 54, 60, 61, 62, 68, 69, 70, 76, 77, 78, 84, 85, 86, 92, 93, 94, 100, 101, 102, 108, 109, 110, 124, 125, 126, 132, 133, 134, 140, 141, 142, 148, 149, and 150. The antibody or antigen-binding fragment thereof according to claim 1, comprising:

4. i. HCDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 113, 41, 17, 25, 33, 49, 57, 65, 73, 81, 89, 97, 105, 121, 129, 137, and 145; ii. HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 114, 158, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 122, 130, 138 and 146; and iii. HCDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 43, 19, 27, 35, 51, 59, 67, 75, 83, 91, 99, 107, 123, 131, 139 and 147, and / or i. LCDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 116, 44, 20, 28, 36, 52, 60, 68, 76, 84, 92, 100, 108, 124, 132, 140, and 148; ii. LCDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 117, 45, 21, 29, 37, 53, 61, 69, 77, 85, 93, 101, 109, 125, 133, 141 and 149; and iii. LCDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 46, 22, 30, 38, 54, 62, 70, 78, 86, 94, 102, 110, 126, 134, 142 and 150, preferably i. HCDR1 containing the amino acid sequence described in SEQ ID NO: 113, HCDR2 containing the amino acid sequence described in SEQ ID NO: 114, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 115; ii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 41, HCDR2 containing the amino acid sequence described in SEQ ID NO: 158, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 43; iii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 17, HCDR2 containing the amino acid sequence described in SEQ ID NO: 18, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 19; iv. HCDR1 containing the amino acid sequence described in SEQ ID NO: 25, HCDR2 containing the amino acid sequence described in SEQ ID NO: 26, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 27; v. HCDR1 containing the amino acid sequence described in SEQ ID NO: 33, HCDR2 containing the amino acid sequence described in SEQ ID NO: 34, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 35; vi. HCDR1 containing the amino acid sequence described in SEQ ID NO: 41, HCDR2 containing the amino acid sequence described in SEQ ID NO: 42, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 43; vii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 49, HCDR2 containing the amino acid sequence described in SEQ ID NO: 50, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 51; viiii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 57, HCDR2 containing the amino acid sequence described in SEQ ID NO: 58, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 59; ix. HCDR1 containing the amino acid sequence described in SEQ ID NO: 65, HCDR2 containing the amino acid sequence described in SEQ ID NO: 66, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 67; x. HCDR1 containing the amino acid sequence described in SEQ ID NO: 73, HCDR2 containing the amino acid sequence described in SEQ ID NO: 74, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 75; xi. HCDR1 containing the amino acid sequence described in SEQ ID NO: 81, HCDR2 containing the amino acid sequence described in SEQ ID NO: 82, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 83; xi. HCDR1 containing the amino acid sequence described in SEQ ID NO: 89, HCDR2 containing the amino acid sequence described in SEQ ID NO: 90, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 91; xiiii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 97, HCDR2 containing the amino acid sequence described in SEQ ID NO: 98, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 99; xiv. HCDR1 containing the amino acid sequence described in SEQ ID NO: 105, HCDR2 containing the amino acid sequence described in SEQ ID NO: 106, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 107; xv. HCDR1 containing the amino acid sequence described in SEQ ID NO: 121, HCDR2 containing the amino acid sequence described in SEQ ID NO: 122, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 123; xvi. HCDR1 containing the amino acid sequence described in SEQ ID NO: 129, HCDR2 containing the amino acid sequence described in SEQ ID NO: 130, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 131; xvii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 137, HCDR2 containing the amino acid sequence described in SEQ ID NO: 138, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 139; or xviiii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 145, HCDR2 containing the amino acid sequence described in SEQ ID NO: 146, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 147, and / or i. LCDR1 containing the amino acid sequence described in SEQ ID NO: 116, LCDR2 containing the amino acid sequence described in SEQ ID NO: 117, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 118; ii. LCDR1 containing the amino acid sequence described in SEQ ID NO: 44, LCDR2 containing the amino acid sequence described in SEQ ID NO: 45, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 46; iii. LCDR1 containing the amino acid sequence described in SEQ ID NO: 20, LCDR2 containing the amino acid sequence described in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 22; iv. LCDR1 containing the amino acid sequence described in SEQ ID NO: 28, LCDR2 containing the amino acid sequence described in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 30; v. LCDR1 containing the amino acid sequence described in SEQ ID NO: 36, LCDR2 containing the amino acid sequence described in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 38; vi. LCDR1 containing the amino acid sequence described in SEQ ID NO: 52, LCDR2 containing the amino acid sequence described in SEQ ID NO: 53, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 54; vii. LCDR1 containing the amino acid sequence described in SEQ ID NO: 60, LCDR2 containing the amino acid sequence described in SEQ ID NO: 61, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 62; viiii. LCDR1 containing the amino acid sequence described in SEQ ID NO: 68, LCDR2 containing the amino acid sequence described in SEQ ID NO: 69, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 70; ix. LCDR1 containing the amino acid sequence described in SEQ ID NO: 76, LCDR2 containing the amino acid sequence described in SEQ ID NO: 77, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 78; x. LCDR1 containing the amino acid sequence described in SEQ ID NO: 84, LCDR2 containing the amino acid sequence described in SEQ ID NO: 85, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 86; xi. LCDR1 containing the amino acid sequence described in SEQ ID NO: 92, LCDR2 containing the amino acid sequence described in SEQ ID NO: 93, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 94; xi. LCDR1 containing the amino acid sequence described in SEQ ID NO: 100, LCDR2 containing the amino acid sequence described in SEQ ID NO: 101, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 102; xiiii. LCDR1 containing the amino acid sequence described in SEQ ID NO: 108, LCDR2 containing the amino acid sequence described in SEQ ID NO: 109, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 110; xiv. LCDR1 containing the amino acid sequence described in SEQ ID NO: 124, LCDR2 containing the amino acid sequence described in SEQ ID NO: 125, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 126; xv. LCDR1 containing the amino acid sequence described in SEQ ID NO: 132, LCDR2 containing the amino acid sequence described in SEQ ID NO: 133, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 134; xvi. LCDR1 containing the amino acid sequence described in SEQ ID NO: 140, LCDR2 containing the amino acid sequence described in SEQ ID NO: 141, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 142; or xvii. LCDR1 containing the amino acid sequence described in SEQ ID NO: 148, LCDR2 containing the amino acid sequence described in SEQ ID NO: 149, and LCDR3 containing the amino acid sequence described in SEQ ID NO:

150. The antibody or antigen-binding fragment thereof according to claim 1, comprising:

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

150. The antibody or antigen-binding fragment thereof according to claim 1, comprising:

6. a) A VH region having the amino acid sequence described in SEQ ID NOs: 154, 156, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143 or 151, or a homologous sequence having at least 80% sequence identity with SEQ ID NOs: 154, 156, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143 or 151, and / or b) A VL region having the amino acid sequence described in SEQ ID NOs: 155, 157, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144 or 152, or a homologous sequence having at least 80% sequence identity with SEQ ID NOs: 155, 157, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144 or 152, and / or c) VH / VL amino acid sequence pairs selected from the group consisting of SEQ ID NOs: 154 / 155, 156 / 157, 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 and 151 / 152 The antibody or antigen-binding fragment thereof according to claim 1, comprising:

7. a) The antibody or its antigen-binding fragment further comprises one or more amino acid residue substitutions or modifications, but still retains binding affinity to LILRB4, optionally i. At least one of the substitutions or modifications is present in one or more CDR sequences of the VH region or VL region, and / or ii. At least one of the substitutions or modifications is present in one or more non-CDR sequences of the VH region or VL region. Furthermore / or b) The antibody or its antigen-binding fragment may optionally be conjugated with one or more non-natural amino acid (NNAA) substitutions. Furthermore / or c) The antibody or its antigen-binding fragment i. The substance must be able to specifically bind to human LILRB4 when measured by the FACS assay; ii. The drug must be able to specifically bind to human LILRB4 and human LILRB3 when measured by the FACS assay; iii. The substance must be able to specifically bind to human LILRB4 and cynomolgus monkey LILRB4 when measured by the FACS assay; iv. Having a potent ADCP effect on human AML cell lines; v. Having a potent ADCC effect on human AML cell lines; vi. Blocking the LILRB4-fibronectin interaction and measuring by the FcγR stimulation assay allows for the reprogramming of tolerogenic dendritic cells into mature dendritic cells that stimulate T cell activation; vii. Ability to induce TNF-α production; viiii. The ability to reprogram tolerogenic DCs to activate T cells; ix. The ability to reverse macrophage-mediated T cell suppression; x. The ability to reverse THP-1-mediated T cell suppression; and xi. Ability to enhance CD8+ T cell-mediated cytotoxicity against THP-1 cells. Having binding properties to one or more LILRB4 selected from the group consisting of, Furthermore / or d) The antibody or its antigen-binding fragment is a chimeric, humanized, or human antibody or its antigen-binding fragment. Furthermore / or e) The antibody or its antigen-binding fragment is a labeled antibody, a bivalent antibody, an anti-idiotype antibody, or a fusion protein. Furthermore / or f) The antibody or its antigen-binding fragment is a diabody, Fab, Fab', F(ab')², Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), a(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. Furthermore / or g) The antibody or its antigen-binding fragment further comprises an Fc region, optionally an Fc region of human immunoglobulin (Ig), or optionally an Fc region of human IgG, optionally the Fc region being derived from human IgG1, IgG2, IgG3, or IgG4, preferably the Fc region comprising the amino acid sequence described in SEQ ID NO:

153. Furthermore / or h) The light chain is a λ light chain or a κ light chain. Furthermore / or i) The antibody or its antigen-binding fragment is a bispecific or polyspecific antibody or its antigen-binding fragment, optionally capable of specifically binding to one or more additional antigens other than LILRB4, or to a second epitope on LILRB4, optionally one or more of the additional antigens other than LILRB4 being CD3, CD16a, CD33, CD38, CD45, CD123, CD146, CD228, CLL-1, Flt3, TAF1, TgPRF, HVCC1, 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, Her 2, 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 (C D252), BCMA (e.g. BCMA02), PSMA, CLDN18 (e.g. CLDN18.2), NKG2C, 4-1BB, L IGHT, PVRIG, 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, TD O, 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,Selected from the group consisting of FLT3L, TLR3, CLEC9A, DEC-205, STING, IL-12, IDO, and TGFβ, Furthermore / or j) The antibody or its antigen-binding fragment is linked to one or more conjugate portions, optionally the conjugate portions comprising clearance modifiers, chemotherapeutic agents, toxins, radioisotopes, lantanides, detectable labels, DNA alkylating agents, topoisomerase inhibitors, tubulin binders, purified portions, or other anticancer agents, optionally the conjugate portions are covalently linked directly or via a linker. The antibody or its antigen-binding fragment according to claim 1.

8. An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof described in claim 1 with respect to binding to LILRB4.

9. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, and one or more pharmaceutically acceptable carriers.

10. A chimeric antigen receptor comprising an antibody or its antigen-binding fragment, a transmembrane region and an intracellular signaling region according to any one of claims 1 to 8, wherein optionally, a) The transmembrane region includes the transmembrane region of CD3, CD4, CD8, or CD28, b) 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 combinations thereof, and / or c) The antigen-binding fragment is scFv, and / or d) Grafted onto allogeneic cells, autologous cells, or heterogeneous cells, and / or e) Grafted onto immune effector cells, and / or f) Grafted onto T cells, natural killer cells, macrophages, or tumor-infiltrating lymphocytes, Chimeric antigen receptor.

11. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and / or an isolated polynucleotide encoding a chimeric antigen receptor according to claim 10.

12. A vector comprising an isolated polynucleotide as described in claim 11.

13. A host expression system comprising the vector described in claim 12, or having the polynucleotide described in claim 11 incorporated into its genome, wherein the host expression system is optionally a microorganism, yeast, or mammalian cell, and optionally the microorganism is selected from the group consisting of Escherichia coli and Bacillus subtilis, the yeast is Saccharomyces, and the mammalian cell is selected from the group consisting of COS, CHO-S, CHO-K1, HEK-293, and 3T3 cells.

14. A virus comprising the vector described in claim 12.

15. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8 and / or a pharmaceutical composition according to claim 9 and / or a chimeric antigen receptor according to claim 10, further optionally comprising a second therapeutic agent, and useful for detecting LILRB4, optionally recombinant LILRB4, LILRB4 expressed on the cell surface, or LILRB4-expressing cells, optionally.

16. A method for expressing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a chimeric antigen receptor according to claim 10, comprising culturing the host expression system according to claim 13 under conditions in which the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the chimeric antigen receptor according to claim 10, is expressed.

17. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a pharmaceutical composition according to claim 9, or a chimeric antigen receptor according to claim 10, for use in a method of treating, preventing, or alleviating a disease, disorder, or condition in a subject, The method comprises administering a therapeutically effective amount of the antibody or its antigen-binding fragment, or the pharmaceutical composition, or the chimeric antigen receptor to the subject, optionally, a) The disease, disorder, or condition is an immune disorder, inflammatory disorder, cancer, or neurological disorder, and optionally, the cancer is a solid tumor or hematological malignancy. Furthermore / or b) The disease, disorder, or condition is Kawasaki disease, T. gondi, 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 radiotherapy-resistant Lewis lung cancer), abdominal cancer, carcinoid cancer, bone cancer, pancreatic cancer, undifferentiated neuroectodermal tumor, skin cancer, gallbladder cancer, head or neck cancer, squamous cell carcinoma, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial carcinoma), anal cancer (e.g., anal squamous cell carcinoma), gastric cancer, or stomach cancerCancer (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatoma, or liver cancer), cholangiocarcinoma, 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 cancer, thyroid cancer, parathyroid cancer, adrenal cancer, nasopharyngeal cancer, soft tissue sarcoma, polycythemia vera, urethral cancer, penile cancer, kidney cancer or ureteral cancer (e.g., renal rhabdoid tumor), cutaneous T-cell lymphoma, medulloblastoma, nephroblastoma, myelodysplastic syndrome, chronic and non-chronic myeloplasty Progenitor dysfunction, choroid plexus papilloma, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasms, soft tissue sarcomas (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi's sarcoma), spinal axial tumors, gliomas (e.g., ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ocular cancers (e.g., retinoblastoma), brainstem gliomas, or mixed gliomas, e.g., oligodendroglioma), brain tumors (e.g., glioblastoma / glioblastoma multiforme (GBM), non-glioblastomatous brain tumors, or meningiomas), melanomas (e.g., cutaneous or intraocular melanoma), thrombocytosis, mesothelioma, mycosis fungoides, Sézary syndrome, idiopathic myelofibrosis, solitary form Sodium cytoma, vestibular schwannoma, Ewing's sarcoma, chondrosarcoma, MYH-associated polyposis, pituitary adenoma, childhood cancers, e.g., childhood sarcomas (e.g., neuroblastoma, rhabdomyosarcoma, and osteosarcoma), hematological cancers, lymphomas, Hodgkin lymphoma, non-Hodgkin 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 Selected from the group consisting of lymphoblastic leukemia, acute lymphoblastic leukemia, hairy cell leukemia (HCL), Burkitt 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 resulting from follicular lymphoma, high-grade B cell lymphoma, primary mediastinal large B cell lymphoma, follicular lymphoma (FL), and primary mediastinal B cell lymphoma. Furthermore / or c) The subject is a human, Furthermore / or d) The administration is carried out via a parenteral route including subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection; or via a route other than a parenteral route including transdermal, oral, intranasal, intraocular, sublingual, rectal, or topical surface, Furthermore / or e) Optionally further comprising administering an additional therapeutic agent to a subject requiring it, wherein the additional therapeutic agent is selected from the group consisting of activators, contrast agents, cytotoxic agents, and angiogenesis inhibitors, kinase inhibitors, costimulatory molecule agonists, coinhibitory molecule blockers, adhesion molecule blockers, anticytokine antibodies or their functional fragments, detectable labels or reporters, antibacterial agents, gene editing agents, beta-agonists, viral RNA inhibitors, polymerase inhibitors, interferons, and microRNAs, or the additional therapeutic agent is administered to the subject requiring it before, after, or concurrently with the antibody or antigen-binding fragment thereof described in any one of claims 1 to 8 and / or the pharmaceutical composition described in claim 9 and / or the chimeric antigen receptor described in claim 10. The antibody or its antigen-binding fragment, or the chimeric antigen receptor, or the pharmaceutical composition.

18. I) Modulating LILRB4 activity in LILRB4-expressing cells, the LILRB4-expressing cells are exposed to an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8, and / or a pharmaceutical composition as described in claim 9, and / or a chimeric antigen receptor as described in claim 10, wherein the LILRB4-expressing cells are preferably dendritic cells, monocytes, macrophages, B cells, Treg cells, progenitor mast cells, endothelial cells, or osteoclasts. II) Exposing the target cells to an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and / or the pharmaceutical composition according to claim 9, and / or the chimeric antigen receptor according to claim 10, which induces phagocytosis of the target cells in vivo or in vitro, preferably the target cells being antigen-presenting cells, cancer cells, or cells infected with a pathogen. III) Exposing tolerogenic dendritic cells that induce TNF-α production to the antibody or antigen-binding fragment thereof described in any one of claims 1 to 8, and / or the pharmaceutical composition described in claim 9, and / or the chimeric antigen receptor described in claim 10. IV) Reprogramming tolerogenic dendritic cells into mature dendritic cells, comprising exposing the tolerogenic dendritic cells to an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and / or the pharmaceutical composition according to claim 9, and / or the chimeric antigen receptor according to claim 10. V) detecting the presence or amount of LILRB4 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and / or a pharmaceutical composition according to claim 9 and / or a chimeric antigen receptor according to claim 10, and determining the presence or amount of LILRB4 in the sample, or VI) Diagnosing a LILRB4-related disease or condition in a subject, a) obtaining a sample from the subject; b) contacting the sample obtained from the subject with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and / or a pharmaceutical composition according to claim 9; c) determining the presence or amount of LILRB4 in the sample; and d) relating the presence or amount of LILRB4 to the presence or condition of the LILRB4-related disease or condition in the subject. method.

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