Anti-lilrb4 antibodies and Anti-lilrb4 / Anti-4-1BB bispecific antibodies and uses thereof

Abstract

The invention relates to an antibody or an antigen-binding fragment thereof specifically binds to LILRB4; pharmaceutical formulations comprising such the antibody or the antigen-binding fragment and uses thereof. In addition, the invention relates to a bispecific antibody or an antigen-binding fragment thereof comprising a first antigen binding site can bind to LILRB4 and a second antigen binding site can bind to 4-1BB; pharmaceutical formulations comprising such bispecific antibodies and uses thereof. In particular, the antibody or the antigen-binding fragment thereof or the bispecific antibody or an antigen-binding fragment thereof can cause activation of T cells and then it can effectively prevent or treat the cancer.

Description

ANTI-LILRB4 ANTIBODIES AND ANTI-LILRB4 / ANTI-4-1BB BISPECIFIC ANTIBODIES AND USES THEREOF

[0001] The present invention pertains to antibodies and bispecific antibodies, compositions comprising such antibodies and uses of such antibodies and compositions. More specifically, provided are anti-LILRB4 antibodies and anti-LILRB4 / anti-4-1BB bispecific antibodies and uses thereof.

[0002] Tumor-associated myeloid cells such as myeloid-derived suppressor cells (MDSC) accumulate in the tumor microenvironment (TME) and suppress anti-tumor immune responses in a broad range of cancers. LILRB4 (Leukocyte Immunoglobulin-Like Receptor Subfamily B Member 4), an emerging myeloid target, is upregulated in tumor-associated myeloid cells and suppresses the immune response, thus promoting cancer progression and metastasis.

[0003] Co-inhibitory or immune checkpoint receptors play a critical role in the maintenance of immune homeostasis: their expression on effector T cells ensures the proper contraction of effector T cell responses, while their expression on regulatory T (Treg) cells guarantees the proper functioning of Tregcells to control effector T cells. Accordingly, their function in regulating pro-inflammatory T cell responses and maintaining self-tolerance has been most widely studied in this context. More recently, the role of co-inhibitory receptors has come to the forefront particularly in cancer, where these receptors are highly expressed and are being targeted clinically to improve anti-tumor capability. While current immunotherapies directed against the coinhibitory receptors CTLA-4 and PD(L)-1 are exhibiting unprecedented efficacy in several cancer indications and chronic viral infections, there are still many patients that do not respond to these therapeutic approaches and some tumor types that remain largely refractory to such therapies.

[0004] In an aspect, the present disclosure provides an anti-LILRB4 antibody or an antigen-binding fragment thereof.

[0005] In another aspect, the present disclosure provides an isolated nucleic acid encoding the anti-LILRB4 antibody or an antigen-binding fragment thereof; a vector comprising the isolated nucleic acid; and a host cell comprising the vector.

[0006] In another aspect, the present disclosure provides a pharmaceutical formulation of the anti-LILRB4 antibody or an antigen-binding fragment thereof.

[0007] In another aspect, the present disclosure provides a method for treating or preventing cancer in a patient in need thereof, comprised of administering to the patient an effective amount of the anti-LILRB4 antibody or an antigen-binding fragment thereof, or the use of the anti-LILRB4 antibody or an antigen-binding fragment thereof in the manufacture of a medicament for treating or preventing cancer.

[0008] In an aspect, the present disclosure provides an anti-LILRB4 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof.

[0009] In another aspect, the present disclosure provides an isolated nucleic acid encoding the anti-LILRB4 / anti-4-1BB bispecific antibody; a vector comprising the isolated nucleic acid; and a host cell comprising the vector.

[0010] In another aspect, the present disclosure provides a pharmaceutical formulation of the anti-LILRB4 / anti-4-1BB bispecific antibody.

[0011] In another aspect, the present disclosure provides a method for treating or preventing cancer in a patient in need thereof, comprised of administering to the patient an effective amount of the anti-LILRB4 / anti-4-1BB bispecific antibody, or the use of the anti-LILRB4 / anti-4-1BB bispecific antibody in the manufacture of a medicament for treating or preventing cancer.

[0012] An aspect of the present disclosure provides an antibody or antigen-binding fragment thereof specifically binds to LILRB4 (leukocyte immunoglobulin like receptor B4).

[0013] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region and a light chain variable region comprising: (a) a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 3; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 5; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 7; (d) a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 11; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 13; and (f) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 15.

[0014] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 19.

[0015] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 24.

[0016] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain framework 1 (H-FR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 20 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 20; a heavy chain framework 2 (H-FR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 21 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 21; a heavy chain framework 3 (H-FR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22; a heavy chain framework 4 (H-FR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 23 23 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 23; a light chain framework 1 (L-FR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 25 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence consisting of SEQ ID NOs: 10 and 25; a light chain framework 2 (L-FR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 26 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 26; a light chain framework 3 (L-FR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 27 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 27; and a light chain framework 4 (L-FR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 28 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 28.

[0017] In an embodiment, an antibody or antigen-binding fragment thereof that specifically binds to LILRB4 (leukocyte immunoglobulin like receptor B4), may comprise at least one antigen binding site comprising a heavy chain and a light chain: wherein the heavy chain comprising: a heavy chain framework 1 (H-FR1) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 20; a heavy chain CDR1 of SEQ ID NO: 3; a heavy chain framework 2 (H-FR2) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 21; a heavy chain CDR2 of SEQ ID NO: 5; a heavy chain framework 3 (H-FR3) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22; a heavy chain CDR3 of SEQ ID NO: 7; a heavy chain framework 4 (H-FR4) selected from the group consisting a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 23; wherein the light chain comprising: a light chain framework 1 (L-FR1) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence consisting of SEQ ID NOs: 10 and 25; a light chain CDR1 of SEQ ID NO: 11; a light chain framework 2 (L-FR2) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 26; a light chain CDR2 of SEQ ID NO: 13; a light chain framework 3 (L-FR3) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 27; a light chain CDR3 of SEQ ID NO: 15; and a light chain framework 4 (L-FR4) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 28.

[0018] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 29 and 30.

[0019] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 31.

[0020] In one embodiment, the antibody or antigen-binding fragment thereof may not detectably bind or bind with at least 5-fold lower affinity to other LILR (The leukocyte immunoglobulin-like receptor) family including LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB5, or a combination thereof.

[0021] An aspect of the present disclosure provides a bispecific antibody or an antigen-binding fragment thereof comprising (i) a first antigen binding site can bind to LILRB4 (leukocyte immunoglobulin like receptor B4) and (ii) a second antigen binding site can bind to 4-1BB (4-1BB ligand).

[0022] In an embodiment, the bispecific antibody may comprise the first antigen binding site comprising a heavy chain variable region and a light chain variable region comprising: (a) a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 3; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 5; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 7; (d) a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 11; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 13; and (f) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 15.

[0023] In an embodiment, the bispecific antibody may comprise the first antigen binding site comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 19.

[0024] In an embodiment, the bispecific antibody may comprise the first antigen binding site comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 24.

[0025] In an embodiment, the bispecific antibody may comprise the first antigen binding site comprising a heavy chain framework 1 (H-FR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 20 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 20; a heavy chain framework 2 (H-FR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 21 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 21; a heavy chain framework 3 (H-FR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22; a heavy chain framework 4 (H-FR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 23 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 23; a light chain framework 1 (L-FR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 25 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 25; a light chain framework 2 (L-FR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 26 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 26; a light chain framework 3 (L-FR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 27 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 27; and a light chain framework 4 (L-FR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 28 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 28.

[0026] In an embodiment, the bispecific antibody may comprise the first antigen binding site comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 29 and 30.

[0027] In an embodiment, the bispecific antibody may comprise the first antigen binding site comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 31.

[0028] In an embodiment, the bispecific antibody may comprise the second antigen binding site comprising a heavy chain variable region and a light chain variable region comprising: (a) a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 34; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 36; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 38; (d) a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 42; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 44; and (f) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 46.

[0029] In an embodiment, the bispecific antibody may comprise the second antigen binding site comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 32.

[0030] In an embodiment, the bispecific antibody may comprise the second antigen binding site comprising a light chain variable region comprising an amino acid sequence of SEQ ID NO: 40.

[0031] In an embodiment, the bispecific antibody may comprise the second antigen binding site comprising a heavy chain framework 1 (H-FR1) comprising an amino acid sequence of SEQ ID NO: 33 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 33; a heavy chain framework 2 (H-FR2) comprising an amino acid sequence of SEQ ID NO: 35 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 35; a heavy chain framework 3 (H-FR3) comprising an amino acid sequence of SEQ ID NO: 37 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 37; a heavy chain framework 4 (H-FR4) comprising an amino acid sequence of SEQ ID NO: 39 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 39; a light chain framework 1 (L-FR1) comprising an amino acid sequence of SEQ ID NO: 41 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 41; a light chain framework 2 (L-FR2) comprising an amino acid sequence of SEQ ID NO: 43 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 43; a light chain framework 3 (L-FR3) comprising an amino acid sequence of SEQ ID NO: 45 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 45; and a light chain framework 4 (L-FR4) comprising an amino acid sequence of SEQ ID NO: 47 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 47.

[0032] In an embodiment, the bispecific antibody may comprise the first antigen binding site comprising a VH CDR1 of SEQ ID NO: 3; a VH CDR2 of SEQ ID NO: 5; a VH CDR3 of SEQ ID NO: 7; a VL CDR1 of SEQ ID NO: 11; a VL CDR2 of SEQ ID NO: 13; and a VL CDR1 of SEQ ID NO: 15; and the second antigen binding site comprises a VH CDR1 of SEQ ID NO: 34; a VH CDR2 of SEQ ID NO: 36; a VH CDR3 of SEQ ID NO: 38; a VL CDR1 of SEQ ID NO: 42; a VL CDR2 of SEQ ID NO: 44; and a VL CDR3 of SEQ ID NO: 46.

[0033] In an embodiment, a bispecific antibody or antigen-binding fragment thereof may comprise: (i) a first antigen binding site that binds to LILRB4 (leukocyte immunoglobulin like receptor B4) comprising a heavy chain and a light chain: (ii) a second antigen binding site that binds to 4-1BB (4-1BB ligand), comprising at least one antigen binding site comprising a heavy chain and a light chain: wherein the heavy chain of first antigen binding site comprising: a heavy chain framework 1 (H-FR1) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 20; a heavy chain CDR1 of SEQ ID NO: 3; a heavy chain framework 2 (H-FR2) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 21; a heavy chain CDR2 of SEQ ID NO: 5; a heavy chain framework 3 (H-FR3) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22; a heavy chain CDR3 of SEQ ID NO: 7; a heavy chain framework 4 (H-FR4) selected from the group consisting a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 23; wherein the light chain of first antigen binding site comprising: a light chain framework 1 (L-FR1) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence consisting of SEQ ID NOs: 10 and 25; a light chain CDR1 of SEQ ID NO: 11; a light chain framework 2 (L-FR2) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 26; a light chain CDR2 of SEQ ID NO: 13; a light chain framework 3 (L-FR3) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 27; a light chain CDR3 of SEQ ID NO: 15; a light chain framework 4 (L-FR4) selected from the group consisting of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 28; and wherein the heavy chain of the second antigen binding site comprises: a heavy chain framework 1 (H-FR1) of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 33; a heavy chain CDR1 of SEQ ID NO: 34; a heavy chain framework 2 (H-FR2) of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 35; a heavy chain CDR2 of SEQ ID NO: 36; a heavy chain framework 3 (H-FR3) of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 37; a heavy chain CDR3 of SEQ ID NO: 38; a heavy chain framework 4 (H-FR4) of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 39; wherein the light chain of second antigen binding site comprising: a light chain framework 1 (L-FR1) of SEQ ID NO: 41 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 41; a light chain CDR1 of SEQ ID NO: 42; a light chain framework 2 (L-FR2) of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 43; a light chain CDR2 of SEQ ID NO: 44; a light chain framework 3 (L-FR3) of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 45; a light chain framework 4 (L-FR4) of a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 47; and a light chain CDR3 of SEQ ID NO: 46.

[0034] In an embodiment, the antibody or the bispecific antibody may independently be a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

[0035] In an embodiment, the antibody or the bispecific antibody may be independently selected from a group consisting of a whole IgG, sdAb (single-domain antibody), Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG and combinations thereof.

[0036] In an embodiment, the antibody or the bispecific antibody may independently comprise an Fc region or a constant region, wherein the Fc region is an Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody, or a hybrid Fc region.

[0037] In an embodiment, the first antigen binding site can bind to LILRB4 is covalently linked to a second antigen binding site can bind to 4-1BB in a bispecific antibody or an antigen-binding fragment thereof. In another embodiment, the first antigen binding site and the second antigen binding site are linked by a peptide linker. In another embodiment, one part or C-terminus of the Fab domain from the anti-LILRB4 antibody can be linked to one part or N-terminus of the Fab domain from the anti-4-1BB antibody.

[0038] In an embodiment, the anti-LILRB4 / anti-4-1BB bispecific antibody may comprise an Fc domain comprising a first sub-unit and a second sub-unit.

[0039] In an embodiment, the bispecific antibody may comprise the first antigen binding site, which is covalently linked to the second antigen binding site.

[0040] In an embodiment, the bispecific antibody may comprise the first antigen binding site and the second antigen binding site, which are linked by a peptide linker.

[0041] In an embodiment, a pharmaceutical formulation may comprise the antibody or the bispecific antibody and a pharmaceutically acceptable carrier.

[0042] In an embodiment, a pharmaceutical composition for use in preventing or treating cancer may comprise the antibody or the bispecific antibody.

[0043] In an embodiment, the cancer may be an immune checkpoint inhibitor-resistant cancer or a LILRB4-positive cancer.

[0044] In an embodiment, the cancer may be a solid cancer or a blood cancer.

[0045] In an embodiment, the cancer may be selected from the group consisting of leukemia, rectal cancer, endometrial cancer, nephroblastoma, basal cell carcinoma, nasopharyngeal cancer, bone tumor, esophageal cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular thyroid cancer, hepatocellular carcinoma, oral cancer, renal cell carcinoma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, mesenchymal tumor, soft tissue sarcoma, liposarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratoma, neuroblastoma, medulloblastoma, glioma, benign skin tumor, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy-cell leukemia, myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, neuroectodermal tumor, epithelial tumor, cutaneous T-cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), pancreas cancer, hematological malignancies, kidney cancer, tumor vasculature, breast cancer, renal cancer, ovarian cancer, epithelial ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), Head and neck squamous cell carcinoma (HNSCC), glioblastoma multiforme (GBM), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.

[0046] In an embodiment, the cancer may be selected from the group consisting of acute myeloid leukemia (AML), and multiple myeloma.

[0047] In an embodiment, the cancer may be selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), and hepatocellular carcinoma.

[0048] In an embodiment, the present invention provides a method for preventing or treating a cancer in a patient in need thereof, which may comprise administering to the patient an effective amount of the antibody or the bispecific antibody.

[0049] In an embodiment, the present invention provides a use of the antibody or the bispecific antibody in the manufacture of a medicament for treating or preventing cancer.

[0050] In an embodiment, the present invention provides a method for restoring T cell in a patient in need thereof, comprising administering to the patient an effective amount of the antibody or the bispecific antibody.

[0051] In an embodiment, the present invention provides an isolated nucleic acid encoding the antibody or the bispecific antibody.

[0052] In an embodiment, the present invention provides a vector comprising the isolated nucleic acid.

[0053] In an embodiment, the present invention provides a host cell comprising the vector.

[0054] In an embodiment, the Fc domain may be a human Fc domain.

[0055] In an embodiment, the present disclosure provides a method for treating or preventing a cancer in a patient in need thereof, comprising administering to the patient an effective amount of the antibody or the bispecific antibody.

[0056] According to one of the embodiments, antibodies or antigen binding fragments thereof and bispecific antibodies or antigen binding fragments thereof may inhibit LILRB4 mediated immunosuppression, restoring T cell activity by antagonizing LILRB4 signaling. Thus, the antibody of the present invention may provide synergistic therapeutic tumor treatment.

[0057] FIG.1 is a graph showing antigen binding affinity of chimeric anti-LILRB4 antibody according to an embodiment by using Biocore.

[0058] FIG.2(a) to FIG.2(b) are graphs showing cross-specificity of chimeric anti-LILRB4 monospecific antibody according to an embodiment by using ELISA.

[0059] FIG.3(a) to FIG.3(k) are graphs showing LILRB4-binding specificity of chimeric anti-LILRB4 monospecific antibody according to an embodiment by using ELISA.

[0060] FIG.4(a) to FIG.4(b) are graphs showing binding confirmation of chimeric anti-LILRB4 antibody according to an embodiment by using FACS analysis.

[0061] FIG.5(a) to FIG.5(b) are graphs showing binding affinity of humanized anti-LILRB4 monospecific antibodies to target protein LILRB4.

[0062] FIG.6(a) to FIG.6(b) are graphs showing cross-specificity of humanized anti-LILRB4 antibody according to an embodiment by using ELISA.

[0063] FIG.7 is a graph showing T cell checkpoint blockade activity of anti-LILRB4 antibody according to an embodiment with dose dependent manner.

[0064] FIG.8(a) to FIG.8(f) are graphs showing effects of chimeric anti-LILRB4 antibody according to an embodiment on tolerogenic dendritic cells.

[0065] FIG.9 is a graph showing AB0224 restores immune cell activity from LILRB4-mediated suppression by fibronectin blockade assay.

[0066] FIG.10 is a graph showing ch6E9C4 restores immune cell activity from LILRB4-mediated suppression by MDSC assay.

[0067] FIG.11 is a graph showing evaluation of the potency of AB0166 byin vitroADCC bioassay.

[0068] FIG. 12 is a graph showing chimeric 6E9C4 has a different binding epitope compared to other anti-LILRB4 antibodies.

[0069] FIG.13(a) to FIG.13(f) are graphs showing cross-specificity of anti-LILRB4 / anti-4-1BB bispecific antibody to human and cynomolgus LILRB4 by ELISA.

[0070] FIG.14(a) and FIG.14(d) are graphs showing cross-specificity of anti-LILRB4 / anti-4-1BB bispecific antibody to Human and Cynomolgus LILRB4 and 4-1BB (SPR data)

[0071] FIG.15(a) to FIG.15(e) are graphs showing binding confirmation of anti-LILRB4 / anti-4-1BB bispecific antibodies by using FACS analysis.

[0072] FIG.16 is a graph showing anti-LILRB4 antibodies and anti-LILRB4 / anti-4-1BB bispecific antibodies restore immune cell activity from LILRB4-mediated suppression.

[0073] FIG.17 is a graph showing anti-LILRB4 antibodies and anti-LILRB4 / anti-4-1BB bispecific antibodies restore immune cell activity from LILRB4-mediated suppression.

[0074] FIG.18(a) to FIG.18(c) are graphs showing AB0228 and AB0229 activate effector cells in LILRB4 expression-dependent manner.

[0075] FIG.19(a) and FIG.19(b) are graphs showing efficacy of the AB0224 with SK-MEL-5 melanoma CDX bearing hematopoietic stem cell (HSC) mice model.

[0076] FIG.20(a) and FIG.20(c) are graphs showing in vivoefficacy test using hLILRB4-luc EL4 (i.v.) in h4-1BB knock-in mice.

[0077] FIG.21(a) and FIG.21(c) are graphs showingin vivoefficacy test using hLILRB4-luc EL4 (i.v.) in hLILRB1,4 / h4-1BB triple TG mice.

[0078] FIG.22(a) and FIG.22(c) are graphs showingin vivoefficacy test using B16-F10-OVA in hLILRB1,4 / h4-1BB triple TG mice.

[0079] FIG. 23(a) is a graph showing LILRB4 Expression on NCI-H929 (MM cell line).

[0080] FIG. 23(b) is a graph showing binding affinity of AB0228 against NCI-H929 (MM cell line).

[0081] FIG. 23(c) is a graph showing multiple myeloma targeted PBMC assay (IFN-γ secretion).

[0082]

[0083] Definitions

[0084] Unless defined otherwise, technical and scientific terms used herein have the same meaning as generally those used in the art to which this invention belongs. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa.

[0085] As used herein, the term "a" or "an" entity may refer to one or more of that entity, for example, "an antibody," is understood to represent one or more antibodies. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0086] As used herein, the term "consisting of a sequence," "consisting essentially of a sequence," or "comprising a sequence" may refer to any case comprising the sequence, but it may not be intended to exclude a case comprising further sequence other than the sequence.

[0087] As used herein, the term "a protein or polypeptide comprising or consisting of an amino acid sequence identified by SEQ ID NO" and "a gene or polynucleotide comprising or consisting of a nucleic acid sequence identified by SEQ ID NO" may refer to a protein (or polypeptide) or gene (or polynucleotide), which consists essentially of the amino acid sequence or nucleic acid sequence, or which has 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 with the amino acid sequence or nucleic acid sequence with maintaining its inherent activity and / or function.

[0088] As used herein, the term "antibody" may encompass various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g., γl-γ4), and light chains are classified as either kappa or lambda (κ,λ). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization.

[0089] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding affinity.

[0090] The term "monospecific" antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

[0091] The term "bispecific" means that the antibody is able to specifically bind to at least two distinct antigenic determinants. For example, two binding sites each formed by any one of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) or both, binding to different antigens or to different epitopes on the same antigen. Such a bispecific antibody may be an 1+1 format (comprising one binding site for a first antigen or epitope and one binding site for a second antigen or epitope), 2+1 or 1+2 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 formats (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). For another example, the bispecific antibody may include a biparatopic antibody. If a biparatopic antibody is included, one binding site has binding affinity to two or more different target.

[0092] The antibody provided herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. Provided herein is a bispecific antibody, with binding specificities for LILRB4 and 4-1BB. Bispecific antibodies of the invention include, for example, multivalent single chain antibodies, diabodies and triabodies, as well as antibodies having the constant domain structure of full length antibodies to which further antigen-binding sites (e.g., single chain Fv, a VH domain and / or a VL domain, Fab, or (Fab)2) are linked via one or more peptide-linkers. The antibodies can be full length from a single species, or be chimerized or humanized. For another example, the multispecific antibody may include a multiparatopic antibody.

[0093] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities, and "knob-in-hole" engineering. Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules; cross-linking two or more antibodies or fragments; using leucine zippers to produce bi-specific antibodies; using "diabody" technology for making bispecific antibody fragments; and using single-chain Fv (sFv) dimers; and preparing trispecific antibodies as described.

[0094] The term "valent" as used within the current application denotes the presence of a specified number of binding domains in an antibody or antibody fragment. As such, the terms "monovalent", "bivalent", "trivalent", "tetravalent", "pentavalent", and "hexavalent" denote the presence of one binding domain, two binding domains, three binding domains, four binding domains, five binding domains, and six binding domains, respectively, in an antibody. The bispecific antibodies according to the invention are at least "bivalent" and may be "trivalent" or "multivalent" (e.g., "tetravalent", "pentavalent" or "hexavalent"). In a particular aspect, the antibodies of the present invention have two or more binding sites and are bispecific. That is, the antibodies may be bispecific even in cases where there are more than two binding sites (i.e., that the antibody is trivalent or multivalent).

[0095] The terms "full length antibody", "Intact antibody", and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures.

[0096] The complementarity determining regions (CDRs) in the variable region allow the antibody to selectively recognize and specifically bind epitopes on antigens. More specifically, in a conventional antibody such as IgG, the antigen-binding site is defined by three CDRs on each of the VH and VL chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist solely of heavy chains, with no light chains.

[0097] The terms "CDR-H", "HCDR" and "CDRH" herein are used interchangeably to refer to a VH chain of the CDR (e.g., CDR-H1, HCDR1 and CDRH1 are refer to a VH1 of the CDR). The terms "CDR-L", "LCDR" and "CDRL" herein are used interchangeably to refer to a VL chain of the CDR (e.g., CDR-L1, LCDR1 and CDRL1 are a refer to a VL1 of the CDR).

[0098] In naturally occurring antibodies, the "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, referred to as "framework" regions, show less inter-molecular variability. The CDRs and the framework regions included in an antibody, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined.

[0099] In the case where there are two or more definitions of a term which is used and / or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated otherwise. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the noncontiguous antigen combining sites found within the variable region of heavy and / or light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) or by Chothia et al., J. MoI. Biol. 196: 901-917 (1987). In addition, the numbering scheme of the international ImMunoGeneTics information system (IMGT), the AbM definition (see bioinfo.org.uk / abs / ), Martin Antibody Numbering (Enhanced Chothia or AbM) or Honneger's numbering scheme (AHo) can be used for the identification of variable regions and CDRs. The definitions of CDR according to such multiple numbering schemes include overlaps or subsets of amino acid residues when compared against each other. Nevertheless, application of any definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0100] The definitions of CDR according to Kabat and Chothia include overlaps or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0101] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence, without reliance on any experimental data beyond the sequence itself.

[0102] Antibodies disclosed herein may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.

[0103] As used herein, the term "heavy chain constant region" includes amino acid sequences derived from an immunoglobulin heavy chain. As set forth above, it will be understood by one of ordinary skill in the art that the heavy chain constant region may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.

[0104] The heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain constant region can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.

[0105] As used herein, the term "light chain constant region" includes amino acid sequences derived from antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or constant lambda domain.

[0106] A "light chain-heavy chain pair" refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0107] An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. An immunologically functional immunoglobulin fragment includes sdAb (single-domain antibody), Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv, scFvFc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG or combinations thereof, but not limited thereto. The term "Fab" used in Fab, Fab', F(ab')2, xFab and scFab may include a traditional Fab fragment and the chimeric Fab-like domain. In addition, it may be derived from any mammal including human, mouse, rat, camelid or rabbit, but not limited thereto. The functional part of the antibody such as one or more CDRs described herein may be linked with a secondary protein or small molecular compound by a covalent bond, thereby being used as a target therapeutic agent to a specific target. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0108] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g.,E. colior phage), as described herein.

[0109] The term "full length IgG" according to the invention is defined as comprising an essentially complete IgG, which however does not necessarily have all functions of an intact IgG. For the avoidance of doubt, a full-length IgG contains two heavy and two light chains. Each chain contains constant I and variable (V) regions, which can be broken down into domains designated CH1, CH2, CH3, VH, and CL, VL. Full length antibodies according to the invention encompass IgG molecules wherein mutations may be present that provide desired characteristics. Such mutations should not be deletions of substantial portions of any of the regions. However, IgG molecules wherein one or several amino acid residues are deleted, without essentially altering the binding characteristics of the resulting IgG molecule, are embraced within the term "full length IgG".

[0110] Full length IgG antibodies are preferred because of their favorable half-life and the need to stay as close to fully autologous (human) molecules for reasons of immunogenicity.

[0111] According to the invention, bispecific IgG antibodies are used.

[0112] Bispecific antibodies that mediate cytotoxicity by recruiting and activating endogenous immune cells are an emerging class of next generation antibody therapeutics. According to the invention, bispecific antibodies are provided wherein both the LILRB4-binding site and the 4-1BB-binding site bind to the antigens on T cells, antigen presenting cells, natural killer cells, etc.- i.e., tumor microenvironment.

[0113] As used herein, the term "antigen binding domain" or "antigen binding site" refers to the part of the antibody or antibody fragment that specifically binds to an antigenic determinant. More particularly, the term "antigen binding domain" refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. In one aspect, the antigen binding domain is able to bind to its antigen and block or partly block its function. Antigen binding domains that specifically bind to LILRB4 and / or to the 4-1BB include antibodies and fragments thereof as further defined herein. In addition, antigen binding domains may include scaffold antigen binding proteins, e.g., binding domains which are based on designed repeat proteins or designed repeat domains.

[0114] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site (e.g., a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex.

[0115] "Specific binding" means that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antibody or antibody fragment to bind to a specific antigen can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g., Surface Plasmon Resonance (SPR) technique (analyzed on a BIAcore instrument), and traditional binding assays.

[0116] "Affinity" or "binding affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).

[0117] An "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, with such alterations resulting in an improvement in the affinity of the antibody for antigen.

[0118] The term "a bispecific antibody that specifically binds LILRB4 and 4-1BB", "bispecific antibody specific for LILRB4 and 4-1BB" or an "anti- LILRB4 / anti-4-1BB antibody" are used interchangeably herein and refer to a bispecific antibody that can bind to LILRB4 and 4-1BB with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting LILRB4 and 4-1BB.

[0119] As used herein, the terms "LILRB4 protein" or "LILRB4 antigen" or "LILRB4" or "Leukocyte immunoglobulin like receptor B4" or "Leukocyte immunoglobulin-like receptor subfamily B member 4" is a protein encoded by the LILRB4 gene in humans. This protein is a unique subtype of LILRB family containing only 2 Ig-like domains and monocyte specifically expresses this protein in normal tissue and this protein was over expressed in monocytic AML (FAB M4 / M5 types), MDSC, TAM, suppressive DC, and on tumor-associated myeloid cells (tolDC, MDSC and M2 Mφ). T cell inhibition mediates by LILRB4 binding to the counterpart on T cell or inhibitory signaling. Myeloid cells suppress antitumor T-cell responses by the interaction of fibronectin with LILRB4. T cell inhibition mediates by Arg-1 upon ApoE binding to LILRB4. This gene is a member of the immunoglobulin-like receptor (LIR) family of leukocytes, which is present in the gene cluster of chromosomal regions 19q 13.4. The encoded proteins belong to the LIR receptor subfamily B class, which contains two or four extracellular immunoglobulin domains, one transmembrane domain, and two to four cytoplasmic Immunoreceptor Tyrosine Inhibitory Motifs (ITIMs). The receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen presenting cells and transduces a negative signal that inhibits stimulation of an immune response. The receptor may also play a role in antigen capture and presentation. It is thought that it may control inflammatory responses and cytotoxicity to help focus immune responses and limit autoreactivity. LILRB4 is also expressed in human gastric cancer cells and can enhance tumor growth (see Zhang et al, 2012). Multiple transcriptional variants of this gene have been found that encode different isoforms. LILRB4 has been shown to interact with PTPN 6.

[0120] As used herein, the term "4-1BB" is a member of TNF-receptor superfamily (TNFRSF) and is a co-stimulatory molecule which is expressed following the activation of immune cells, both innate and adaptive immune cells. 4-1BB plays important role in modulate the activity of various immune cells. 4-1BB agonists enhance immune cell proliferation, survival, secretion of cytokines and cytolytic activity CD8+T cells. Many other studies showed that activation of 4-1BB enhances immune response to eliminate tumors in mice. Therefore, it suggests that 4-1BB is a promising target molecule in cancer immunology. Despite of their anti-tumor efficacy, anti-4-1BB antibody induced severe liver toxicity in clinical application.

[0121] The term "4-1BB" refers to CD137, or TNFRSF9 (TNF Receptor 25 Superfamily Member 9), is a member of TNF-receptor superfamily (TNFRSF) and is a co-stimulatory molecule which is expressed following the activation of immune cells, both innate and adaptive immune cells. As used herein, 4-1BB may be originated from a mammal, for example, Homo sapiens (human) (NCBI Accession No. NP_001552.2).

[0122] As described herein, the term "4-1BB" includes variants, isoforms, homologs, orthologs, and paralogs. For example, antibodies specific for a human 4-1BB protein may, in certain cases, cross-react with a 4-1BB protein from a species other than human. In other embodiments, the antibodies specific for a human 4-1BB protein may be completely specific for the human 4-1BB protein and may exhibit species or other types of cross-reactivity, or may cross-react with 4-1BB from certain other species but not all other species (e.g., cross-react with monkey 4-1BB, but not mouse 4-1BB). The term "human 4-1BB" refers to human sequence 4-1BB, such as the complete amino acid sequence of human 4-1BB having NCBI Accession No. NP_001552.2. The term "mouse 4-1BB" refers to mouse sequence 4-1BB, such as the complete amino acid sequence of mouse 4-1BB having NCBI Accession No. NP 033430.1. 4-1BB also can be known in the art as, for example, CD137. The human 4-1BB sequence in the disclosure may differ from human 4-1BB of NCBI Accession No. NP_001552.2 by having, e.g., conserved mutations or mutations in non-conserved regions and the 4-1BB in the disclosure has substantially the same biological function as the human 4-1BB of NCBI Accession No. NP_001552.2.

[0123] The terms "anti-LILRB4 antibody", "an antibody that binds to LILRB4" and "an antibody comprising an antigen binding domain that binds to LILRB4" refer to an antibody that can bind to LILRB4, especially a LILRB4 polypeptide expressed on a cell surface, with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting LILRB4. The term "anti- LILRB4 antibody" also encompasses bispecific antibodies that can bind to LILRB4 and a different antigen.

[0124] The terms "anti-4-1BB antibody", "an antibody that binds to 4-1BB" and "an antibody comprising an antigen binding domain that binds to 4-1BB" refer to an antibody that can bind to 4-1BB, especially a 4-1BB polypeptide expressed on a cell surface, with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting 4-1BB.

[0125] The term "restoration" refers to repairing a function of effector T cell (cytotoxic T cell) which was affected by cancer cells or other cells. For example, inhibition of LILRB4, an immune checkpoint inhibitors expressed by effector T cells, binding using antagonistic antibodies has shown potential to restore T-cell function.

[0126] The term "Immune　checkpoint" refers to inhibitory pathways hardwired into the　immune　system that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological　immune　responses in peripheral tissues in order to minimize collateral tissue damage.　Immune　checkpoint molecules can be stimulatory or inhibitory to an　immune　checkpoint. The present disclosure and claims refer to inhibitory molecules of　immune　checkpoints as "immune　checkpoint molecules". Preliminary clinical findings with agents that block　immune　checkpoint molecules, suggest opportunities to enhance antitumor immunity with the potential to produce effective clinical responses. The present application discloses that combining　immune　checkpoint blockade using　immune　checkpoint inhibitor(s) with HSCT and / or HSC mobilization treatment enhances treatment efficacy in a subject having a cancer or an infectious disease.　Immune　Checkpoint Inhibitors and　Immune　Checkpoint Blockade. An　immune　checkpoint inhibitor is a type of drug that blocks the signaling of　immune　checkpoint molecule(s) made by some types of　immune　system cells, such as T cells and some cancer cells.　Immune　checkpoint inhibitors therefore can cause　immune　checkpoint blockade.　Immune　checkpoint molecules help keep　immune　responses in check and can keep T cells from killing cancer cells. When these molecules are blocked, the "brakes" on the　immune　system are released (inhibition of the　immune　system is reduced or blocked) and T cells are able to kill cancer cells better. In some embodiments,　immune　checkpoint molecules are proteins. In some embodiments,　immune　checkpoint molecules are nucleic acids that encode the proteins. In some embodiments,　immune　checkpoint inhibitors bind to and / or antagonize　immune　checkpoint molecules. In some embodiments,　immune　checkpoint inhibitors are used in combination with hematopoietic stem cell transplantation and / or hematopoietic stem cell mobilizing agent treatment to treat a subject having cancer. In some embodiments,　immune　checkpoint inhibitors are used in combination with hematopoietic stem cell transplantation and / or hematopoietic stem cell mobilizing agent treatment to treat a subject having an infectious disease.

[0127] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic linker peptides are, for example, (GS)n, (G4S)n, (SG4)nor G4(SG4)npep peptide linkers, wherein "n" is generally a number between 1 and 10, typically between 2 and 4, in particular 2, i.e. the peptides selected from the group consisting of (GS)9: SEQ ID NO: 50 and (GGGGS)4": SEQ ID NO: 51.

[0128] As used herein, the terms "engineer, engineered, engineering" are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, the glycosylation pattern, or the side chain group of individual amino acids, as well as combinations of these approaches.

[0129] The term "amino acid mutation" as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide.

[0130] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with, any of these terms. Considering the purpose of the present invention, the useful polypeptide fragment includes an immunological functional fragment of an antibody comprising an antigen-binding domain. In the case of LILRB4 or 4-1BB binding antibody, such a useful fragment includes a CDR sequence comprising 1, 2, or 3 of heavy chains and / or light chains, or all or a portion of the antibody chain comprising a variable region or constant region of a heavy chain or light chain, but not limited thereto.

[0131] As used herein, "variant" of a polypeptide such as, for example, an antigen-binding fragment, a protein or an antibody, is a polypeptide in which one or more amino acid residues are inserted, deleted, added and / or substituted, as compared to another polypeptide sequence, and includes a fusion polypeptide. In addition, a protein variant includes one modified by protein enzyme cutting, phosphorylation or other posttranslational modification, but maintains biological activity of the antibody disclosed herein, for example, specific binding to LILRB4 and / or 4-1BB and biological activity.

[0132] As used herein, "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0133] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.

[0134] As used herein, "percent (%) sequence identity" and "homology" with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0135] An "effective amount" of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.

[0136] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.

[0137] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered.

[0138] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0139] As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies or bispecific antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

[0140]

[0141] Anti-LILRB4 antibody

[0142] An anti-LILRB4 antibody may comprise an anti-LILRB4 antibody or an antigen-binding fragment thereof as a LILRB4 targeting moiety. The anti-LILRB4 antibody or antigen binding fragment thereof may exhibit potent binding and inhibitory activities to LILRB4, and be useful for therapeutic uses.

[0143] In one embodiment, the anti-LILRB4 antibody or antigen-binding fragment thereof can specifically bind to a human and monkey LILRB4 protein.

[0144] In an embodiment, the anti-LILRB4 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region and a light chain variable region comprising: (a) a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 3; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 5; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 7; (d) a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 11; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 13; and (f) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 15.

[0145] The CDR sequences of chimeric anti-LILRB4 (ch6E9C4) to be comprised in heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment according to one embodiment of the present invention are shown in table 1 below.

[0146] RegionSequenceSEQ ID NO:Heavy chainvariable regionEVQLQQSGPELVKPGASVKISCKASGYTFTDYNIHWVKQSHGKSLEWIGYIYPYNGGTGYNQKFKNKATMTVDNFSSTAYMELRSLTSEDSVVYYCARSEYYGYVGYAMDYWGQGTSVTVSS1HFR1EVQLQQSGPELVKPGASVKISCKASGYTFT2VH CDR1DYNIH3HFR2WVKQSHGKSLEWIG4VH CDR2YIYPYNGGTGYNQKFKN5HFR3KATMTVDNFSSTAYMELRSLTSEDSVVYYCAR6VH CDR3SEYYGYVGYAMDY7HFR4WGQGTSVTVSS8Light chainvariable regionDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVVWYQQKSGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQFNSSPYTFGGGTKLEMK9LFR1DIVMTQSQKFMSTSVGDRVSVTC10VL CDR1KASQNVGTNVV11LFR2WYQQKSGQSPKALIY12VL CDR2SASYRYS13LFR3GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC14VL CDR3QQFNSSPYT15LFR4FGGGTKLEMK16

[0147] The CDR sequences of humanized anti-LILRB4 (h6E9C4) to be comprised in heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment according to one embodiment of the present invention are shown in table 2 below.

[0148] RegionSequenceSEQ ID NO:Heavy chainvariable regionQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNIHWVRQAPGQGLEWMGYIYPYNGGTGYNQKFKNRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSEYYGYVGYAMDYWGQGTLVTVSS19HFR1QVQLVQSGAEVKKPGASVKVSCKASGYTFT20VH CDR1DYNIH3HFR2WVRQAPGQGLEWMG21VH CDR2YIYPYNGGTGYNQKFKN5HFR3RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR22VH CDR3SEYYGYVGYAMDY7HFR4WGQGTLVTVSS23Light chainvariable regionDIQMTQSPSSVSASVGDRVTITCKASQNVGTNVVWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQFNSSPYTFGGGTKLEIK24LFR1DIQMTQSPSSVSASVGDRVTITC25VL CDR1KASQNVGTNVV11LFR2WYQQKPGKAPKALIY26VL CDR2SASYRYS13LFR3GVPSRFSGSGSGTDFTLTISSLQPEDFATYFC27VL CDR3QQFNSSPYT15LFR4FGGGTKLEIK28

[0149] In some embodiments, the antibody or antigen-binding fragment thereof may include no more than one, no more than two, or no more than three substitutions.

[0150] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 19 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 19.

[0151] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 24 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 24.

[0152] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain framework 1 (H-FR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 20 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 20; a heavy chain framework 2 (H-FR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 21 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 21; a heavy chain framework 3 (H-FR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22; a heavy chain framework 4 (H-FR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 23 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 23; a light chain framework 1 (L-FR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 25 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 25; a light chain framework 2 (L-FR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 26 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 26; a light chain framework 3 (L-FR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 27 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 27; and a light chain framework 4 (L-FR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 28 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 28.

[0153] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 29 and 30 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 29 and 30.

[0154] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 31 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 31.

[0155] In other embodiment, the variable regions of heavy chain and light chain disclosed in this invention can be combined freely for preparation of various forms of antibodies.

[0156] The present invention may comprise one or more amino acid sequences having substantial sequence identities with one or more amino acid sequences disclosed herein. The substantial identity means maintaining the effect disclosed herein in which the sequence variation is present.

[0157] In some embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof may be a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0158] In one embodiment, the anti-LILRB4 antibody or antigen-binding fragment thereof can fuse with polypeptide(s) to form a fusion protein. The polypeptide(s) may be an antibody or antigen. In one embodiment, the fusion protein may be in the form of a multi specific antibody (e.g., bispecific antibody), optionally including biparatopic antibody.

[0159] In one embodiment, the disclosure provides "monovalent", "bivalent", "trivalent", "tetravalent", "pentavalent", and "hexavalent" denote the presence of one binding domain, two binding domains, three binding domains, four binding domains, five binding domains, and six binding domains, respectively, in an antibody. The antibodies according to the invention may comprise "bivalent" "trivalent" or "multivalent" (e.g., "tetravalent", "pentavalent" or "hexavalent") antibodies. In a particular aspect, the antibodies of the present invention may have two or more binding sites and are bispecific. That is, the antibodies may be bispecific even in cases where there are more than two binding sites (i.e., that the antibody is trivalent or multivalent).

[0160] In one embodiment, the anti-LILRB4 antibody or antigen-binding fragment thereof may not detectably bind or bind with at least 5-fold lower affinity to other LILR (The leukocyte immunoglobulin-like receptor) family including LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB5, or a combination thereof, relative to the binding affinity to LILRB4. LILR family is classified into two subfamilies: activating (LILRA) and inhibitory (LILRB). LILRA family members share high homology with LILRB receptors and can compete for the same HLA ligands. Thus, in regard to their therapeutic potential, the high homology among certain LILR family must be taken into consideration. Thus, the anti-LILRB4 antibody or antigen-binding fragment can may reduce side effects and treat or prevent cancer by not binding to other LILR family but specifically binding with at least 5-fold, at least 7-fold, at least 10-fold or more higher affinity to LILRB4.

[0161] The term "does not detectably bind" herein means the binding is undetectable by ELISA with an OD 450 to 650 nm reading below (specific threshold, e.g., 0.3). In other words, the binding is below the detection limit of ELISA.

[0162] In one embodiment, the anti-LILRB4 antibody or antigen-binding fragment thereof may bind to LILRB4 (for example, human LILRB4) with a greater affinity than to any one or a combination of LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, or LILRB5.

[0163] In one embodiment, the anti-LILRB4 antibody or antigen-binding fragment thereof may bind to LILRB4 with at least 5-fold, at least 7-fold, at least 10-fold, or more greater affinity relative to any one or a combination thereof LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, or LILRB5.

[0164] According to another embodiment of the present invention, there is provided an antibody-drug conjugate (ADC) comprising the antibody provided in the present invention and a drug.

[0165] As used herein, the term "antibody-drug conjugate (ADC)" refers to a form in which the drug and the antibody are chemically linked to each other without degrading biological activity of the antibody and the drug.

[0166] As used herein, the term "drug" may mean any substance having a certain biological activity for a cell, which is a concept including chemical compound, DNA, RNA, or a peptide.

[0167]

[0168] Anti-4-1BB antibody

[0169] An anti-4-1BB antibody may comprise an anti-4-1BB antibody or an antigen-binding fragment thereof as a 4-1BB targeting moiety. The anti-4-1BB antibody or antigen-binding fragment thereof may exhibit potent binding and inhibitory activities to 4-1BB, and be useful for therapeutic uses.

[0170] In an embodiment, the anti-4-1BB antibody or fragment thereof can specifically bind to 4-1BB (e.g., human 4-1BB) protein.

[0171] For example, the human 4-1BB protein may be selected from the group consisting of proteins represented by NCBI Accession No. NP_001552.2, etc., but may not be limited thereto. These anti-4-1BB antibodies or antigen-binding fragments thereof can enhance immune response and / or treating tumor (cancer) in a mammal. The anti-4-1BB antibody or an antigen-binding fragment thereof is characterized by being localized and / or activated in tumor microenvironment (TME) and / or considerably reducing liver toxicities compared to pre-existing anti-4-1BB antibodies, with maintaining the efficacies of enhancing immune response enhancement and / or tumor treatment.

[0172] In an embodiment, the anti-4-1B antibody or antigen-binding fragment thereof may comprise a heavy chain variable region and a light chain variable region comprising: (a) a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 34; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 36; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 38; (d) a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 42; I a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 44; and (f) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 46.

[0173] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 32 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 32.

[0174] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising an amino acid sequence of SEQ ID NO: 40 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 40.

[0175] In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain framework 1 (H-FR1) comprising an amino acid sequence of SEQ ID NO: 33 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 33; a heavy chain framework 2 (H-FR2) comprising an amino acid sequence of SEQ ID NO: 35 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 35; a heavy chain framework 3 (H-FR3) comprising an amino acid sequence of SEQ ID NO: 37 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 37; a heavy chain framework 4 (H-FR4) comprising an amino acid sequence of SEQ ID NO: 39 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 39; a light chain framework 1 (L-FR1) comprising an amino acid sequence of SEQ ID NO: 41 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 41; a light chain framework 2 (L-FR2) comprising an amino acid sequence of SEQ ID NO: 43 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 43; a light chain framework 3 (L-FR3) comprising an amino acid sequence of SEQ ID NO: 45 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 45; and a light chain framework 4 (L-FR4) comprising an amino acid sequence of SEQ ID NO: 47 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 47.

[0176] The CDR sequences of humanized anti-4-1BB (1A10M12) to be comprised in heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment according to one embodiment of the present invention are shown in table 3 below.

[0177] RegionSequenceSEQ ID NO:Heavy chainvariable regionEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS32HFR1EVQLLESGGGLVQPGGSLRLSCAASGFTFS33VH CDR1SYDMS34HFR2WVRQAPGKCLEWVS35VH CDR2WISYSGGSIYYADSVKG36HFR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR37VH CDR3DAQRNSMREFDY38HFR4WGQGTLVTVSS39Light chainvariable regionQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL40LFR1QSVLTQPPSASGTPGQRVTISC41VL CDR1SGSSSNIGNNYVT42LFR2WYQQLPGTAPKLLIY43VL CDR2ADSHRPS44LFR3GVPDRFSGSKSGTSASLAISGLRSEDEADYYC45VL CDR3ATWDYSLSGYV46LFR4FGCGTKLTVL47

[0178] In some embodiments, an antibody or antigen-binding fragment thereof may include no more than one, no more than two, or no more than three substitutions.In other embodiments, the variable regions of heavy chain and light chain disclosed above can be combined freely for preparation of various forms of antibodies.

[0179] In some embodiments, the antigen-binding fragment of the anti-4-1BB antibody may be any fragment comprising heavy chain CDRs and / or light chain CDRs of the antibody.

[0180] The present invention comprises one or more amino acid sequences having substantial sequence identities with one or more amino acid sequences disclosed herein. The substantial identity means maintaining the effect disclosed herein in which the sequence variation is present.

[0181] In one embodiment, the disclosure provides fusion proteins comprising (i) one or more single domain antibodies, or antigen-binding fragments thereof, described herein (e.g., one or more CDRs described herein), and (ii) one or more additional polypeptides. For example, a fusion protein can include one or more single domain antibodies described herein and a constant region or Fc region described herein. In one embodiment, one or more single domain antibodies, or antigen-binding fragments thereof, described herein (e.g., one or more CDRs described herein) can be conjugated noncovalently or covalently, e.g., fused, to an antibody or antigen.

[0182]

[0183] Anti-LILRB4 / anti-4-1BB bispecific antibody

[0184] An anti-LILRB4 / anti-4-1BB bispecific antibody may comprise the anti-LILRB4 antibody or antigen-binding fragment thereof; and anti-4-1BB antibody or antigen binding fragment thereof. The anti-LILRB4 / anti-4-1BB bispecific antibody may be useful for therapeutic uses.

[0185] In one embodiment, the bispecific antibody comprises the LILRB4 targeting moiety and the 4-1BB targeting moiety.

[0186] In one embodiment, the anti- LILRB4 antibody or antigen-binding fragment thereof and the anti-4-1BB antibody or antigen-binding fragment thereof may be fused, directly or via a peptide linker.

[0187] In one embodiment, each of the anti-LILRB4 antibody or antigen-binding fragment thereof and the anti-4-1BB antibody or antigen-binding fragment thereof may independently be a scFv or a Fab molecule.

[0188] In one embodiment, the bispecific antibodies or antigen binding fragments thereof are a First-in-Class bispecific antibody designed to simultaneously target LILRB4 and 4-1BB, enabling LILRB4 expression-dependent 4-1BB activation. By antagonizing LILRB4 signaling, the bispecific antibodies or antigen binding fragments thereof inhibited LILRB4 mediated immunosuppression, restoring T cell activity. In addition, the bispecific antibodies or antigen binding fragments thereof inhibited fibronectin, a potential LILRB4 ligand, -mediated suppression of myeloid cells. To evaluate the efficacy of the bispecific antibodies or antigen binding fragments thereof, we established EL4 murine tumor model overexpressing LILRB4 (EL4 / LILRB4) in h4-1BB transgenic mice and hLILRB1 / 4-h4-1BB triple transgenic mice. We observed LILRB4 was overexpressed in M-MDSC in the blood and liver-metastasized tumors. A significant decrease of M-MDSC population and tumor growth inhibition was observed in mice treated with anti-LILRB4 antibody and the bispecific antibodies or antigen binding fragments thereof. the bispecific antibodies or antigen binding fragments thereof treatment resulted in a proportion increment of T cells. However, the percentage of Tregcells and M-MDSC in the liver and blood was reduced by the bispecific antibodies or antigen binding fragments thereof treatment in hLILRB1 / 4-h4-1BB triple transgenic mice. Mice with complete remission (CR) were further protected from the rechallenge of previously exposed tumor after 3 months of cessation of the bispecific antibodies or antigen binding fragments thereof treatment, implicating that immunological memory might be generated. In conclusion, our findings suggest that the bispecific antibodies or antigen binding fragments thereof, LILRB4x4-1BB bispecific antibody, may be a promising strategy for the treatment of cancer. It can be applied as "a "LILRB4 antagonist" as well as "a "LILRB4-dependent 4-1BB agonist" across a broad spectrum of cancers.

[0189] In one embodiment, the bispecific antibodies or antigen-binding fragment thereof may comprise "monovalent", "bivalent", "trivalent", "tetravalent", "pentavalent", and "hexavalent" denote the presence of one binding domain, two binding domains, three binding domains, four binding domains, five binding domains, and six binding domains, respectively, in an antibody. The bispecific antibodies according to the invention are at least "bivalent" and may be "trivalent" or "multivalent" (e.g., "tetravalent" "pentavalent" or "hexavalent"). In a particular aspect, the antibodies of the present invention have two or more binding sites and are bispecific. That is, the antibodies may be bispecific even in cases where there are more than two binding sites (i.e., that the antibody is trivalent or multivalent).

[0190] In one embodiment, the bispecific antibody or antigen-binding fragment thereof further comprising at least an antigen binding site, wherein the antigen binding site specifically binds to Tumor-associated antigens (TAA), Tumor-specific antigens (TSA), T-cell surface antigens.

[0191]

[0192] Fc Domain

[0193] The Fc domain confers to the antibody or bispecific antibody favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. Wild-type Fc domain is involved in antibody dependent cellular cytotoxicity (ADCC) which is an Fc-dependent effector function of IgG important for anti-viral immunity and anti-tumor therapies. On the other hand, it may lead to undesirable targeting of the antibody or bispecific antibody to cells expressing Fc receptors rather than to the preferred antigen-bearing cells.

[0194] In one embodiment, the Fc domain of the present invention is wild-type Fc domain. In another embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to a Fc receptor and / or effector function may be introduced to the antibody. For example, the Fc domain may comprise an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). For example, the further amino acid substitution may be E233P, L234A, L235A, L235E, N297A, N297D or P331S.

[0195]

[0196] Therapeutic Methods and Compositions

[0197] Any of the antibodies or bispecific antibodies provided herein may be used in therapeutic methods. Antibodies or bispecific antibodies of the invention may be used as immunotherapeutic agents, for example in the treatment of cancers.

[0198] In one aspect, antibodies or bispecific antibodies of the invention for use as a medicament are provided. In further aspects, antibodies or bispecific antibodies of the invention for use in treating a disease are provided. In certain embodiments, antibodies or bispecific antibodies of the invention for use in a method of treatment are provided.

[0199] In one embodiment, the invention provides an antibody or bispecific antibodies as described herein for use in the treatment of a cancer in an individual in need thereof.

[0200] In one embodiment, the cancer may be a solid cancer or a blood cancer.

[0201] In one embodiment, the cancer may be selected from the group consisting of leukemia, rectal cancer, endometrial cancer, nephroblastoma, basal cell carcinoma, nasopharyngeal cancer, bone tumor, esophageal cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular thyroid cancer, hepatocellular carcinoma, oral cancer, renal cell carcinoma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, mesenchymal tumor, soft tissue sarcoma, liposarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratoma, neuroblastoma, medulloblastoma, glioma, benign skin tumor, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy-cell leukemia, myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, neuroectodermal tumor, epithelial tumor, cutaneous T-cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), pancreas cancer, haematological malignancies, kidney cancer, tumor vasculature, breast cancer, renal cancer, ovarian cancer, epithelial ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), Head and neck squamous cell carcinoma (HNSCC), glioblastoma multiforme (GBM), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.

[0202] In an embodiment, the cancer may be selected from the group consisting of acute myeloid leukemia (AML), and multiple myeloma.

[0203] In an embodiment, the cancer may be selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), and hepatocellular carcinoma.

[0204] LILRB4 expression can be observed in certain types of blood cancer (hematologic malignancy), such as acute myeloid leukemia (AML) and multiple myeloma (MM). Considering the tumor microenvironment, it may exhibit antitumor effects in certain types of solid tumors, such as melanoma, NSCLC, and hepatocellular carcinoma. Tumor cells may infiltrate into the liver. Thus, many MDSC expressing LILRB4 are observed in the tumor microenvironment. Thus, an anti-LILRB4 antibody or an antigen-binding fragment thereof may show therapy effect on not only A549 mouse model but also hepatocellular carcinoma.

[0205] LILRB4 is over expressed in monocytic AML (FAB M4 / M5 types), MDSC, TAM, suppressive DC, and on tumor-associated myeloid cells (tolDC, MDSC and M2 Mφ). LILRB4 is also expressed in human gastric cancer cells and can enhance tumor growth (see Zhang et al, 2012). T cell inhibition mediates by LILRB4 binding to the counterpart on T cell or inhibitory signaling. myeloid cells suppress antitumor T-cell responses by the interaction of fibronectin with LILRB4. It is thought that it may control inflammatory responses and cytotoxicity to help focus immune responses and limit autoreactivity. Anti-LILRB4 antibody may restore of T cell activity by blocking ApoE and LILRB4 interaction or by blocking LILRB4 and the counterpart interaction or inhibitory signaling. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased anti-tumor T-cell responses. Thus, the antibodies or antigen-binding fragment thereof and the bispecific antibodies according to the present invention can prevent and treat all type of cancers by specifically binding to LILRB4.

[0206]

[0207] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only for describing the present invention in more detail, and it will be apparent to those skilled in the art that according to the gist of the present invention, the scope of the present invention is not limited to these examples.

[0208]

[0209] Examples

[0210] Example 1. Production of anti-LILRB4 / anti-4-1BB bispecific antibodies

[0211] 1-1. Generation of anti-LILRB4 antibody

[0212] 1-1-1. Mouse monoclonal antibody generation by immunization

[0213] Monoclonal antibodies against human LILRB4 were newly generated by mouse hyperimmunization. Specifically, SJL and Balb / C mice (GenScript, New Jersey, USA) were hyperimmunized with recombinant human and cynomolgus LILRB4-ECD protein. Mice were injected with recombinant human and cynomolgus LILRB4-ECD protein (25 microgram each via subcutaneous) followed by 2 times of boost with recombinant human and cynomolgus LILRB4-ECD (15 microgram each via intraperitoneal). Serum titers were evaluated twice by standard enzyme linked immunosorbent assay (ELISA) and FACS after 1stboost and 2ndboost. 4 days post final boost, Splenic B cells harvested from sera LILRB4 positive mice were fused with mouse myeloma cells by electrofusion. After 10-14 days, hybridoma supernatants were screened for antibody secretion by ELISA. All positive clones were then expanded and re-screened for binding to human and cynomolgus LILRB4 by ELISA and FACS.

[0214] One hybridoma clone was identified: 6E9C4. It was positively bound to human and cynomolgus LILRB4-ECD protein and LILRB4 expressed cell line (human LILRB4 CHOK1 and cynomolgus LILRB4 CHOK1) (data shown below). Variable regions and CDRs of the identified mouse clones are shown as Table 4 below.

[0215] RegionSequenceSEQ ID NO:Heavy chainvariable regionEVQLQQSGPELVKPGASVKISCKASGYTFTDYNIHWVKQSHGKSLEWIGYIYPYNGGTGYNQKFKNKATMTVDNFSSTAYMELRSLTSEDSVVYYCARSEYYGYVGYAMDYWGQGTSVTVSS1HFR1EVQLQQSGPELVKPGASVKISCKASGYTFT2VH CDR1DYNIH3HFR2WVKQSHGKSLEWIG4VH CDR2YIYPYNGGTGYNQKFKN5HFR3KATMTVDNFSSTAYMELRSLTSEDSVVYYCAR6VH CDR3SEYYGYVGYAMDY7HFR4WGQGTSVTVSS8Light chainvariable regionDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVVWYQQKSGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQFNSSPYTFGGGTKLEMK9LFR1DIVMTQSQKFMSTSVGDRVSVTC10VL CDR1KASQNVGTNVV11LFR2WYQQKSGQSPKALIY12VL CDR2SASYRYS13LFR3GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC14VL CDR3QQFNSSPYT15LFR4FGGGTKLEMK16

[0216]

[0217]

[0218] 1-1-2. Cloning and chimerization of mouse monoclonal antibody

[0219] Mouse monoclonal antibodies 6E9C4 was cloned and chimerized as follows: Total RNA was extracted from hybridoma cells producing murine 6E9C4 using standard methods. The variable light (VL) and variable heavy (VH) domains were amplified using RT-PCR with degenerate primers to the heavy and light chains. The forward primers were specific for the N-terminal amino acid sequence of the VL and VH regions. Respectively, the LC and HC reverse primers were designed to anneal to a region in the constant light (CL) and constant heavy domain 1 (CH1), which are highly conserved across species. The polynucleotide sequence of the inserts was determined using routine sequencing methods. Antibody was chimerized by cloning the mouse heavy chain variable region onto a human IgG1 heavy chain constant region and cloning the light chain variable region onto a human kappa light chain constant region. The antibody of the chimeric 6E9C4 clone has the heavy chain of SEQ ID NO: 17 and the light chain of SEQ ID NO: 18. The sequences of the chimeric 6E9C4 clone are shown as Table 5 below.

[0220] RegionSequenceSEQ ID NO:Chimeric 6E9C4 Heavy chain _ N297A mutated FcEVQLQQSGPELVKPGASVKISCKASGYTFTDYNIHWVKQSHGKSLEWIGYIYPYNGGTGYNQKFKNKATMTVDNFSSTAYMELRSLTSEDSVVYYCARSEYYGYVGYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK17Chimeric 6E9C4 Light chain _ kappaDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVVWYQQKSGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQFNSSPYTFGGGTKLEMKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC18

[0221]

[0222] 1-1-3. Humanization of Mouse Monoclonal Antibody

[0223] The structure of parental antibody was modelled by computer-aided homology modelling program. Humanized antibodies were designed using CDR grafting plus back mutation. Briefly, the CDRs of parental antibodies were grafted into the human acceptors to obtain humanized light chains. The sequences of humanized heavy and light chain of 6E9C4 clone are shown as Table 3 below. The antibody of the humanized 6E9C4 clone has the heavy chain of SEQ ID NOs: 29 or 30 and the light chain of SEQ ID NO: 31. The sequences of the humanized 6E9C4 clone are shown as Table 6 below.

[0224] RegionSequenceSEQ ID NO:Heavy chainvariable regionQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNIHWVRQAPGQGLEWMGYIYPYNGGTGYNQKFKNRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSEYYGYVGYAMDYWGQGTLVTVSS19HFR1QVQLVQSGAEVKKPGASVKVSCKASGYTFT20VH CDR1DYNIH3HFR2WVRQAPGQGLEWMG21VH CDR2YIYPYNGGTGYNQKFKN5HFR3RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR22VH CDR3SEYYGYVGYAMDY7HFR4WGQGTLVTVSS23Light chainvariable regionDIQMTQSPSSVSASVGDRVTITCKASQNVGTNVVWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQFNSSPYTFGGGTKLEIK24LFR1DIQMTQSPSSVSASVGDRVTITC25VL CDR1KASQNVGTNVV11LFR2WYQQKPGKAPKALIY26VL CDR2SASYRYS13LFR3GVPSRFSGSGSGTDFTLTISSLQPEDFATYFC27VL CDR3QQFNSSPYT15LFR4FGGGTKLEIK28Humanized 6E9C4 Heavy chain _ Wild type FcQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNIHWVRQAPGQGLEWMGYIYPYNGGTGYNQKFKNRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSEYYGYVGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK29Humanized 6E9C4 Heavy chain _ N297A mutated FcQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNIHWVRQAPGQGLEWMGYIYPYNGGTGYNQKFKNRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSEYYGYVGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK30Humanized 6E9C4 Light Chain _ kappaDIQMTQSPSSVSASVGDRVTITCKASQNVGTNVVWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQFNSSPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC31

[0225]

[0226] 1-2. Generation of anti-4-1BB Antibody

[0227] 1-2-1. Preparation of full human anti-4-1BB monoclonal antibodies

[0228] Full human anti-4-1BB monoclonal antibodies were prepared as disclosed in WO2020 / 111913. Specifically, for panning of a phage library (obtained from Kbio Health) against target molecules, A total of four rounds of panning were carried out using 4-1BB (NCBI Accession No. NP_001552.2) coated immunotubes. Bacterial colonies from the 3 rounds of panning output were grown in SB-Carbenicilin in 96 deep well plate until turbid, at which point 1011 pfu of VCSM13 helper phage was added to each well. After 1 h infection at 37°C with gentle shaking (80 rpm), 70 μg / mL of kanamycin was added, and the cells were cultured overnight at 30℃ with shaking at 200 rpm. Next day, the plates were centrifuged and the supernatants containing the phages were added to 4-1BB antigen-coated ELISA plates blocked with 3% BSA in PBST. After 1 h incubation at room temperature, the plates were washed three times with PBST and anti M13 antibody was added. The plates were incubated for 1 h, washed three times with PBST, and the binding affinity was measured using tetramethylbenzidine (TMB).

[0229] The 4-1BB specific binders were amplified for plasmid DNA sequencing. Ig light chain variable (VL) genes and Ig heavy chain (VH) genes were analyzed to identify unique sequences and determine sequence diversity.

[0230]

[0231] 1-2-2. Preparation of anti-4-1BB scFv antibodies

[0232] Anti-4-1BB scFv antibodies with a structure of (N')-VL-linker [(GGGGS)4SEQ ID NO:51] VH-(C') were prepared using the variable regions of the full human monoclonal antibodies against 4-1BB obtained in Example 1-2-1 above, wherein the amino acid residue "G" at the position 44 of a heavy chain variable region was substituted with "C", and the amino acid residue "G" at the position 103 of a light chain variable region was substituted with "C". Such amino acid substitution from "G" to "C" in scFv can contribute to increase in stabilities of bispecific antibodies comprising the scFv as one target specific moiety. The amino acid sequences of the prepared anti-4-1BB scFvs are provided in Table 7, while skilled persons in the art may apply changes or modifications of amino acid sequences to meet specific purposes, including applying various types of peptide linkers. The anti-4-1BB scFv antibodies have the variable heavy domain of SEQ ID NO: 32 and the variable light domain of SEQ ID NO: 40.

[0233] RegionSequenceSEQ ID NO:Heavy chainvariable regionEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS32HFR1EVQLLESGGGLVQPGGSLRLSCAASGFTFS33VH CDR1SYDMS34HFR2WVRQAPGKCLEWVS35VH CDR2WISYSGGSIYYADSVKG36HFR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR37VH CDR3DAQRNSMREFDY38HFR4WGQGTLVTVSS39Light chainvariable regionQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL40LFR1QSVLTQPPSASGTPGQRVTISC41VL CDR1SGSSSNIGNNYVT42LFR2WYQQLPGTAPKLLIY43VL CDR2ADSHRPS44LFR3GVPDRFSGSKSGTSASLAISGLRSEDEADYYC45VL CDR3ATWDYSLSGYV46LFR4FGCGTKLTVL47

[0234]

[0235] 1-3. Generation of anti-LILRB4 / anti-4-1BB Bispecific antibody

[0236] The anti-LILRB4 and anti-4-1BB clones prepared in Example 1-1 and Example 1-2, respectively, were exemplarily selected, to prepare anti-LILRB4 / anti-4-1BB bispecific antibodies, in which a scFv antibody fragment of one antigen being fused to the C-terminal of IgG of another antigen. When LILRB4 is placed in full IgG part, wild type of IgG1 or IgG1 with ADCC reduced mutant backbone (N297A mutation / NA mutation) were used.

[0237] Hereinafter, humanized 6E9C4 monospecific antibodies with are also referred to as AB0166 (hIgG1 WT) and AB0224 (hIgG1 NA), and humanized 6E9C4 x 1A10M12 bispecific antibodies are also referred to as AB0228 (hIgG1 WT) and AB0229 (hIgG1 NA).

[0238] Cloning of the bispecific antibody was performed as follows: A DNA segment 1 having a nucleotide sequence encoding a heavy chain of an IgG antibody of the anti-LILRB4 / anti-4-1BB bispecific antibody was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 1), and a DNA segment 2 having a nucleotide sequence encoding a light chain of an IgG antibody of the anti-LILRB4 / anti-4-1BB bispecific antibody was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 2). Thereafter, a DNA segment 3 encoding a scFv was fused at a part of the DNA segment 1 corresponding to the c-terminus of the Fc region of the IgG antibody inserted into the plasmid 1, using a DNA segment 4 encoding a linker peptide having 16 amino acid lengths consisting of (GGGGS)4: SEQ ID NO: 51 or using a DNA segment 5 encoding a linker peptide having 18 amino acid lengths of (GS)9: SEQ ID NO: 50 to construct vectors for the expression of bispecific antibodies. Furthermore, in order to stabilize scFv, as described in Example 1-2, additional modification was applied to generate disulfide bridge fusing VL103-VH44 (VL103: VL having G→C mutation at the position 103; VH 44: VH having G→C mutation at position 44) to C-terminus of light chain and C-terminus of heavy chain, respectively. The anti-LILRB4 / anti-4-1BB bispecific antibodies have the heavy chain of SEQ ID NOs: 48 or 49.

[0239] The constructed vectors were transiently expressed in ExpiCHO-S™ cells (Thermo Fisher, A29127) using ExpiFectamine™ CHO Kit (Thermo Fisher, A29129), cultured in ExpiCHO™ Expression medium (Thermo Fisher, A2910002) under the conditions of 37℃ for 7 days in a CO2incubator equipped with rotating shaker. Plasmid DNA (250 μg) and ExpiFectamin™ CHO Reagent (800 μL) were mixed with Opti-MEM® I medium (20 mL final volume) and allowed to stand at room temperature for 5 minutes. The mixed solution was added to 1.5x109ExpiCHO™ cells cultured in 250mL of ExpiCHO™ Expression Medium and gently mixed in a shaker incubator at 37°C with a humidified atmosphere of 8% CO2in air. At 18 hours post-transfection, 1.5 mL of ExpiFectamin™ CHO Transfection Enhancer and 60 mL of ExpiFectamin™ CHO Transfection Feed were added to each flask. After transfection for 7 days, cells were harvested, and the supernatant was used for purification.

[0240] Each bispecific antibody was purified from the cell culture supernatant by recombinant Protein A affinity chromatography (MabSelect™ SuRe™, Cytiva, 17-5438-03) and gel filtration chromatography with a HiLoad 26 / 600 Superdex-200 prep grade column (Cytiva, 28-9893-36). SDS-PAGE (NuPAGE 4-12% Bis-Tris gel, NP0321) and size exclusion HPLC (Agilent, 1200 series) analysis with SE-HPLC column (TSKgel G3000SWXL ID 7.8 mm, 30 cm, 5 μm, TOSOH, 0008541) were performed to detect and confirm the size and purity of each bispecific antibody. The purified proteins were buffer-exchanged and concentrated with 20 mM Histidine-HCl, pH 6.0, 8% (w / v) Sucrose buffer using an Amicon Ultra -15, 30K centrifugal filter unit (Millipore, UFC903096) and then formulation was performed by spiking the 20 mM Histidine-HCl, pH 6.0, 8% (w / v) Sucrose, 2% (w / v) Polysorbate 80 stock solution to become the final 20 mM Histidine-HCl, pH 6.0, 8%(w / v) Sucrose, 0.02% (w / v) Polysorbate 80. And then, protein concentrations were estimated using a Nanodrop One (Thermo Fisher).

[0241] When a two-vector system is applied, the ratio between light to heavy chain could be 2:1 by weight. Alternatively, a one-vector system that contains both chains in one single vector can also be used.

[0242] The information of the monoclonal antibodies is show in Table 8 below.

[0243] AntibodyCloneCodeBackbonemAb (Monospecific antibody)LILRB46E9C4AB0166hIgG1 WT6E9C4AB0244hIgG1 NA4-1BB1A10M12--BsAb (Bispecific antibody)LILRB4: 6E9C44-1BB: 1A10M126E9C4 x 1A10M12AB0228hIgG1 WTLILRB4: 6E9C44-1BB: 1A10M126E9C4 x 1A10M12AB0229hIgG1 NA

[0244]

[0245] Example 2. Evaluation of anti-LILRB4 monospecific antibody

[0246] 2-1. Antigen binding affinity of chimeric anti-LILRB4 antibody (SPR)

[0247] The binding affinity of chimeric anti-LILRB4 antibody was tested by surface plasmon resonance (SPR) using Biacore 8K (GE Healthcare). The assay was performed at 25℃ and the running buffer was HBS-EP. Diluted antibody was injected over the surface during the capture phase, followed by the injection of diluted hLILRB4 antigens over the surface during the association phase, and finally, the running buffer was injected during the dissociation phase. Running configuration was listed. As shown in Fig. 1 and Table 9 below, ch6E9C4 showed high binding affinity to hLILRB4 antigens.

[0248] Affinity measurement of hLILRB4 to antibodiesLigandAnalyteChi2(RU2)ka(1 / Ms)Kd(1 / s)KD(M)Rmax(RU)6E9C4hLILRB46.51E-021.21E+052.48E-042.04E-0959.6bufferhLILRB4NANANANANA

[0249]

[0250] 2-2. Cross-specificity of chimeric anti-LILRB4 monospecific antibody (ELISA)

[0251] To evaluate the antigen binding affinity of the chimeric antibody, ch6E9C4 was subjected to ELISA. Briefly, 96-well ELISA plates were coated with Fc fused human or cynomolgus LILRB4 protein at 1 μg / ml in PBS(100 μl / well) at 4℃ overnight, then blocked with 200 μl / well of 1% BSA in PBS at 37℃ for 2 hours. Four-fold dilutions of monoclonal antibody starting from 15μg / ml were added to each well and incubated for 1 hour at 37℃. The plates were washed with PBST (0.05% Tween 20 in PBS) and then incubated with HRP (Horse Radish Peroxidase) conjugated Anti-human Fab secondary antibody (Thermo Fisher, cat. 31414) for 1 hour at 37℃. The plates were washed with PBST (0.05% Tween 20 in PBS) developed with TMB substrate and analyzed by spectrophotometer (Molecular Devices) at OD 450 nm. As shown in Fig. 2(a) and Fig. 2(b), ch6E9C4 bound to both human and cynomolgus LILRB4 in a dose-dependent manner.

[0252]

[0253] 2-3. LILRB4-binding specificity of chimeric anti-LILRB4 monospecific antibody (ELISA)

[0254] To evaluate the binding specificity of the chimeric antibody to LILRB4 compared to other LILRA / B family proteins, ch6E9C4 was subjected to ELISA. Briefly, 96-well ELISA plates were coated with human Fc or his-tag fused LILRA1(Sino biological, 17220-H08H), LILRA2(R&D systems, 9040-T4-050), LILRA3(Sino biological, 13549-H08H), LILRA4(Sino biological, 16058-H08H), LILRA5(Sino biological, 16059-H08H), LILRA6(R&D systems, 9088-T4-050) and LILRB1(Sino biological, 16014-H08H), LILRB2(Sino biological, 14132-H08H), LILRB3(Sino biological, 11978-H08H), LILRB4(Sino biological, 16742-H08H), LILRB5(Sino biological, 17221-H08H) proteins at 0.4 μg / ml in PBS(150 μl / well) at 4℃ overnight, then blocked with 200 μl / well of 1% BSA in PBS at 37℃ for 2 hours. For the experimental control of each family protein, mouse anti-human LILRA1 / LILRB1 antibody (R&D systems, MAB30851), mouse anti-human LILRA2 antibody (R&D systems, MAB6364), mouse anti-human LILRA3 antibody (R&D systems, MAB2574), mouse anti-human LILRA4 antibody (R&D systems, MAB6287), mouse anti-human LILRA5 antibody (R&D systems, MAB6754), mouse anti-human LILRA6 antibody (R&D systems, MAB8656) were used. Six-fold dilutions of control mouse anti-human LILRA / B family antibodies and chimeric 6E9C4 antibody starting from 0.4μg / ml were added to each well and incubated for 1 hour at 37℃. The plates were washed with PBST (0.05% Tween 20 in PBS) and then incubated with HRP (Horse Radish Peroxidase) conjugated goat anti-mouse IgG(H+L) secondary antibody (Invitrogen, 31432) and HRP conjugated goat anti-human IgG F(ab')2 secondary antibody (Thermo Fisher, 31414) for 1 hour at 37℃. The plates were washed with PBST (0.05% Tween 20 in PBS) developed with TMB substrate and analyzed by spectrophotometer (Molecular Devices) at OD 450 nm-650 nm. As shown in Figs. 3(a) to 3(k), ch6E9C4 bound to only human LILRB4 in a dose-dependent manner.

[0255]

[0256] 2-4. Binding confirmation of chimeric anti-LILRB4 antibody by using FACS analysis

[0257] To evaluate the antigen binding property, ch6E9C4 was analyzed for its binding to LILRB4-expressing cells (human / cynomolgus LILRB4 CHOK1). Briefly, each cell was treated with four-fold dilutions of monoclonal antibodies starting from 10 μg / ml of Isotype control and ch6E9C4 at 4℃ for 1 hour. After washing by FACS buffer (1% BSA in PBS), the cells were incubated with the FITC-conjugated goat anti-human IgG F(ab')2secondary antibody (Thermo Fisher, 31628) at 4℃ for 1 hour, and then subjected to FACS analysis. As a result, ch6E9C4 antibodies are specifically bound to human and cynomolgus LILRB4 expressing CHOK1 cells. As shown in Fig. 4(a) and Fig. 4(b), ch6E9C4 showed binding affinity against hLILRB4 antigens.

[0258]

[0259] 2-5. Binding affinity of humanized anti-LILRB4 monospecific antibodies to target protein LILRB4 (SPR)

[0260] The binding affinity of purified antibodies to Human LILRB4 was individually determined using Biacore™ T200. Antibodies were immobilized on the sensor chip through an Fc capture method. The LILRB4 antigen was used as the analyte. The data of dissociation (kd) and association (ka) rate constants were obtained using Biacore™ T200 evaluation software. The equilibrium dissociation constants (KD) were calculated as the ratio of kdto ka. As shown in Fig. 5(a), Fig. 5(b) and Table 10 below, humanized 6E9C4 showed high binding affinity for hLILRB4 antigens.

[0261] The affinity of Human LILRB4 to selected humanized IgGs batchLigandAnalyteka(1 / Ms)kd(1 / s)KD(M)Rmax(RU)Chi2(RU2)Chimeric 6E9C4hLILRB42.54E+051.44E-045.66E-1041.890.183humanized 6E9C4hLILRB42.94E+051.04E-043.55E-1058.880.019

[0262]

[0263] 2-6. Cross-specificity of humanized anti-LILRB4 antibody (ELISA)

[0264] To evaluate the antigen binding affinity of humanized 6E9C4 (WT / NA) antibodies were subjected to ELISA. Briefly, 96-well ELISA plates were coated with Fc fused human or cynomolgus LILRB4 protein at 1 μg / ml in PBS(100 μl / well) at 4℃ overnight, then blocked with 200 μl / well of 1% BSA in PBS at 37℃ for 2 hours. Four-fold dilutions of monoclonal antibodies starting from 100nM were added to each well and incubated for 1 hour at 37℃. The plates were washed with PBST (0.05% Tween 20 in PBS) and then incubated with HRP (Horse Radish Peroxidase) conjugated Anti-human Fab secondary antibody (Thermo Fisher, cat. 31414) for 1 hour at 37℃. The plates were washed with PBST (0.05% Tween 20 in PBS) developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in Fig. 6(a), Fig. 6(b) and Table 11 below, humanized 6E9C4 (WT / NA) bound to human and cynomolgus LILRB4 in a dose-dependent manner.

[0265] Protein binding(ELISA)SACE EC50 (nM)hLILRB4cyLILRb4AB01660.0550.141AB02240.0420.110

[0266]

[0267] Example 3. Activity and efficacy of anti-LILRB4 monospecific antibody

[0268] 3-1. T cell checkpoint blockade activity of anti-LILRB4 antibody with dose dependent manner

[0269] In order to characterize the T cell checkpoint blockade activity of the LILRB4 antibodies in stimulated human peripheral blood mononuclear cells (PBMCs) response, the concentration of IFN-gamma(γ) in the supernatant secreted from T cell was measured. LILRB4 inhibition of T-cell activation was conducted using plate-immobilized recombinant LILRB4 protein and PBMC from a healthy donor. Various concentrations of isotype control (hIgG1) and ch6E9C4 (chimeric anti-LILRB4 antibody) were incubated with PBMC in the presence of anti-human CD3 to be tested. After culturing in a humidified chamber with 5% CO2at 37℃ for 72 hours, the concentration of IFN-gamma in the supernatant was measured by Human IFN-gamma Quantikine Kit (R&D system, SIF50).

[0270] As shown in Fig. 7, ch6E9C4 blocked the T cell checkpoint blockade activity of LILRB4, which can be supported by the increase in IFN-γ secretion level compared with the isotype control treated condition. That is, in a LILRB4 mediated immune suppressive condition, such immune suppression could be overcome by ch6E9C4. In other words, ch6E9C4 restored immune cell activity from LILRB4-mediated immune suppression.

[0271]

[0272] 3-2. Effects of chimeric anti-LILRB4 antibody on tolerogenic dendritic cells

[0273] Primary human monocytes were isolated from cryopreserved peripheral blood mononuclear cells by negative selection using a Miltenyi Pan Monocyte Isolation Kit (Miltenyi Biotec, 130-096-537), according to the manufacturer's instructions. To generate immature dendritic cells, human monocytes were plated at 5x105cells / mL in RPMI1640 media containing 1000 IU / mL recombinant human GM-CSF and 1000 IU / mL recombinant human IL-4 (both from Peprotech) and cultured for 5 days, with the rhGM-CSF / rhIL-4 media change on days 2 and 3. To generate tolerogenic dendritic cells, immature dendritic cells were harvested and plated at a density of 7x105cells / mL in 96-well plates. The cells were cultured in the presence of 1000 IU / mL rhGM-CSF, 1000 IU / mL rhIL-4, 4 ng / mL rhIL-10, 1 μg / mL LPS, along with three-fold dilutions of antibodies starting from 3 μg / mL and incubated for 2 days. These tolerogenic dendritic cells were harvested and analyzed by the flow cytometry to confirm the cell membrane protein expression. The concentration of TNF-alpha in the supernatant was measured by Human TNF-alpha ELISA Kit (R&D system, DuoSet, DY210).

[0274] As shown in Fig. 8(a) to Fig. 8(f), the secretion of TNF-alpha in tolerogenic dendritic cells was increased in a concentration-dependent manner by the ch6E9C4 antibody. Additionally, the expression of dendritic cell activation markers HLA-DR, CD86, and CD83 was increased upon treatment with ch6E9C4. These results suggest that the immunosuppressive properties of tolerogenic dendritic cells can be reversed by the anti-LILRB4 antibody, ch6E9C4, leading to immune-activation.

[0275]

[0276] 3-3. anti-LILRB4 antibody restores immune cell activity from LILRB4-mediated suppression

[0277] THP-1 is a human monocytic cell line established from acute monocytic leukemia cells and was obtained from ATCC. Activation of monocytes can be achieved by crosslinking Fc receptor (FcR) on the cell surface, which leads to cytokine production, for example, TNF-α and / or IL-8. Studies have shown that LILRB4 is a potent inhibitor of FcR-mediated cytokine production in monocytes. THP-1 cells were used in an assay to investigate the interaction between LILRB4 and fibronectin in a myeloid cell.

[0278] 96-well ELISA plates were co-coated with fibronectin at 5 μg / mL (Millipore, FC010) and human IgG1 antibody at 1 μg / mL. Plates were incubated at room temperature for 2 hours, washed twice with PBS, and blocked with RPMI 1640 containing 10% FBS. THP-1 cells (2x105cells / well) were added to the wells in the presence of anti-LILRB4 antibodies (N297A mutated; serially diluted from 100nM), Benchmark antibody 1 (NGM bio's US 20210221887A1, SEQ ID NOs: 54 and 55), Benchmark antibody 2 (Merck's US 20190153093A1, SEQ ID NOs: 52 and 53) or human IgG1 antibody (N297A mutated; four-fold serially diluted from 100nM). The plates were incubated overnight at 37℃. And cell-free culture supernatants were collected for TNF-α measurement by ELISA Kit (R&D systems, DuoSet).

[0279] As shown in Fig. 9, the reduced TNF-α secretion caused by the LILRB4 / fibronectin-induced inhibition of FcR activation in THP-1 cells was recovered by anti-LILRB4 antibody. This result means that the anti-LILRB4 antibody can block the interaction between LILRB4 and fibronectin and thus improve TNF-α secretion. In particular, AB0224 (humanized anti-LILRB4 monospecific antibody) showed superior effect in recovering TNF-α secretion compared to other benchmark antibodies.

[0280] The amino acid sequences of the benchmark antibodies are provided in Table 12. Benchmark antibody 1 (NGM bio's US 20210221887A1) comprises a heavy chain domain comprising SEQ ID NO: 54 and a light chain domain comprising SEQ ID NO: 55. Benchmark antibody 2 (Merck's US 20190153093A1) comprises a heavy chain domain comprising SEQ ID NO: 52 and a light chain domain comprising SEQ ID NO: 53.

[0281] Benchmark antibodiesRegionSequenceSEQ ID NO:NGM bio(US 20210221887A1)Heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATISGGGSYTNYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARREWRYTLYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK54Light chainDIQLTQSPSFLSASVGDRVTITCRASESVESYGSSFMHWYQQKPGKAPKWYLTSNLESGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQNNEDPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC55Merck's(US 20190153093A1)Heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK52Light chainDIQLTQSPSSLSASVGDRVTITCRASEKVDSFGQSFMHWYQQKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC53

[0282]

[0283] 3-4. Anti-LILRB4 monospecific antibody restores immune cell activity from LILRB4-mediated suppression

[0284] Chimeric 6E9C4's ability to reduce suppressive capacity of Myeloid Derived Suppressor Cells (MDSCs) was assessed using an MDSC assay.

[0285] A model was established by co-culturing human PBMCs with SK-MEL-5 tumor cells in vitro, followed by purification of MDSCs and evaluation of their ability to suppress the proliferation of autologous CD8+T cells. This model enabled investigation of the role of LILRB4 in MDSC-mediated immune suppression. The purpose of this experiment was to show whether 6E9C4, even after humanization, is able to impair the acquisition (or maintenance) of the T cell-suppressive phenotype.

[0286] To generate MDSCs, PBMCs (1x107cells) from healthy donors (human) were cultured with SK-MEL-5 cells (0.85x105cells) and 10 ng / mL GM-CSF for 7 days. CD33+cells were collected by positive antibody based magnetic bead selection and then co-cultured with purified autologous CD8+T cells at the indicated ratios for 3 days in the presence of a polyclonal stimulus. Chimeric 6E9C4 antibody (1 μg / mL) was included in both the co-culture and T cell suppression steps. The T cell suppression assay was conducted with a T cell-to-MDSC ratio of 4 : 1, and the suppression was evaluated by measuring the amount of interferon gamma (IFN-γ) production. As shown in Fig. 10, the chimeric 6E9C4 in the MDSC model effectively blocked LILRB4 on MDSCs and restored T cell inhibition by reducing the suppressive capacity of MDSCs.

[0287]

[0288] 3-5. Evaluation of the potency of AB0166 by in vitro ADCC bioassay

[0289] LILRB4-dependent FcγRIIIa activity was evaluated using an antibody-dependent cellular cytotoxicity (ADCC) reporter cell assay (Promega) in the presence of human LILRB4 expressing cells. AB0166 was analyzed for its in vitro ADCC activity by using the Promega propagation system (Promega, G7102), in comparison with Benchmark antibody (Immune-Onc's WO2021183839A2, SEQ ID NOs: 56 and 57) and isotype control (hIgG1).

[0290] Human LILRB4-expressing CHOK1 cells and normal CHOK1 cells (7.5x104cells / well) were seeded into a white 96-well assay plate (Costar, 90020) in 100 μL of culture medium and incubated overnight at 37℃ in a 5% CO2humidified incubator. After overnight culture, 100 μL of culture medium was removed, and 25 μL of Assay medium (RPMI1640(Gibco, 22400-089 + 4% Ultra Low IgG FBS (Gibco, 16250-078) was dispensed to the pre-plated target cells. 25 μL of each tested antibody dilution (starting from 200nM or 100nM diluted with 4-fold dilutions) was added to the plate. ADCC Bioassay Effector Cells were harvested and resuspended in Assay medium. 25 μL of the ADCC Bioassay Effector Cells was dispensed per well to make 1.5x106cells / well, then cultured for 6 hours at 37℃ in a 5% CO2humidified incubator. During incubation time, Bio-Glo™ reagent was reconstituted according to the manufacturer's instruction. After 6 hours incubation, 75 μL of Bio-Glo™ reagent was added per well to the assay plate. After a 5-minute waiting period, luminescence was measured using a microplate reader. A four-parameter logistic curve analysis was performed using GraphPad software.

[0291] As shown in Fig. 11, the humanized anti-LILRB4 monospecific antibody AB0166 induced FcγRIIIa activity in a dose-dependent manner in the presence of Human LILRB4-expressingCHOK1 cells, whereas no FcγRIIIa activity was observed in the absence of Human LILRB4-expressing CHOK1 cells. In addition, AB0166 showed higher FcγRIIIa activation in the presence of Human LILRB4-expressing CHOK1 cells compared to Benchmark antibody.

[0292] The amino acid sequences of the benchmark antibody is provided in Table 13. The benchmark antibody (Immune-Onc's WO 2021183839 A2) comprising a heavy chain domain comprising SEQ ID NO: 56 and a light chain domain comprising SEQ ID NO: 57.

[0293] Benchmark antibodyRegionSequenceSEQ ID NO:Immune-Onc's (WO 2021183839 A2)Heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFSLSSSYWISWVRQAPGKGLEWIGSIDSGSVGITYYATWVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGDNWALDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK56Light chainDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHGYIRGDLDNVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC57

[0294]

[0295] 3-6. Validation of binding epitope

[0296] 200 μL of 10X kinetic buffer (Fortebio, Cat.18-5032) was dispensed into each well of a 96-well black microplate (Greiner Bio-One, Cat.655209). The HIS1K biosensors (Fortebio, Cat.18-5120) were passively hydrated on the lab bench for at least 10 minutes. To prepare of antibody and antigen, chimeric 6E9C4, benchmark antibodies (from Immune-Onc, Merck, and NGM Bio), and an hIgG1 isotype control (Biolegend, Cat.403502) as a negative control were diluted to 100 nM in 10X kinetic buffer. These antibodies were used as the saturating antibody (1st Ab) and the competing antibody (2nd Ab) in the epitope binning assay, and their binding interactions were evaluated in all sequence combinations.　Recombinant Human LILRB4-His tag (Sino biologics, Cat.16742-H08H) was diluted to 2 μg / mL in 10X kinetic buffer. Epitope binning was conducted with the Bio-Layer Interferometry (BLI)-based Octet RED96e system (Sartorius, S / N: FB-90356). Epitope Binning Assay was performed in a tandem format using HIS1K biosensor. Initially, the antigen (2 μg / mL) was captured on the surface of the HIS1K biosensor for 300 seconds, reaching a loading threshold of approximately 0.2 nm. After antigen capture, the biosensor was incubated with the saturating antibody (1st Ab) for 600 seconds to achieve a saturation binding signal. The competing antibody (2nd Ab) was then associated with the antigen-1st Ab complex for an additional 600 seconds to evaluate competitive binding between the two antibodies. Prior to each association phase, a baseline was established by immersing the biosensor in 10X kinetic buffer for 30 seconds. All assay steps were performed with shaking at a speed of 1000 rpm. Data analysis was performed using Octet data analysis software (HT 11.0 ver.). Binding interactions were visualized as sensorgram, with time plotted on the x-axis and the change in wavelength (nm) on the y-axis. In the sensorgram, a significant binding signal of the 2nd Ab in the presence of the 1st Ab indicated that the two antibodies bound to distinct, non-overlapping epitopes. Conversely, a minimal or absent binding signal of the 2nd Ab suggested that the antibodies competed for the same or overlapping epitopes. These competitive binding relationships were then summarized in a heat map shown in Fig. 12.

[0297] As shown in Fig. 12, the tandem epitope binning assay using the Octet RED96e system was conducted to compare the binding epitopes of anti-LILRB4 antibodies. Chimeric 6E9C4 bound to a completely distinct, non-overlapping epitope compared to the benchmark antibodies, indicating that it recognized a unique antigenic site. Among the benchmark antibodies, Merck and NGM Bio, shared the same antigen-binding site, suggesting overlapping epitopes. In contrast, Immune-Onc's antibody bound to a different epitope that does not overlap with those of Merck and NGM Bio.

[0298] Notably, as evidenced by the results in Fig. 9, the anti-LILRB4 antibody(6E9C4), which recognizes the same D2 domain, has demonstrated significantly superior results in this regard compared to benchmark antibodies. This superiority can likely be attributed to the fact that the anti-LILRB4 antibody (6E9C4) targets a distinct epitope from benchmark antibodies. In conclusion, possessing a different epitope may enable a higher level of immune activation compared to benchmark antibodies.

[0299]

[0300] Example 4. Evaluation of anti-LILRB4 / anti-4-1BB bispecific antibody

[0301] 4-1. Cross-specificity of anti-LILRB4 / anti-4-1BB bispecific antibody to Human and Cynomolgus LILRB4 (ELISA)

[0302] To evaluate the antigen binding affinity, the bispecific antibodies AB0228 and AB0229 (h6E9C4 x 1A10M12, WT and NA) were subjected to Single Antigen Capture ELISA (SACE).

[0303] Briefly, 96-well ELISA plates were coated with Fc-fused human or cynomolgus LILRB4 and 4-1BB protein at 1 μg / ml in PBS (100 μl / well) at 4℃ overnight, then blocked with 200 μl / well of 1% BSA in PBS at 37℃ for 2 hours. Four-fold dilutions of the antibodies, starting from 100nM, were added to each well and incubated for 1 hour at 37℃. The plates were washed with PBST (0.05% Tween 20 in PBS) and then incubated with HRP (Horse Radish Peroxidase) conjugated Anti-human Fab antibody for 1 hour at 37℃. The plates were washed with PBST (0.05 Tween 20 in PBS), developed using TMB substrate, and analyzed by spectrophotometer at OD 450 nm-650 nm. As shown in Fig. 13(a) to Fig. 13(d) and Table 11 below, AB0228 and AB0229 exhibited dose-dependent binding to both human and cynomolgus LILRB4.

[0304]

[0305] To further evaluate the antigen binding affinity, the bispecific antibodies AB0228 and AB0229 (h6E9C4 x 1A10M12, WT and NA) were subjected to Dual Antigen Capture ELISA (DACE).

[0306] Briefly, microtiter plates were coated with human and cynomolgus 4-1BB-Fc protein at 1 μg / ml in PBS (100 μl / well) at 4℃ overnight, then blocked with 200 μl / well of 1% BSA in PBS at 37℃ for 2 hours. Four-fold dilutions of the antibodies, starting from 100 nM, were added to each well and incubated for 1 hour at 37℃. The plates were washed with PBST (0.05% Tween 20 in PBS) and then incubated with 1% BSA in PBS containing human or cynomolgus LILRB4-His protein at 1 μg / ml for 1 hour at 37℃. The plates were washed with PBST (0.05% Tween 20 in PBS) and then incubated with HRP (Horse Radish Peroxidase) conjugated anti-His antibody for 1 hour at 37℃. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at 450 nm-650 nm. As shown in Fig. 13(e), Fig. 13(f) and Table 14 below, AB0228 and AB0229 exhibited dose-dependent binding to both of human and cynomolgus LILRB4, as well as human and cynomolgus 4-1BB, at the same time.

[0307] Protein binding(ELISA)SACE EC50 (nM)SACE EC50 (nM)DACE EC50 (nM)hLILRB4cyLILRB4h4-1BBcy4-1BBhLILRB4h4-1BBcyLILRB4cy4-1BBAB02280.0780.1690.0120.0110.0800.047AB02290.0800.1890.0130.0120.0960.046IgG1(WT)x4-1 BB-0.0100.009-

[0308]

[0309] 4-2. Cross-specificity of anti-LILRB4 / anti-4-1BB bispecific antibody to Human and Cynomolgus LILRB4 and 4-1BB (SPR)

[0310] Using surface plasmon resonance (SPR) analysis, the binding affinity of AB0228 to LILRB4 and / or 4-1BB from human or monkey (cynomolgus) was measured. The antigens used here are as follows: LILRB4 (Human(Sino biological, 16742-H08H), Monkey (cynomolgus)(Acro biosystems, CDK-C5227)), 4-1BB (Human(R&D systems, 9220-4B-100), Monkey (cynomolgus)(Sino biological, 90847-K08H)). All binding affinities were measured and evaluated in Biacore™ T200 (Cytiva).

[0311] To evaluate the binding affinity of AB0228, LILRB4 and / or 4-1BB were diluted to 0 / 6.25 / 12.5 / 25 / 50 / 100 nM (for human or monkey (cynomolgus) LILRB4), or to 0 / 15.625 / 31.25 / 62.5 / 125 / 250 nM (for human or monkey (cynomolgus) 4-1BB) as the final concentrations, using HBS-EP (1x). And then AB0228 was captured on the surface of a Protein A chip (Cytiva, 29127556) at about 100 RU (for measuring LILRB4 binding affinity) or about 300 RU (for measuring 4-1BB binding affinity). Serially diluted analytes (LILRB4 or 4-1BB) were injected for 60 seconds (association) on the surface that AB0228 was captured. HBS-EP (1x) was flowed for 180 seconds to monitor the complex dissociation. To recover the chip surface, Glycine 1.5 (Cytiva, BR100354) was injected for 30 seconds. All measurements were performed twice independently at 25℃ with a flow rate of 30 μL / min. The kinetic parameters of AB0228 to LILRB4 and / or 4-1BB, were calculated using a 1:1 binding model with Biacore쪠 T200 evaluation software (ver.3.2.1).

[0312] As shown in Table 14 and Figs. 14(a) to 14(d), AB0228 exhibited dose-dependent binding to both of human and cynomolgus LILRB4, as well as human and cynomolgus 4-1BB, at the same time.

[0313] Kinetic parameters of AB0228TargetSpeciesRun(N)Ka(1 / Ms, x105)Kd(1 / s, x10-4)KD(M, x10-9)Rmax(RU)Chi2(RU2)LILRB4Human23.775± 0.0911.559± 0.1670.414± 0.05426.87± 0.620.0107 - 0.0230Monkey(cyno)22.437± 0.0691.534± 0.0580.630± 0.00624.76± 0.690.0163 - 0.01674-1BBHuman22.155± 0.0239.794± 0.2334.546± 0.15553.07± 0.660.0327 - 0.0540Monkey(cyno)21.404± 0.0019.639± 0.0596.866± 0.09057.17± 0.640.0170 - 0.0484

[0314]

[0315] 4-4. Binding confirmation of anti-LILRB4 / anti-4-1BB bispecific antibodies by using FACS Analysis

[0316] To evaluate antigen binding property, the bispecific antibodies AB0228 and AB0229 (h6E9C4 x 1A10M12, WT and NA) were analyzed for their bindings to LILRB4-expressing cells (human / cynomolgus LILRB4-expressing CHOK1, THP-1, and MV-4-11) and human 4-1BB -expressing Jurkat cells.

[0317] Briefly, each cell line was treated with four-fold dilutions of the antibodies, starting from 100 nM, at 4℃ for 1 hour. After washing by FACS buffer (1% BSA in PBS), cells were incubated with Alexa Fluor 647-conjucated anti-human Fab antibody at 4℃ for 1 hour, and then subjected to flow cytometry analysis (FACS).

[0318] As shown in Fig. 15(a) to Fig 15(e) and Table 16, AB0228 and AB0229 exhibited binding to cells expressing LILRB4 and 4-1BB.

[0319] Cell binding(Flow Cytometry)EC50 (nM)hLILRB4 / CHOK1cyLILRB4 / CHOK1THP-1MV-4-11H4-1BB / JurkatAB02281.4761.3400.7731.3592.065AB02291.5101.3901.1261.6592.249

[0320]

[0321] Example 5. Activity and efficacy of anti-LILRB4 / anti-4-1BB bispecific antibody

[0322] 5-1. The anti-LILRB4 antibodies and the anti-LILRB4 / anti-4-1BB bispecific antibodies restore immune cell activity from LILRB4-mediated suppression

[0323] Tolerogenic dendritic cells (tolDCs) used in this assay were prepared as described in Example 3-2. Specifically, tolDCs were differentiated from normal PBMCs with GM-CSF, IL-4, IL-10 and LPS. An increment of TNF-alpha secretion was observed in tolDCs treated with the anti-LILRB4 antibodies (AB0166 and AB0224) and the anti-LILRB4 / anti-4-1BB bispecific antibodies (AB0228 and AB0229).

[0324] As shown in Fig. 16, the secretion of TNF-alpha in tolDCs was increased in a concentration-dependent manner upon treatment with both the monospecific humanized 6E9C4 antibodies and the bispecific antibodies. These results indicate that both the monospecific antibodies and the bispecific antibodies activate tolDCs, suggesting that the immunosuppressive properties of tolDCs can be reversed by the anti-LILRB4 antibody.

[0325]

[0326] 5-2. The anti-LILRB4 antibody and the anti-LILRB4 / anti-4-1BB bispecific antibody restores immune cell activity from LILRB4-mediated suppression

[0327] The interaction between LILRB4 and fibronectin is known to reduce TNF-alpha secretion in THP-1 monocytes. To evaluate whether the anti-LILRB4 monospecific antibody and the anti-LILRB4 / anti-4-1BB bispecific antibody can block the interaction and improve TNF-alpha secretion, in vitro LILRB4-Fibronection interaction blockade assays were conducted.

[0328] 96-well ELISA plates were co-coated with fibronectin at 5 μg / mL (Millipore) and human IgG1 antibody at 1 μg / mL. The plates were incubated at room temperature for 2 hours, washed twice with PBS, and blocked with RPMI 1640 containing 10% FBS. THP-1 cells (2x105cells / well) were added to the wells in the presence of AB0224 (N297A mutated; serially diluted from 100nM), AB0229 (N297A mutated; serially diluted from 100nM) or human IgG1 antibody (N297A mutated; four-fold serially diluted from 100nM). The plates were incubated overnight at 37℃. And cell-free culture supernatants were collected, and TNF-α level was measured by ELISA Kit (R&Dsystems, DuoSet).

[0329] As shown in Fig. 17, the LILRB4 / fibronectin-induced inhibition of FcR activation in THP-1 cells was effectively suppressed or blocked by AB0224 and AB0229 at a similar level. These results showed that anti-LILRB4 and anti-LILRB4 / 4-1BB bispecific antibodies can inhibit the immunosuppressive effects mediated by LILRB4-fibronectin interaction.

[0330]

[0331] 5-3. The anti-LILRB4 / anti-4-1BB bispecific antibodies activate effector cells in LILRB4 expression-dependent manner

[0332] Anti-LILRB4 / 4-1BB bispecific antibodies AB0228 and AB0229 were analyzed for their in vitro 4-1BB activity using 4-1BB Bioassay (Promega, J2332)

[0333] In brief, cell lines (2.5 x 104cells per well) (CHOK1, human LILRB4-expressing CHOK1, and cynomolgus LILRB4-expressing CHOK1) were plated in a white 96-well assay plate in 100 μL culture medium each. Cells were cultured overnight at 37℃ in a 5 % CO2humidified incubator. After overnight culture, 100 μL of culture medium was removed and 25 μL of assay medium (RPMI1640 with 1% FBS) was added to pre-plated target cells. 25 μL of AB0228 and AB0229 (starting from 1 nM diluted for 3-fold) or BMUR (starting from 100 nM diluted for 5-fold) were added to the plate. GloResponse™ NFκB-luc2 / 4-1BB Jurkat cell line was harvested and resuspended in assay Medium. 25 μL of GloResponse™ NFκBluc2 / 4-1BB Jurkat cell line was added to each well to make 2.5x104cells per well to plate. Cells were cultured for 6 hours at 37℃ in 5% CO2 humidified incubator. During the incubation, Bio-Glo™ reagent was reconstituted according to the manufacturer's instruction. After 6 hours of incubation, 75 μL per well of Bio-Glo™ Reagent was added to the assay plate. After 5 minutes, luminescence was measured using microplate reader. A four-parameter logistic curve analysis was performed using GraphPad software.

[0334] As shown in Fig. 18(a) to 18(c), AB0228 and AB0229 activated 4-1BB signaling in a dose-dependent manner, but only in the presence of human and cynomolgus LILRB4. AB0228 and AB0229 did not activate 4-1BB signaling in the absence of LILRB4 expression. On the other hand, in the BMUR treatment group, T-cell stimulatory activity was increased in both LILRB4-positive and LILRB4-negative cell lines. The above results indicate that our anti-LILRB4 / 4-1BB bispecific antibodies especially increase T-cell activity in the LILRB4-expressing tumor environment, in contrast to BMUR.

[0335]

[0336] Example 6.In vivoefficacy test of anti-LILRB4 / anti-4-1BB bispecific antibody

[0337] 6-1. Efficacy of the AB0224 with SK-MEL-5 Melanoma CDX bearing Hematopoietic Stem Cell (HSC) mice model

[0338] To test thein vivoefficacy of AB0224 antibody (humanized 6E9C4 (N297A)), HSC mice were subcutaneously injected with SK-MEL-5 Melanoma tumor cells (1Х107cells) suspended in 0.1 mL PBS : Matrigel (1:1) into the right front flank for tumor development. Tumor-bearing mice were randomly assigned to three study groups once the mean tumor volume reached 150-200 mm3. Each group consisted of 4 mice. The three groups were G1 (Vehicle, 10 ml / kg), G2 (AB0224, 20 mg / kg), G3 (Benchmark antibody (Merck's US 20190153093A1, SEQ ID NOs: 52 and 53), 20 mg / kg). All test antibodies were Intraperitoneally administrated to tumor-bearing mice at a frequency of twice a week for total six times.

[0339] The tumor volume (TV) was measured and recorded twice per week. The study was terminated 35 days post last dosing. On Day 35 post grouping, in G1, G2 and G3 group, the mean tumor volume was 1020.7 mm3, 734.5 mm3with a TGI of 36.7%, and 728.5 mm3with a TGI of 36.1%, respectively. As shown in Fig. 19(a), both AB0224 and Benchmark antibody exhibited tumor growth inhibition, demonstrating anti-tumor activity on tumor volume.

[0340] In addition, flow cytometry analysis of blood and tumor-infiltrating lymphocytes (TILs) was performed at the study endpoints. As shown in Fig. 19(b), among human CD45+cells, the proportion of M-MDSCs (CD33+CD66lowcells) was decreased in both the AB0224 and Benchmark antibody treatment groups compared to the control group.

[0341]

[0342] 6-2.In vivoefficacy test using hLILRB4-luc EL4 (i.v.) in h4-1BB Knock-in Mice

[0343] In vivoefficacy evaluation of AB0228 in the Treatment of the B-hLILRB4 luc EL4 mouse lymphoma Model in B-h4-1BB Mice was conducted.

[0344] 49 female B-h4-1BB mice were injected through the tail vein with B-hLILRB4-luc EL4 mouse lymphoma cells (1Х105 / mouse) for tumor development. 35 tumor-bearing mice were randomly assigned to five study groups once the mean tumor flux reached approximately 106p / sec. Each group consisted of 7 mice. The five groups were G1: hIgG1 (10 mg / kg), G2: Benchmark antibody (Immune-Onc's WO2021183839A2, SEQ ID NOs: 56 and 57) (10 mg / kg), G3: Isotype control (13.3 mg / kg), G4: Urelumab (10 mg / kg) and G5: AB0228 (13.3 mg / kg). All test antibodies were intraperitoneally administered at a frequency of once every three days. The hIgG1 was administered seven times in total, and the remaining test antibodies were administered eight times in total. The tumor signal intensity and body weight were measured twice per week. The study was terminated 121 days post grouping. Once any single mouse in any group reached the humane endpoint, anticoagulated blood (containing EDTA-K2, 100 μL / mouse) was collected from G1, G4 and G5 mice for FACS analysis (Live / Dead, mCD45, mCD3, mCD4, mCD8, mCD25, and mFoxP3) to evaluate the activity of AB0228 to induce immune cell population change in blood. In addition, the tumor growth inhibition (TGI) was analyzed, and animal survival curves were plotted.

[0345] The result of the in vivo efficacy study is shown in Fig. 20(a) and Table 17 below. On Day 14, the mean tumor signal intensity of G1 to G5 was 2.95Х109± 1.24Х108p / s, 2.98Х108± 1.98Х108p / s, 1.81Х109± 3.70Х108p / s, 7.80Х108± 3.24Х108p / s and 2.40Х107± 1.54Х107p / s respectively. AB0228 showed a tumor growth inhibition rate of 99.2%, which was superior to even the benchmark antibody.

[0346] hLILRB4 / EL4TGI(%)h4-1BB Knock-in MiceUrelumab73.6Isotype control38.7Benchmark's89.9AB022899.2

[0347]

[0348] The activity of AB0228 in inducing immune cell population change in the blood is shown in Fig. 20(b). Compared to the hIgG1 control, AB0228 treatment resulted in a significant increase in the percentage and cell counts of T cells, including mCD4+T cells, and a significant decrease in the percentage of Tregcells.

[0349]

[0350] A tumor rechallenge study was initiated 100 days after the final dose was administered in the in vivo efficacy study. Seven naive B-h4-1BB mice were ordered on Day 49 of the in vivo efficacy study. At the start of the rechallenge study, five mice were randomly selected by weight from the seven naive B-h4-1BB mice. These five mice, along with four tumor CR B-h4-1BB mice from Group 5 (G5) of thein vivoefficacy study, were divided into two rechallenge study groups. The two groups were G1: naive B-h4-1BB mice, G2: tumor CR mice from 13.3 mg / kg of AB0228 treatment group. On grouping day (Day 0), enrolled mice were injected through the tail vein with B-hLILRB4-luc EL4 mouse lymphoma cells (1Х105) for tumor development. The tumor signal intensity and body weight were measured twice per week. The rechallenge study was terminated on Day 28.

[0351] On Day 20, the mean tumor signal intensity of G1 and G2 was 3.79Х1010±2.60Х1010p / s and 4.21Х105±4.98Х103p / s, respectively. From the imaging images, as shown in Fig. 20(c), it was found that the tumor signal was completely lost in mice injected with AB0228, compared to Naive mice group.

[0352] In this example, AB0228 demonstrated significant anti-tumor activity and significantly extended survival times in B-hLILRB4-luc EL4 tumor-bearing mice. Treatment with AB0228 alone cured tumor in half of the tumor-bearing mice. The tumor rechallenge study showed that the mice treated with AB0228 successfully established immune memory against B-hLILRB4-luc EL4 cells. No significant body weight loss or obvious adverse effects were observed. These findings suggest that the anti-LILRB4 / anti-4-1BB bispecific antibody has the potential to provide durable anti-tumor immunity and may be a promising therapeutic agent.

[0353]

[0354] 6-3.In vivoefficacy test using hLILRB4-luc EL4 (i.v.) in hLILRB1,4 / h4-1BB triple TG mice

[0355] Efficacy evaluation of AB0228 in the treatment of the B-hLILRB4-luc EL4 mouse lymphoma model in B-Tg (hLILRB1 / hLILRB4) / h4-1BB mice was conducted.

[0356] 20 female B-Tg (hLILRB1 / hLILRB4) / h4-1BB mice were injected through the tail vein with B-hLILRB4-luc EL4 mouse lymphoma cells (1Х105 / mouse) for tumor development. 14 tumor-bearing mice were randomly assigned to 2 study groups once the mean tumor flux reached approximately 106p / sec. Each group consisted of 7 mice. The 2 groups were G1: hIgG1 (10 mg / kg) and G2: AB0228 (13.3 mg / kg). All test antibodies were intraperitoneally administered at a frequency of once every three days for total four times. The tumor signal intensity and body weight were measured twice per week. The study was terminated 12 days post grouping. At the study endpoint, anticoagulant blood (300 μL / mouse) and liver of G1-G2 mice were collected for FACS analysis (Live / dead, mCD45, mCD11b, mLy6C, mLy6G, mCD3, mCD4, mCD8, mCD25, mFoxP3, mTCR Vβ12, hLILRB4, and h4-1BB).

[0357] At the study endpoint, the mean tumor signal intensity of G1 and G2 was 3.41Х1010± 1.08Х1010p / s and 5.60Х108± 5.29Х108p / s, respectively, and a TGI of G2 was 98.4%. Tumor signals in G1 and G2 were statistically analyzed. Compared with hIgG1, AB0228 significantly inhibited tumor growth (TGI 97.2%, p=0.0208) as shown in Fig. 21(a).

[0358] The injected hLILRB4-luc EL4 cells mostly infiltrated the liver. Compared to the hIgG1 (10 mg / kg), AB0228 (13.3 mg / kg) treatment significantly decreased the percentage of mCD11b+cells, G-MDSC, and M-MDSC cells in the liver and blood. It also significantly increased the percentage of T cells, CTLs, and Th cells in the liver and blood, and significantly decreased the cell counts of B-hLILRB4-luc EL4 cells, mCD11b+cells, G-MDSC, and M-MDSC cells in the liver and blood. For the liver and blood, the FACS analysis showed that M-MDSCs highly expressed hLILRB4. As shown in Figs.21(b) to 21(c), the treatment with AB0228 induced a decrease in infiltrated EL4 cells and Tregcells in the liver, a reduction of MDSCs (granulocytic and monocytic) in both blood and liver, and an increase in Pan T cells (CTLs and Th cells) in all tested tissues.

[0359]

[0360] 6-4.In vivoefficacy test using B16-F10-OVA in hLILRB1,4 / h4-1BB triple TG mice

[0361] Efficacy evaluation of AB0228 in the treatment of the subcutaneous B16-F10-OVA melanoma model in B-Tg (hLILRB1 / hLILRB4) / h4-1BB mice was conducted.

[0362] 42 female B-Tg (hLILRB1 / hLILRB4) / h4-1BB mice were subcutaneously injected with B16-F10-OVA tumor cells (1Х105 / mouse) in the right front flank for tumor development. 7 days post inoculation, 21 tumor-bearing animals were randomly assigned to three study groups once the mean tumor size reached 83 mm3. Each group consisted of 7 mice. The three groups were G1: Human IgG1 (7.5 mg / kg), G2: Benchmark antibody (Immune-Onc's WO2021183839A2, SEQ ID NOs: 56 and 57) (7.5 mg / kg) and G3: AB0228 (10 mg / kg). Bispecific antibody of hIgG1x1A10 and h4-1BB Tg mice were also used for reference.

[0363] Treatment began on the day of grouping (Day 0). All test antibodies were intraperitoneally administered at a frequency of once every three days. Human IgG1 was injected a total of six times, and Benchmark's and AB0228 were injected a total of eight times each. The tumor volume and body weight were measured three times per week. The study was terminated on Day 62. At the end of this experiment, tumors were collected from euthanized animals, weighed and photographed.

[0364] On Day 13, the mean tumor volume of G1 was 2578±268 mm3, and the mean tumor volume of G2 and G3 was 2500±384 mm3and 356±194 mm3, with TGITVof 3.1% and 89.1%, respectively. The median survival days of G1 to G3 were 15, 15 and 36 days, respectively. Mice in G3 had significantly longer survival times after receiving treatment compared to those in G1.

[0365] As shown in Fig. 22(a), hIgG1x1A10 did not show any efficacy in the h4-1BB Tg mouse model, suggesting that the bispecific antibody without anti-LILRB4 antibody component cannot inhibit tumor growth.

[0366] As shown in Fig. 22(b) and 22(c), in the efficacy test on the same cell line in hLILRB1,4 / h4-1BB triple TG mice, hIgG1x1A10, which can only act as a 4-1BB agonist, was ineffective, whereas AB0228 showed excellent efficacy, indicating that the LILRB4 antagonist is a suitable bispecific antibody partner for the 4-1BB agonist.

[0367] As shown in Table 18, AB0228 showed significant efficacy (TGI 87.2%) in the hLILRB1,4 / h4-1BB triple TG mice model.

[0368] B16-F10-OVATGI (%)hLILRB1, 4 / h4-1BB Knock-in MiceBenchmark's3.8AB022887.2

[0369]

[0370] 6-5.In vivoefficacy test of AB0228 against multiple myeloma

[0371] 6-5-1. LILRB4 expression on NCI-H929 (MM cell line)

[0372] To prepare cell, NCI-H929 cells (ATCC, cat. CRL-3580) were cultured in RPMI 1640 medium containing 10% FBS in a 5% CO2, 37℃ incubator. The required cells (0.2 Х 105cells / test) were transferred into a 50 mL tube and centrifuged at 1,200 RPM for 3 minutes to pellet the cells. After centrifugation, the supernatant was discarded. The pelleted cells were resuspended in 10 mL of FACS buffer (1X PBS with 2% FBS) and centrifuged at 1,200 RPM for 3 minutes. The supernatant was discarded, and the cells were resuspended in FACS buffer before adding the Fc block. The Fc block bound to the Fc receptor on the cell surface and prevented nonspecific binding of detection antibodies. The Fc block (BioLegend, cat. 422302) was added at 5 μL per 106cells. After incubation at room temperature for 10 minutes, FACS buffer was added without washing to adjust the cell concentration to 1 Х 106 cells / mL. The cells were then dispensed at 100 μL per well into a 96-well RV plate. To stain cells, various antibody staining procedures were performed on NCI-H929 cells. Control wells included mouse IgG1,k isotype control-APC (Invitrogen, cat. 14-4714-82) and a cell-only well. Experimental well was stained with an anti-LILRB4 antibody-APC (Invitrogen, clone ZM4.1, cat. 17-5139-42). For antibody binding reaction, 1 μL (0.5 mg) of mouse IgG1,k isotype control and 1 μL (0.025 mg) of anti-LILRB4 antibody solution were added to each well and incubated at 4℃ for 30 minutes. The cells were then centrifuged at 2,000 RPM for 3 minutes, and the supernatant was discarded. Each well was washed with 200 μL of FACS buffer, followed by centrifugation at 2,000 RPM for 3 minutes to remove the supernatant. This washing step was repeated twice. The pelleted cells were resuspended in 150 μL of 2% formaldehyde and stored at 4℃ for 10 minutes. After that, cells were analyzed using a cell analyzer (BD, LSRFortessa X-20).

[0373] As shown in Fig. 23(a), the expression of LILRB4 was evaluated in the NCI-H929 cell line, a Multiple Myeloma (MM) cell line, using flow cytometry. The mean fluorescence intensity (MFI) of LILRB4 in NCI-H929 cells was approximately 8.64-fold higher than that of the mouse IgG1 isotype control, indicating significant expression of LILRB4 in this cell line.

[0374]

[0375] 6-5-2. Binding affinity of AB0228 against NCI-H929 (MM cell line)

[0376] To prepare cell, NCI-H929 cells (ATCC, cat. CRL-3580) were cultured in RPMI 1640 medium containing 10% FBS in a 5% CO2, 37℃ incubator. The required cells (0.1 Х 105cells / test) were transferred into a 50 mL tube and centrifuged at 1,200 RPM for 3 minutes to pellet the cells. To dilute antibody, antibodies (AB0228 and isotype control) were diluted in FACS buffer (1X PBS with 2% FBS). Antibodies were prepared at diluted in a 4-fold serial dilution with a final concentration of 100 nM. To stain cells, cells were stained with antibodies in a 96-well plate. Each row contained wells with specific antibodies: Isotype control and AB0228. Additionally, control wells were included for cell-only conditions, secondary antibody-only conditions. For primary antibody binding, 100 μL of 4-fold serial diluted AB0228 and isotype control solutions were added to each well and incubated at 4℃ for 1 hour. The cells were then centrifuged at 2,000 RPM for 3 minutes at 4℃, and the supernatant was discarded. Each well was washed with 200 μL of FACS buffer, followed by centrifugation at 2,000 RPM for 3 minutes, and the supernatant was discarded. This washing step was repeated twice. For secondary antibody binding, 100 μL of 500-fold diluted FITC conjugated goat anti-human IgG (Sigma, cat. F9512) solution was added to each well and incubated at 4℃ for 1 hour. The washing process was repeated twice using 200 μL of FACS buffer and centrifugation at 2,000 RPM for 3 minutes at 4℃. The pelleted cells were resuspended in 150 μL of 2% formaldehyde and stored at 4℃ for 10 minutes. After that, cells were analyzed using a cell analyzer (BD, LSRFortessa X-20).

[0377] To assess the binding affinity of AB0228 for LILRB4, EC50 values were determined using the same cell line. As shown in Fig. 23(b), The EC50 of AB0228 was measured at 0.972 nM, demonstrating specific binding at a sub-nanomolar level. These results suggest that the expression of LILRB4 in MM cells supports the potential expansion of anti-LILRB4 / anti-4-1BB bispecific antibody as a therapeutic candidate for multiple myeloma.

[0378]

[0379] 6-5-3. Multiple myeloma targeted PBMC assay (IFN-γsecretion)

[0380] To coat plate with anti-CD3 antibody, a solution of anti-CD3 antibody (BioLegend, UCHT1 clone, cat.300438) was prepared in sterile cold PBS at a concentration of 5 μg / mL. A total of 50 μL of the anti-CD3 antibody solution was dispensed into each well of the 96-well assay plates following the plate layout. The plates were incubated at 37℃ for at least 2 hours in a 5% CO2incubator. The antibodies were prepared at 4X in assay buffer (RPMI1640 with 10% FBS), with the highest dilution point at 400 nM and the final concentration set to 100 nM. Antibodies included isotype control, AB0166 and AB0228. Serial dilutions were prepared in 4-fold steps down to 0.002 nM in the deep well plate (BIONEER, cat.90060). PBMCs were thawed in a 37℃ water bath for 1-2 minutes until only a small amount of ice remained. The cells were transferred to a 50 mL conical tube and resuspended in 9 mL of media per 1 mL of PBMC. Cell viability was assessed using Trypan Blue staining. Target cells were also counted and assessed for viability. Cells were pelleted at 130 Х g for 10 minutes at ambient temperature, washed with DPBS, and resuspended in RPMI1640 at a density of 0.6 Х 106 cells / mL. After incubation, anti-CD3 antibody-coated assay plates were retrieved, and the anti-CD3 antibody solution was aspirated. Without washing, 50 μL (3 x 104cells) of effector cells were added per well, followed by 50 μL of diluted antibodies. For control wells, 50 μL of assay media was added. Finally, 100 μL (6 x 104cells) of target cells were added to each well, reaching a final volume of 200 μL per well. The assay plates were covered with breathable sealing tape and incubated for 48 hours at 37℃ in a 5% CO2incubator. 24 μL Supernatants from each well were transferred to a new 96-well plate for cytokine analysis and diluted with 96 μL PBS buffer containing 1% BSA. The concentration of IFN-γ was measured using the Duoset ELISA kit (R&D Systems, cat. DY285B) according to the manufacturer's protocol. Cytokine quantification was calculated through standard curve fit using the 'InterpX' function of Softmax 7 pro software (Molecular Devices, LLC.). Then, the obtained values were then converted to actual concentrations by multiplying by the reciprocal of the dilution factor. The calculated concentrations were visualized using Prism (GraphPad Software).

[0381] As shown in Fig. 23(c), AB0166 showed no significant increase in IFN-γ secretion, similar to the isotype control, whereas AB0228 induced a concentration-dependent increase in IFN-γ levels. These findings suggest that the anti-LILRB4 / anti-4-1BB bispecific antibody can enhance T cell immune activation against multiple myeloma, potentially leading to improved antitumor efficacy.

Claims

1.An antibody or antigen-binding fragment thereof that specifically binds to LILRB4 (leukocyte immunoglobulin like receptor B4), comprising at least one antigen binding site comprising:(a) a heavy chain CDR1 of SEQ ID NO: 3;(b) a heavy chain CDR2 of SEQ ID NO: 5;(c) a heavy chain CDR3 of SEQ ID NO: 7;(d) a light chain CDR1 of SEQ ID NO: 11;(e) a light chain CDR2 of SEQ ID NO: 13; and(f) a light chain CDR3 of SEQ ID NO: 15.2.The antibody or antigen-binding fragment thereof according to claim 1,wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 1 and 19.3.The antibody or antigen-binding fragment thereof according to claim 1,wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of SEQ ID NOs: 9 and 24.4.The antibody or antigen-binding fragment thereof according to claim 1,wherein the antibody or antigen-binding fragment thereof comprises:a heavy chain framework 1 (H-FR1) selected from the group consisting of SEQ ID NOs: 2 and 20 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 20;a heavy chain framework 2 (H-FR2) selected from the group consisting of SEQ ID NOs: 4 and 21 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 21;a heavy chain framework 3 (H-FR3) selected from the group consisting of SEQ ID NOs: 6 and 22 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22;a heavy chain framework 4 (H-FR4) selected from the group consisting of SEQ ID NOs: 8 and 23 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 23;a light chain framework 1 (L-FR1) selected from the group consisting of SEQ ID NOs: 10 and 25 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence consisting of SEQ ID NOs: 10 and 25;a light chain framework 2 (L-FR2) selected from the group consisting of SEQ ID NOs: 12 and 26 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 26;a light chain framework 3 (L-FR3) selected from the group consisting of SEQ ID NOs: 14 and 27 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 27; anda light chain framework 4 (L-FR4) selected from the group consisting of SEQ ID NOs: 16 and 28 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 28.5.The antibody or antigen-binding fragment thereof according to claim 1,wherein the antibody or antigen-binding fragment thereof comprises a heavy chain selected from the group consisting of SEQ ID NOs: 17, 29 and 30.6.The antibody or antigen-binding fragment thereof according to claim 1,wherein the antibody or antigen-binding fragment thereof comprises a light chain selected from the group consisting of SEQ ID NOs: 18 and 31.7.The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6,wherein the antibody or antigen-binding fragment thereof is a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody.8.The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6,wherein the antibody or antigen-binding fragment thereof is selected from a group consisting of a whole IgG, sdAb (single-domain antibody), Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG and combinations thereof.9.The antibody or antigen-binding fragment thereof or according to any one of claims 1 to 6,wherein the antibody or antigen-binding fragment thereof comprises an Fc region or a constant region,wherein the Fc region is an Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody, or a hybrid Fc region.10.The antibody or antigen-binding fragment thereof according to claim 1 to 6,wherein the antibody or antigen-binding fragment thereof does not detectably bind or binds with at least 5-fold lower affinity to other LILR family including LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB5, or a combination thereof.11.A bispecific antibody or antigen-binding fragment thereof comprising:(i) a first antigen binding site that binds to LILRB4 (leukocyte immunoglobulin like receptor B4) and(ii) a second antigen binding site that binds to 4-1BB (4-1BB ligand).12.The bispecific antibody or antigen-binding fragment thereof according to claim 11,wherein the first antigen binding site comprises:(a) a heavy chain CDR1 of SEQ ID NO: 3;(b) a heavy chain CDR2 of SEQ ID NO: 5;(c) a heavy chain CDR3 of SEQ ID NO: 7;(d) a light chain CDR1 of SEQ ID NO: 11;(e) a light chain CDR2 of SEQ ID NO: 13; and(f) a light chain CDR3 of SEQ ID NO: 15.13.The bispecific antibody or antigen-binding fragment thereof according to claim 11,wherein the first antigen binding site comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 1 and 19.14.The bispecific antibody or antigen-binding fragment thereof according to claim 11,wherein the first antigen binding site comprises a light chain variable region selected from the group consisting of SEQ ID NOs: 9 and 24.15.The bispecific antibody or antigen-binding fragment thereof according to claim 11,wherein the first antigen binding site comprising:a heavy chain framework 1 (H-FR1) selected from the group consisting of SEQ ID NOs: 2 and 20 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 20;a heavy chain framework 2 (H-FR2) selected from the group consisting of SEQ ID NOs: 4 and 21 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 21;a heavy chain framework 3 (H-FR3) selected from the group consisting of SEQ ID NOs: 6 and 22 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22;a heavy chain framework 4 (H-FR4) selected from the group consisting of SEQ ID NOs: 8 and 23 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 23;a light chain framework 1 (L-FR1) selected from the group consisting of SEQ ID NOs: 10 and 25 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 25;a light chain framework 2 (L-FR2) selected from the group consisting of SEQ ID NOs: 12 and 26 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 26;a light chain framework 3 (L-FR3) selected from the group consisting of SEQ ID NOs: 14 and 27 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 27; anda light chain framework 4 (L-FR4) selected from the group consisting of SEQ ID NOs: 16 and 28 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 28.16.The bispecific antibody or antigen-binding fragment thereof according to claim 11,wherein the first antigen binding site comprises a heavy chain selected from the group consisting of SEQ ID NOs: 17, 29 and 30.17.The bispecific antibody or antigen-binding fragment thereof according to claim 11,wherein the first antigen binding site comprises a light chain selected from the group consisting of SEQ ID NOs: 18 and 31.18.The bispecific antibody or antigen-binding fragment thereof according to any one of claims 11 to 17,wherein the second antigen binding site comprises:(a) a heavy chain CDR1 of SEQ ID NO: 34;(b) a heavy chain CDR2 of SEQ ID NO: 36;(c) a heavy chain CDR3 of SEQ ID NO: 38;(d) a light chain CDR1 of SEQ ID NO: 42;(e) a light chain CDR2 of SEQ ID NO: 44; and(f) a light chain CDR3 of SEQ ID NO: 46.19.The bispecific antibody or antigen-binding fragment thereof according to claim 18,wherein the second antigen binding site comprises a heavy chain variable region of SEQ ID NO: 32.20.The bispecific antibody or antigen-binding fragment thereof according to claim 18,wherein the second antigen binding site comprises a light chain variable region of SEQ ID NO: 40.21.The bispecific antibody or antigen-binding fragment thereof according to claim 18,wherein the second antigen binding site comprising:a heavy chain framework 1 (H-FR1) of SEQ ID NO: 33 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 33;a heavy chain framework 2 (H-FR2) of SEQ ID NO: 35 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 35;a heavy chain framework 3 (H-FR3) of SEQ ID NO: 37 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 37;a heavy chain framework 4 (H-FR4) of SEQ ID NO: 39 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 39;a light chain framework 1 (L-FR1) of SEQ ID NO: 41 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 41;a light chain framework 2 (L-FR2) of SEQ ID NO: 43 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 43;a light chain framework 3 (L-FR3) of SEQ ID NO: 45 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 45; anda light chain framework 4 (L-FR4) of SEQ ID NO: 47 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 47.22.The bispecific antibody or antigen-binding fragment thereof according to claim 18,wherein the first antigen binding site comprises:a heavy chain CDR1 of SEQ ID NO: 3;a heavy chain CDR2 of SEQ ID NO: 5;a heavy chain CDR3 of SEQ ID NO: 7;a light chain CDR1 of SEQ ID NO: 11;a light chain CDR2 of SEQ ID NO: 13;a light chain CDR3 of SEQ ID NO: 15; andthe second antigen binding site comprisesa heavy chain CDR1 of SEQ ID NO: 34;a heavy chain CDR2 of SEQ ID NO: 36;a heavy chain CDR3 of SEQ ID NO: 38;a light chain CDR1 of SEQ ID NO: 42;a light chain CDR2 of SEQ ID NO: 44; anda light chain CDR3 of SEQ ID NO: 46.23.The bispecific antibody or antigen-binding fragment thereof according to any one of claims 11 to 22,wherein the bispecific antibody is a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody.24.The bispecific antibody or antigen-binding fragment thereof according to any one of claims 11 to 22,wherein the bispecific antibody is independently selected from a group consisting of a whole IgG, sdAb (single-domain antibody), Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG and combinations thereof.25.The bispecific antibody or antigen-binding fragment thereof according to any one of claims 11 to 22,wherein the bispecific antibody comprises an Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody, or a hybrid Fc region or a constant region.26.The bispecific antibody or antigen-binding fragment thereof according to claim 25,wherein the first antigen binding site is covalently linked with the second antigen binding site.27.The bispecific antibody or antigen-binding fragment thereof according to claim 25,wherein the first antigen binding site or the second antigen binding site are linked with a peptide linker.28.The bispecific antibody or antigen-binding fragment thereof according to claim 27,wherein the peptide linker consists of SEQ ID NOs: 50 or 51.29.A pharmaceutical formulation comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 11 to 28 and a pharmaceutically acceptable carrier.30.A pharmaceutical composition for use in a method of preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 11 to 28.31.The pharmaceutical composition of claim 30,wherein the cancer is immune checkpoint inhibitor-resistant cancer.32.The pharmaceutical composition of claim 30,wherein the cancer is LILRB4-positive cancer.33.The pharmaceutical composition of claim 30,wherein the cancer is selected from the group consisting of leukemia, rectal cancer, endometrial cancer, nephroblastoma, basal cell carcinoma, nasopharyngeal cancer, bone tumor, esophageal cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular thyroid cancer, hepatocellular carcinoma, oral cancer, renal cell carcinoma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, mesenchymal tumor, soft tissue sarcoma, liposarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratoma, neuroblastoma, medulloblastoma, glioma, benign skin tumor, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy-cell leukemia, myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, neuroectodermal tumor, epithelial tumor, cutaneous T-cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), pancreas cancer, hematological malignancies, kidney cancer, tumor vasculature, breast cancer, renal cancer, ovarian cancer, epithelial ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), Head and neck squamous cell carcinoma (HNSCC), glioblastoma multiforme (GBM), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.34.The pharmaceutical composition of claim 33,wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), and multiple myeloma.35.The pharmaceutical composition of claim 33,wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), and hepatocellular carcinoma.36.A method for preventing or treating a cancer in a patient in need thereof, comprising administering to the patient an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 11 to 28.37.A use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 11 to 28 in the manufacture of medicament for treating or preventing a cancer.38.A method for restoring T cell in a patient in need thereof, comprising administering to the patient an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 11 to 28.39.An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 11 to 28.40.A vector comprising the isolated nucleic acid according to claim 39.41.A host cell comprising the vector according to claim 40.42.An antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 11 to 28; and a drug.