Human CXCL16 antibodies and uses thereof

Abstract

This application discloses CXCL16 antibody for cancer treatment. In some embodiments, the CXCL16 antibody disclosed herein can inhibit the growth of cancer, such as thyroid cancer, breast cancer and prostate cancer. Also described is the combination of CXCL16 antibody with targeted anticancer drug and / or immune checkpoint inhibitor for cancer treatment.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Korean Patent Application No. 10-2021-0164723, filed with the Korean Intellectual Property Office on November 25, 2021. The entire contents of said application are incorporated herein by reference for all purposes.

[0002] Field The present invention relates to therapeutic antibodies used to treat cancer. [Background technology]

[0003] background Cancer is currently one of the leading causes of death worldwide, and as the age at onset of cancer gradually decreases and life expectancy gradually increases, the incidence of cancer is expected to continue to increase. Cancer develops when genetic and environmental factors cause abnormal cell division.

[0004] Anaplastic thyroid cancer, unlike well-differentiated papillary or follicular thyroid cancer, is a fatal disease due to its rapid proliferation and frequent local recurrence, with a 5-year survival rate of 1.0% to 7.1%. The average survival period is known to be 4 to 12 months. No treatment has shown satisfactory results. Various treatments have been attempted to date, but they have not shown satisfactory results in terms of clinical outcomes and prognosis. Anaplastic thyroid cancer is extremely rare, typically accounting for less than 5% to 10% of all thyroid cancers. Because anaplastic thyroid cancer grows rapidly over a short period of time, at the time of diagnosis, tumors are often large, with severe local invasion and distant metastasis, making surgical treatment impossible. Therefore, a multimodal approach rather than a single treatment is required for the treatment of anaplastic thyroid cancer.

[0005] Triple-negative breast cancer (TNBC) lacks the receptors commonly found in breast cancer. It does not express estrogen or progesterone receptors and is HER2-negative (ER- / PR- / HER2-). TNBC is resistant to many conventional breast cancer treatments, including taxol, tamoxifen, and the anti-HER2 receptor antibody (trastuzumab). Triple-negative breast cancer occurs in younger individuals, is more common in premenopausal women, has a higher rate of local and distant recurrence, is more likely to spread hematogenously than to lymph nodes, has higher nuclear and histologic grades than other types, and is larger in size. All of these characteristics are associated with a poor prognosis. However, compared to other breast cancers, there are currently no effective targeted therapies or anticancer drugs for triple-negative breast cancer, and extensive tumor biology research is warranted.

[0006] Recent studies have shown that anticancer drugs with specific targets are more likely to develop resistance (acquired resistance) than non-targeted drugs. To prevent acquired resistance and maximize the efficacy of anticancer drugs, combination therapy with drugs that inhibit factors that induce acquired resistance has emerged. Furthermore, the range of application of targeted anticancer drugs is often limited, and coadministration with inhibitors of other factors can expand the range of application. Furthermore, such combination therapy can enhance the efficacy of anticancer drugs and reduce the dose required, not only when resistance to the anticancer drug is demonstrated, but also when efficacy is demonstrated. This can enhance the efficacy of anticancer drugs while minimizing the toxicity and / or side effects of the drug on various organs in the body. Summary of the Invention

[0007] Quick Overview The present application discloses CXCL16 antibodies for cancer treatment. In some embodiments, the CXCL16 antibodies disclosed herein can inhibit the growth of cancers, such as thyroid cancer, breast cancer, and prostate cancer. Also described is the combination of CXCL16 antibodies with targeted anticancer drugs and / or immune checkpoint inhibitors for cancer treatment.

[0008] In one embodiment, provided herein is an antibody that binds to CXCL16, comprising a heavy chain variable region comprising an HCDR1 in SEQ ID NO: 12 or any one of its variants having 1, 2, 3, 4, or 5 amino acid substitutions relative to the sequence; an HCDR2 in SEQ ID NO: 13 or any one of its variants having 1, 2, 3, 4, or 5 amino acid substitutions relative to the sequence; and an HCDR3 in SEQ ID NO: 14 or any one of its variants having 1, 2, 3, 4, or 5 amino acid substitutions relative to the sequence; an LCDR1 in SEQ ID NO: 15 or any one of its variants having 1, 2, 3, 4, or 5 amino acid substitutions relative to the sequence; an LCDR2 in SEQ ID NO: 16 or any one of its variants having 1, 2, or 3 amino acid substitutions relative to the sequence; and an LCDR3 in SEQ ID NO: 17 or any one of its variants having 1, 2, 3, 4, or 5 amino acid substitutions relative to the sequence.

[0009] In some embodiments, the antibody comprises one or more of the following: HC-FR1 that is at least 80% identical to SEQ ID NO: 22; HC-FR2 that is at least 80% identical to SEQ ID NO: 23; HC-FR3 that is at least 80% identical to SEQ ID NO: 24; HC-FR4 that is at least 80% identical to SEQ ID NO: 25; LC-FR1 that is at least 80% identical to SEQ ID NO: 26; LC-FR2 that is at least 80% identical to SEQ ID NO: 27; LC-FR3 that is at least 80% identical to SEQ ID NO: 28; and LC-FR4 that is at least 80% identical to SEQ ID NO: 29.

[0010] In some embodiments, the antibody comprises: HC-FR1 having the amino acid sequence of SEQ ID NO: 22; HC-FR2 having the amino acid sequence of SEQ ID NO: 23; HC-FR3 having the amino acid sequence of SEQ ID NO: 24; HC-FR4 having the amino acid sequence of SEQ ID NO: 25; LC-FR1 having the amino acid sequence of SEQ ID NO: 26; LC-FR2 having the amino acid sequence of SEQ ID NO: 27; LC-FR3 that is at least 80% identical to SEQ ID NO: 28; and LC-FR4 that is at least 80% identical to SEQ ID NO: 29.

[0011] In some embodiments, the antibody comprises one or more of the following: HC-FR1 having the amino acid sequence of SEQ ID NO: 22; HC-FR2 having the amino acid sequence of SEQ ID NO: 23; HC-FR3 having the amino acid sequence of SEQ ID NO: 24; HC-FR4 having the amino acid sequence of SEQ ID NO: 25; LC-FR1 having the amino acid sequence of SEQ ID NO: 26; LC-FR2 having the amino acid sequence of SEQ ID NO: 27; LC-FR3 that is at least 80% identical to SEQ ID NO: 28; and LC-FR4 that is at least 80% identical to SEQ ID NO: 29. In some embodiments, the antibody competes with CXCR6 (SEQ ID NO: 21) for binding to CXCL16.

[0012] In some embodiments, the antibody comprises a heavy chain variable region comprising an HCDR1 in SEQ ID NO: 12 or any one of its variants having 1, 2, 3, 4, or 5 amino acid substitutions relative to the sequence; an HCDR2 in SEQ ID NO: 13 or any one of its variants having 1, 2, 3, 4, or 5 amino acid substitutions relative to the sequence; and an HCDR3 in SEQ ID NO: 14 or any one of its variants having 1, 2, 3, 4, or 5 amino acid substitutions relative to the sequence; an LCDR1 in SEQ ID NO: 15 or any one of its variants having 1, 2, 3, 4, or 5 amino acid substitutions relative to the sequence; an LCDR2 in SEQ ID NO: 16 or any one of its variants having 1, 2, or 3 amino acid substitutions relative to the sequence; and an LCDR3 in SEQ ID NO: 17 or any one of its variants having 1, 2, 3, 4, or 5 amino acid substitutions relative to the sequence.

[0013] In some embodiments, the antibody comprises all six CDRs of SEQ ID NOs: 12-17.

[0014] In some embodiments, the antibody comprises a VH region comprising the VH amino acid sequence of any one of SEQ ID NOs: 1-5, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the VH amino acid sequence; and / or the antibody comprises a VL region comprising the VL amino acid sequence of any one of SEQ ID NOs: 6-10, and an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the VL amino acid sequence.

[0015] In some embodiments, the antibody comprises a VH of an antibody or variant thereof selected from the group consisting of HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5.

[0016] In some embodiments, the antibody comprises a VL of an antibody selected from the group consisting of HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5. In some embodiments, the antibody comprises both a VH and a VL of an antibody selected from the group consisting of HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5.

[0017] In some embodiments, the antibody comprises a heavy chain of SEQ ID NO:42 and a light chain of SEQ ID NO:43.

[0018] In some embodiments, at least one or two of the substitutions are conservative; at least 50% of the substitutions are conservative; or all of the substitutions are conservative.

[0019] In another aspect, provided herein is an isolated antibody or antibody fragment disclosed herein.

[0020] In some embodiments, the antibody is a humanized antibody, a chimeric antibody, a multispecific antibody, a bispecific antibody, an scFv, or a Fab. In some embodiments, the CXCL16 antibody is a humanized antibody and comprises a human IgG1 isotype constant domain, SEQ ID NO: 48. In some embodiments, the humanized CXCL16 antibody comprises SEQ ID NO: 49.

[0021] In some embodiments, the antibody competes for binding with a CXCL16 antibody disclosed herein.

[0022] In some embodiments, the antibody comprises a VH region comprising the VH amino acid sequence of any one of SEQ ID NOs: 1-5 and / or a VL region comprising the VL amino acid sequence of any one of SEQ ID NOs: 6-10, or a VH region having at least 70% identity to the VH amino acid sequence and a VL region having at least 70% identity to the VL amino acid sequence, wherein changes to the corresponding VH or VL region are present only in the framework regions.

[0023] In some embodiments, the FW region of the VL region of an antibody is at least 80% identical to the FW region present in the VL region of any one of the corresponding antibodies.

[0024] In another aspect, provided herein is an immunoconjugate comprising an antibody and a cytotoxic agent.

[0025] In another aspect, provided herein is a polypeptide comprising: (1) a VH sequence having at least 70% amino acid sequence identity to the VH amino acid sequence of any one of SEQ ID NOs: 1 to 5, and / or a VL sequence having at least 70% amino acid sequence identity to the VL amino acid sequence of any one of SEQ ID NOs: 6 to 10.

[0026] In another aspect, provided herein is a polynucleotide encoding the polypeptide disclosed above.

[0027] In another aspect, provided herein is an expression vector comprising a polynucleotide encoding the VH and / or VL region of the antibody disclosed above.

[0028] In another aspect, provided herein is a host cell comprising the expression vector of Example 20. In some embodiments, the host cell comprises a polynucleotide encoding the VH region and / or the VL region of the antibody disclosed above.

[0029] In another aspect, provided herein is a pharmaceutical composition comprising (i) the antibody described above or an immunoconjugate of the antibody, and (ii) a pharmaceutically acceptable carrier.

[0030] In another aspect, provided herein is a method for inducing an immune response and / or treating cancer, comprising administering the antibody or pharmaceutical composition disclosed above.

[0031] In another aspect, provided herein is a method for inhibiting tumor metastasis, comprising administering the antibody or pharmaceutical composition disclosed above. In some embodiments, the tumor metastasis is bone metastasis. In some embodiments, the antibody is administered intravenously.

[0032] In another aspect, provided herein is a method for treating a cancer patient having tumor tissue that can be bound by an antibody that binds to CXCL16, comprising administering to the patient the antibody disclosed above. In some embodiments, the cancer is thyroid cancer or breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the antibody is administered intravenously.

[0033] In some embodiments, the method further comprises administering chemotherapy and / or radiation therapy. In some embodiments, the chemotherapy is paclitaxel. In some embodiments, the method further comprises administering an agent that targets an immunological checkpoint antigen.

[0034] In some embodiments, the agent is a monoclonal antibody. In some embodiments, the monoclonal antibody inhibits PD-1 ligand binding to PD-1. In some embodiments, the monoclonal antibody is an anti-PD-1 antibody.

[0035] In yet another aspect, provided herein is a method for identifying a patient having a tumor suitable for treatment with an antibody that binds to CXCL16, the method comprising contacting a tumor sample from the patient with the antibody disclosed above and detecting binding of the antibody to the tumor sample, wherein detection of binding indicates a patient having a tumor suitable for treatment with an antibody that binds to CXCL16.

[0036] In yet another aspect, provided herein is a method of producing an antibody, comprising culturing the host cell disclosed above under conditions in which a polynucleotide encoding the heavy chain and a polynucleotide encoding the light chain are expressed.

[0037] In yet another aspect, provided herein is a method for identifying an antibody having tumor-targeting activity, comprising: mutating a polynucleotide encoding the VH or VL CDR3 of the antibody disclosed above; expressing an antibody comprising the mutated VH or VL CDR3; and selecting an antibody that inhibits tumor growth in vivo or reduces tumor size, tumor invasion, and / or metastasis.

[0038] In yet another aspect, provided herein is the use of the antibody disclosed above for a method of treating cancer. In some embodiments, the cancer is associated with increased CXCL16 expression. In some embodiments, the cancer is breast cancer, thyroid cancer, cervical cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, colon cancer, colorectal cancer, bone cancer, skin cancer, head cancer, cervical cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, liver cancer, brain cancer, bladder cancer, blood cancer, gastric cancer, perianal cancer, breast cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis cancer, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, or brainstem glioma.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS [Brief explanation of the drawings]

[0040] [Figure 1A]Figures 1A and 1B show the results of binding of humanized CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, and HC5LC5) to human CXCL16 (Figure 1A) and mouse CXCL16 (Figure 1B). [Figure 1B] Figures 1A and 1B show the results of binding of humanized CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, and HC5LC5) to human CXCL16 (Figure 1A) and mouse CXCL16 (Figure 1B). [Figure 2A] Figures 2A, 2B, 2C, and 2D show the effects of humanized CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, and HC5LC5) in analyzing cancer cell chemotaxis and migration. The ability of humanized CXCL16 antibodies to inhibit cancer cell chemotaxis and migration was evaluated in several cell lines: CXCR6-overexpressing CHO-K1 cells (Figure 2A), the thyroid cancer cell line BHP10-3M (Figure 2B), the breast cancer cell line MDA-MB-231 (Figure 2C), and the monocytic cell line THP-1 (Figure 2D). [Figure 2B] Figures 2A, 2B, 2C, and 2D show the effects of humanized CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, and HC5LC5) in analyzing cancer cell chemotaxis and migration. The ability of humanized CXCL16 antibodies to inhibit cancer cell chemotaxis and migration was evaluated in several cell lines: CXCR6-overexpressing CHO-K1 cells (Figure 2A), the thyroid cancer cell line BHP10-3M (Figure 2B), the breast cancer cell line MDA-MB-231 (Figure 2C), and the monocytic cell line THP-1 (Figure 2D). [Figure 2C] Figures 2A, 2B, 2C, and 2D show the effects of humanized CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, and HC5LC5) in analyzing cancer cell chemotaxis and migration. The ability of humanized CXCL16 antibodies to inhibit cancer cell chemotaxis and migration was evaluated in several cell lines: CXCR6-overexpressing CHO-K1 cells (Figure 2A), the thyroid cancer cell line BHP10-3M (Figure 2B), the breast cancer cell line MDA-MB-231 (Figure 2C), and the monocytic cell line THP-1 (Figure 2D). [Figure 2D]Figures 2A, 2B, 2C, and 2D show the effects of humanized CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, and HC5LC5) in analyzing cancer cell chemotaxis and migration. The ability of humanized CXCL16 antibodies to inhibit cancer cell chemotaxis and migration was evaluated in several cell lines: CXCR6-overexpressing CHO-K1 cells (Figure 2A), the thyroid cancer cell line BHP10-3M (Figure 2B), the breast cancer cell line MDA-MB-231 (Figure 2C), and the monocytic cell line THP-1 (Figure 2D). [Figure 3A] Figures 3A, 3B, 3C, and 3D show the effects of humanized CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, and HC5LC5) on Akt activation. The ability of humanized CXCL16 antibodies to inhibit CXCL16-induced Akt activation in cancer cells was evaluated in several cell lines: CXCR6-overexpressing CHO-K1 cells (Figure 3A), the thyroid cancer cell line BHP10-3M (Figure 3B), the breast cancer cell line MDA-MB-157 (Figure 3C), and the prostate cancer cell line PC3 (Figure 3D). [Figure 3B] Figures 3A, 3B, 3C, and 3D show the effects of humanized CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, and HC5LC5) on Akt activation. The ability of humanized CXCL16 antibodies to inhibit CXCL16-induced Akt activation in cancer cells was evaluated in several cell lines: CXCR6-overexpressing CHO-K1 cells (Figure 3A), the thyroid cancer cell line BHP10-3M (Figure 3B), the breast cancer cell line MDA-MB-157 (Figure 3C), and the prostate cancer cell line PC3 (Figure 3D). [Figure 3C] Figures 3A, 3B, 3C, and 3D show the effects of humanized CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, and HC5LC5) on Akt activation. The ability of humanized CXCL16 antibodies to inhibit CXCL16-induced Akt activation in cancer cells was evaluated in several cell lines: CXCR6-overexpressing CHO-K1 cells (Figure 3A), the thyroid cancer cell line BHP10-3M (Figure 3B), the breast cancer cell line MDA-MB-157 (Figure 3C), and the prostate cancer cell line PC3 (Figure 3D). [Figure 3D] Figures 3A, 3B, 3C, and 3D show the effects of humanized CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, and HC5LC5) on Akt activation. The ability of humanized CXCL16 antibodies to inhibit CXCL16-induced Akt activation in cancer cells was evaluated in several cell lines: CXCR6-overexpressing CHO-K1 cells (Figure 3A), the thyroid cancer cell line BHP10-3M (Figure 3B), the breast cancer cell line MDA-MB-157 (Figure 3C), and the prostate cancer cell line PC3 (Figure 3D). [Figure 4A] Figures 4A and 4B show the results of tumor growth (Figure 4A) or tumor weight (Figure 4B) in mice bearing tumors derived from the triple-negative breast cancer cell line MDA-MB-231. These mice were treated with either anti-mouse CXCL16 antibody alone or a combination of anti-mouse CXCL16 antibody and paclitaxel (PTX). The results show that anti-mouse CXCL16 antibody significantly reduced tumor volume and weight in this breast cancer mouse model. [Figure 4B] Figures 4A and 4B show the results of tumor growth (Figure 4A) or tumor weight (Figure 4B) in mice bearing tumors derived from the triple-negative breast cancer cell line MDA-MB-231. These mice were treated with either anti-mouse CXCL16 antibody alone or a combination of anti-mouse CXCL16 antibody and paclitaxel (PTX). The results show that anti-mouse CXCL16 antibody significantly reduced tumor volume and weight in this breast cancer mouse model. [Figure 5] Figure 5 shows tumor growth in mice bearing thyroid cancer treated with a combination of lenvatinib (a targeted anti-cancer drug) and an anti-mouse CXCL16 antibody. The results show that the combination of lenvatinib and an anti-mouse CXCL16 antibody significantly reduced tumor volume in a mouse model of thyroid cancer. [Figure 6] Figure 6 shows tumor growth in mice with thyroid cancer treated with a combination of lenvatinib (a targeted anticancer drug), a PD-L1 inhibitor (an immune checkpoint inhibitor), and an anti-mouse CXCL16 antibody. [Figure 7] Figure 7 shows tumor growth in breast cancer-bearing mice treated with a combination of paclitaxel, a PD-L1 inhibitor (immune checkpoint inhibitor), and an anti-mouse CXCL16 antibody. [Figure 8A] Figure 8A shows a two-chamber system that mimics the bone metastatic niche. [Figure 8B] FIG. 8B shows the effect of CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, and HC5LC5) on the migration of CD11b+ cells. [Figure 9A] Figures 9A, 9B, and 9C show that higher concentrations of CXCL16 were detected in bone marrow serum of zolendronic acid (ZA)-resistant bone metastases. Figure 9A shows bioluminescence imaging (BLI) of bone tumors in the tibia at 5 and 7 weeks. Figure 9B is a schematic diagram showing the development of the ZA-R model of bone metastasis. Figure 9C shows data indicating CXCL16 concentrations in bone marrow serum. [Figure 9B] Figures 9A, 9B, and 9C show that higher concentrations of CXCL16 were detected in bone marrow serum of zolendronic acid (ZA)-resistant bone metastases. Figure 9A shows bioluminescence imaging (BLI) of bone tumors in the tibia at 5 and 7 weeks. Figure 9B is a schematic diagram showing the development of the ZA-R model of bone metastasis. Figure 9C shows data indicating CXCL16 concentrations in bone marrow serum. [Figure 9C] Figures 9A, 9B, and 9C show that higher concentrations of CXCL16 were detected in bone marrow serum of zolendronic acid (ZA)-resistant bone metastases. Figure 9A shows bioluminescence imaging (BLI) of bone tumors in the tibia at 5 and 7 weeks. Figure 9B is a schematic diagram showing the development of the ZA-R model of bone metastasis. Figure 9C shows data indicating CXCL16 concentrations in bone marrow serum. [Figure 10A]Figures 10A, 10B, and 10C show that anti-CXCL16 antibodies reduced tumor growth of bone metastases. Figure 10A shows the experimental design. Figure 10B shows a representative BLI image indicating the extent of bone metastasis. Figure 10C includes dot plots showing the bioluminescence (BLI) results. [Figure 10B] Figures 10A, 10B, and 10C show that anti-CXCL16 antibodies reduced tumor growth of bone metastases. Figure 10A shows the experimental design. Figure 10B shows a representative BLI image indicating the extent of bone metastasis. Figure 10C includes dot plots showing the bioluminescence (BLI) results. [Figure 10C] Figures 10A, 10B, and 10C show that anti-CXCL16 antibodies reduced tumor growth of bone metastases. Figure 10A shows the experimental design. Figure 10B shows a representative BLI image indicating the extent of bone metastasis. Figure 10C includes dot plots showing the bioluminescence (BLI) results. [Figure 11A] Figures 11A, 11B, and 11C show that treatment with anti-CXCL16 antibody reduced tumor growth of ZA-resistant bone metastases. Figure 11A shows the experimental design. Figure 11B shows a representative BLI image indicating the extent of bone metastases. Figure 11C includes dot plots showing the bioluminescence (BLI) results. [Figure 11B] Figures 11A, 11B, and 11C show that treatment with anti-CXCL16 antibody reduced tumor growth of ZA-resistant bone metastases. Figure 11A shows the experimental design. Figure 11B shows a representative BLI image indicating the extent of bone metastases. Figure 11C includes dot plots showing the bioluminescence (BLI) results. [Figure 11C] Figures 11A, 11B, and 11C show that treatment with anti-CXCL16 antibody reduced tumor growth of ZA-resistant bone metastases. Figure 11A shows the experimental design. Figure 11B shows a representative BLI image indicating the extent of bone metastases. Figure 11C includes dot plots showing the bioluminescence (BLI) results. DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description The present disclosure provides a pharmaceutical composition for preventing or treating cancer, comprising a CXCL16 antibody. In some embodiments, the pharmaceutical composition further comprises one or more of a targeted anti-cancer drug and an immune checkpoint inhibitor. The CXCL16 antibody can be provided in the form of a full-length antibody or a fragment thereof.

[0042] term As used herein, the singular forms "a," "an," and "the" include plural referents unless the content dictates otherwise. Thus, for example, reference to "an antibody" includes a combination of two or more such molecules, and the like.

[0043] As used herein, the term "about" refers to a normal error range for the respective value, readily known to one of ordinary skill in the art, such as ±20%, ±10%, or ±5%, which is within the intended meaning of the stated value.

[0044] As used herein, the term "antibody" refers to an isolated or recombinant binding agent that contains the necessary variable region sequences for specific binding to an antigen epitope. Thus, as used herein, "antibody" refers to any form of antibody, or fragment thereof, of any class or subclass, that exhibits the desired biological activity, e.g., binding to a specific target antigen. Thus, it is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including, but not limited to, scFv, Fab, etc., so long as they exhibit the desired biological activity. In this application, the terms "anti-CXCL16 antibody" and "CXCL16 antibody" are used interchangeably.

[0045] An "antibody fragment" comprises a portion of an intact antibody, e.g., the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, Fab', F(ab')2, Fv fragments; diabodies; linear antibodies (e.g., Zapata et al., Protein Eng. 8(10):1057-1062(1995)); single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment (a designation reflecting its ability to be readily crystallized). Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites that is still capable of cross-linking antigen. In the context of this invention, the binding domain comprises an Fv, which comprises a variable light chain (VL) and a variable heavy chain (VH). These are generally organized either as scFv domains comprising (N- to C-terminally) VL-scFv linker-VH or VH-scFv linker-VL on a single polypeptide chain, or as Fab fragments with VH-CH1 and VL-CL linked together on two different polypeptide chains.

[0046] Single-chain Fv or scFv refers to an antibody fragment containing the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For a general description of scFvs, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also WO88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.

[0047] As used herein, "V region" or "variable region" or "variable domain" refers to an antibody variable region domain comprising framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4 segments. The heavy chain V region, VH, is the result of rearrangement of the V gene (HV), D gene (HD), and J gene (HJ) during B cell differentiation in a manner known as V(D)J recombination. The light chain V region, VL, is the result of rearrangement of the antibody V gene (LV) and J gene (LJ), and refers to the amino-terminal domain of the heavy or light chain of an antibody. The heavy chain variable region is referred to as "VH" or "V H " and the light chain variable region is represented as "VL" or "V L These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0048] As used herein, "complementarity determining region (CDR)" refers to the three hypervariable regions (HVRs) in each chain that interrupt the four "framework" regions established by the light and heavy chain variable regions. CDRs are the primary contributors to binding to an epitope of an antigen. The CDRs of each chain are numbered sequentially from the N-terminus and are referred to as CDR1, CDR2, and CDR3, and are also identified by the chain in which the CDR is located. Thus, V H CDR3 (HCDR3) is found in the variable domain of the heavy chain of the antibody in which it is found, while V L CDR3 (LCDR3) is the CDR3 of the variable domain of the light chain of the antibody in which it is located. The term "CDR" is used interchangeably with "HVR" when referring to the sequence of a CDR.

[0049] The amino acid sequences of the CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, the International ImMunoGeneTics database (IMGT), and AbM (see, for example, Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, Structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol. 1997, 273(4)).The definition of antigen binding sites is also described in: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1; 29(1):207-9 (2001); MacCallum et al., Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262(5), 732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al., Methods Enzymol., 203, 121-153 (1991); Pedersen et al. al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172, 1996). References to CDRs determined by Kabat numbering are based on, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Chothia CDRs are determined as defined by Chothia (see, for example, Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The numbering and arrangement of CDRs may vary depending on the numbering system employed. It is understood that the disclosure of variable heavy chain and / or variable light chain sequences includes the disclosure of the associated CDRs, regardless of the numbering system employed.Unless otherwise indicated, the CDRs of this application, e.g., those shown in Table 3, are defined by a combination of IMGT and Kabat. As one illustrative example, the CDR1 region of SEQ ID NO: 1 is GFTFSNAVMN, which is a combination of residues from GFTFSNAV (according to IMGT) and NAVMN (according to Kabat).

[0050] "Fc region" refers to the constant region of an antibody, excluding the first constant region immunoglobulin domain. In human immunoglobulins, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. In the case of IgA and IgM, Fc may include the J chain. In the case of IgG, Fc includes immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although it is understood in the art that the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined as including residues C226 or P230 through the carboxyl terminus, using numbering according to the EU index, as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). The term "Fc region" can refer to this region alone or in the context of an antibody or antibody fragment. "Fc region" includes naturally occurring allelic variants of the Fc region as well as modifications that modulate effector function. The Fc region also includes variants that do not alter biological function. For example, one or more amino acids can be deleted from the N- or C-terminus of the Fc region of an immunoglobulin without substantially impairing biological function. Such variants can be selected according to general rules known in the art to minimize impact on activity (see, e.g., Bowie, et al., Science 247:306-1310, 1990). For example, in an IgG4 antibody, a single amino acid substitution (S228P according to Kabat numbering; designated IgG4Pro) can be introduced to eliminate the heterogeneity observed in recombinant IgG4 antibodies (see, e.g., Angal, et al., Mol Immunol 30:105-108, 1993). In certain embodiments, the Fc region comprises substitutions that improve the pharmacokinetic properties of the antibody, eg, substitutions that increase serum half-life.Non-limiting examples of Fc region substitutions can be found in US Pat. No. 8,088,376, the contents of which are incorporated by reference in their entirety.

[0051] As used herein, the term "heavy chain" refers to a full-length heavy chain comprising a variable region domain VH and three constant domains CH1, CH2, and CH3, which contains an amino acid sequence having sufficient variable region sequence to confer specificity for an antigen, and all fragments thereof.

[0052] Furthermore, the term "light chain" as used herein refers to both a full-length light chain and fragments thereof comprising a variable region domain VL and a constant region CL, which contain an amino acid sequence having sufficient variable region sequence to confer specificity for an antigen.

[0053] The term "identical" or percent "identity," in the context of two or more polypeptide sequences, refers to two or more sequences or subsequences that are identical or have a certain percentage of identical amino acid residues (e.g., at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) over the length of the two sequences when compared and aligned for maximum correspondence over a particular region, e.g., a comparison window or designated region. Alignment to determine percent identity of amino acid sequences can be performed in a variety of ways, including using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. An example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which is described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990). Thus, for purposes of the present invention, BLAST 2.0 can be used with default parameters to determine percent sequence identity.

[0054] The antibodies or fragments thereof of the present invention can be produced using methods known in the art, such as phage display or yeast cell surface expression systems. For example, the methods described in U.S. Patent Nos. 4,946,778 and 5,258,498 can be used to prepare scFv, and the methods described in WO92 / 22324 can be used to produce recombinant Fab, Fab', and F(ab')2 fragments.

[0055] The antibodies of the present invention may be derived from any animal, including mammals, including humans, birds, etc. Preferably, the antibodies are human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken antibodies.

[0056] Human antibodies are antibodies having the amino acid sequence of a human immunoglobulin and include either antibodies isolated from a human immunoglobulin library or antibodies isolated from animals transfected with one or more human immunoglobulins that do not express endogenous immunoglobulins (see U.S. Pat. No. 5,939,598).

[0057] The antibodies or antigen-binding fragments thereof of the present invention include all variants that achieve the desired effects of the present invention through mutations such as one or more substitutions, deletions, inversions or translocations in the antibodies defined by the above sequences.

[0058] The terms "nucleic acid" and "polynucleotide" are used interchangeably and, as used herein, refer to both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic and mixed polymers of the above. In certain embodiments, nucleotides refer to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide, and combinations thereof. The term also includes, but is not limited to, single- and double-stranded forms of DNA. Furthermore, polynucleotides, such as cDNA or mRNA, can contain either or both naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. As will be readily understood by those skilled in the art, nucleic acid molecules may be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels, methylation, substitution of one or more analogs of naturally occurring nucleotides, uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridines, psoralens, etc.), chelators, alkylators, and internucleotide modifications such as modified linkages (e.g., α-anomeric nucleic acids, etc.). The above terms are also intended to encompass all topological conformations, including single-stranded, double-stranded, partially double-stranded, triple-stranded, hairpinned, circular, and padlock conformations. A reference to a nucleic acid sequence includes its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand with its complementary sequence. The term also includes codon-optimized nucleic acids encoding the same polypeptide sequence.

[0059] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which it is introduced. As used herein, "vector" refers to a recombinant construct in which a nucleic acid sequence of interest has been inserted into the vector. Certain vectors are capable of directing the expression of a nucleic acid to which it is operably linked. Such vectors are referred to herein as "expression vectors."

[0060] As used herein, a "substitution" indicates that one or more amino acids or nucleotides are replaced with different amino acids or nucleotides, respectively.

[0061] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0062] An "isolated nucleic acid encoding an antibody or fragment thereof" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, wherein such nucleic acid molecules are contained in a single vector or in separate vectors and are present in one or more locations within a host cell.

[0063] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Thus, a host cell is a recombinant host cell, and includes the primary transformed cell and progeny derived therefrom, regardless of the number of transfers.

[0064] As used herein, a "variant" of a polypeptide is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. As used herein, "variant" refers to a sequence that has been altered rather than the naturally occurring sequence.

[0065] In the context of describing the binding strength of two antibodies to the same target, the term "equivalent" refers to the difference between the two dissociation constants (K D ) are within three times of each other. D (K of the binding reaction between the first antibody and the target) D ) and the second K D (K of the binding reaction between the second antibody and the target) D ) is in the range of 1:3 or 3:1, not including the endpoints. D A lower value indicates stronger binding. For example, an antibody variant with stronger binding than a reference antibody will have a K value lower than that measured for the reference antibody against the same target. D At least 1 / 3 of the K D binds to the target.

[0066] As used herein, a "therapeutic agent" refers to an agent that, when administered in a therapeutically effective dose to a patient suffering from a disease, cures or at least partially arrests the symptoms of the disease and complications associated with the disease.

[0067] As used herein, the term "individual" refers to a subject in need of treatment for a disease, e.g., a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. In some embodiments, the individual is a mammal.

[0068] As used herein, the term "administering" refers to providing a given composition of the present invention to a subject by any suitable method.

[0069] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of the target disease.

[0070] As used herein, the term "treatment" means that the target disease and its metabolic abnormalities are ameliorated.

[0071] As used herein, the term "amelioration" refers to any effect that reduces a parameter associated with a desired disease, e.g., the severity of symptoms, by administration of a composition disclosed herein.

[0072] As used herein, the term "bone metastasis" refers to the spread of cancer cells from their original site to the bone. Nearly all types of cancer, such as breast, thyroid, and prostate cancer cells, have the potential to spread (metastasize) to the bone. CXCL16

[0073] CXCL16 (NM_022059) is the gene encoding chemokine (C-X-C motif) ligand 16 (CXCL16). The CXCL16 protein is a small cytokine belonging to the C-X-C chemokine family. It consists of a C-X-C chemokine domain, a mucin-like stalk, a transmembrane domain, and a cytoplasmic tail containing a potential tyrosine phosphorylation region capable of binding to SH2. CXCL16 expression is induced by the proinflammatory cytokines IFN-gamma and TNF-alpha. CXCL16 has been reported as a marker for predicting the prognosis of thyroid cancer. See Korean Patent No. 10-2019-0145732.

[0074] The human CXCL16 protein (SEQ ID NO: 11) contains 254 amino acids and can bind to the chemokine receptor CXCR6. Kim et al., Scientific Reports, 9:13288 | https: / / doi.org / 10.1038 / s41598-019-49613-z. Unlike other chemokines, CXCL16 is expressed not only as a membrane-bound molecule but also as a soluble chemokine. Abel et al., J. Immunol. 172, 6362-6372 (2004). CXCL16 is produced by macrophages and dendritic cells and regulates the chemotaxis of immune cells toward CXCL16-rich environments. Jin et al., Oncol. Rep. 37, 3279-3286 (2017).

[0075] Human CXCL16: MGRDLRPGSRVLLLLLLLLLVYLTQPGNGNEGSVTGSCYCGKRISSDSPPSVQFMNRLRKHLRAYHRCLYYTRFQLLSWSVCGGNKDPWVQELMSCLDLKECGHAYSGIVAHQKHLLPTSPPISQASEGASSDIHTPAQMLLSTLQSTQRPTLPVGSLSSDKELTRPNETTIHTAGHSLAAGPEAGENQKQPEKNAGPTARTSATVPVLCLLAIIFILTAALSYVLCKRRRGQSPQSSPDLPVHYIPVAPDSNT (SEQ ID NO: 11)

[0076] CXCL16 antibody In some embodiments, the CXCL16 antibody disclosed herein binds to CXCL16 (SEQ ID NO: 11) and comprises a heavy chain variable region comprising an HCDR1 comprising SEQ ID NO: 12, or a variant HCDR1 with 1, 2, 3, 4, or 5 amino acid substitutions relative to this sequence; an HCDR2 comprising SEQ ID NO: 13, or a variant HCDR2 with 1, 2, 3, 4, or 5 amino acid substitutions relative to this sequence; and an HCDR3 comprising SEQ ID NO: 14, or a variant HCDR3 with 1, 2, 3, 4, or 5 amino acid substitutions relative to this sequence. In some embodiments, the antibody comprises a light chain variable region comprising an LCDR1 comprising SEQ ID NO: 15, or any one of variant LCDR1 with 1, 2, 3, 4, or 5 amino acid substitutions relative to this sequence; an LCDR2 comprising SEQ ID NO: 16, or any one of variant LCDR2 with 1, 2, or 3 amino acid substitutions relative to this sequence; and an LCDR3 comprising SEQ ID NO: 17, or any one of variant LCDR3 with 1, 2, 3, 4, or 5 amino acid substitutions relative to this sequence.

[0077] In some embodiments, the antibody that binds to CXCL16 comprises a V comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 1-5. H and V comprising an amino acid sequence having 95% identity with any one of SEQ ID NOs: 6 to 10. L Includes:

[0078] In some embodiments, a CXCL16 antibody has at least one mutation in the VL amino acid sequence compared to the VL sequence set forth in Table 3, and has no more than 10, 20, 30, 40, or 50 mutations. In some embodiments, the VL amino acid sequence can comprise an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the VL sequence set forth in Table 3. In some embodiments, the VL amino acid sequence can comprise a deletion or insertion relative to the CDR sequence set forth in Table 2, e.g., a deletion or insertion of 1, 2, 3, 4, 5, 6, or 7 amino acids. In some embodiments, a CXCL16 antibody of the disclosure comprises an LCDR1, LCDR2, and LCDR3 that each have at least 70% identity to the LCDR1, LCDR2, and LCDR3 as set forth in Table 2. In some embodiments, CXCL16 antibodies of the invention comprise LCDR1, LCDR2, and LCDR3, each having at least 80% identity to LCDR1, LCDR2, and LCDR3, as shown in Table 2. In some embodiments, CXCL16 antibodies of the invention comprise one, two, or all three of LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 15-17, respectively.

[0079] In some embodiments, the CXCL16 antibody has at least one mutation in the VH amino acid sequence compared to the VH sequence set forth in Table 3, and has no more than 10, 20, 30, 40, or 50 mutations. In some embodiments, the VH amino acid sequence may comprise an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the VH sequence set forth in Table 3. In some embodiments, the VH amino acid sequence may comprise a deletion or insertion relative to a CDR sequence shown in Table 1, for example, a deletion or insertion of 1, 2, 3, 4, 5, 6, or 7 amino acids. In some embodiments, the VH region comprises an HCDR1 with one or two substitutions compared to the HCDR1 sequence shown in Table 1. In some embodiments, the HCDR1 has three, four, or five substitutions compared to the HCDR1 sequence shown in Table 1. In some embodiments, the VH region comprises a CDR2 with one or two substitutions; or one, two, or three substitutions compared to the HCDR2 sequence shown in Table 1. In some embodiments, the VH region comprises an HCDR3 with one, two, or three; or one, two, three, or four substitutions compared to the HCDR3 sequence shown in Table 1. In some embodiments, a CXCL16 antibody of the disclosure comprises an HCDR1, an HCDR2, and an HCDR3 that are at least 70% identical to CDR1, CDR2, and CDR3, respectively, as shown in Table 1. In some embodiments, a CXCL16 antibody of the invention comprises an HCDR1, an HCDR2, and an HCDR3 that are at least 80% identical to HCDR1, HCDR2, and HCDR3, respectively, as shown in Table 1. In some embodiments, an anti-tumor antibody of the invention comprises one, two, or all three of HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs: 12-14, respectively.

[0080] In some embodiments, the V of a CXCL16 antibody described herein H The FR1 region of the region is at least 80% or at least 90% identical to SEQ ID NO: 22. In some embodiments, the V HThe FR1 region of the region has the sequence of SEQ ID NO:22.

[0081] In some embodiments, the V of a CXCL16 antibody described herein H The FR2 region of the region is at least 80% or at least 90% identical to SEQ ID NO: 23. In some embodiments, the V H The FR2 region of the region has the sequence of SEQ ID NO:23.

[0082] In some embodiments, the V of a CXCL16 antibody described herein H The FR3 region of the region is at least 80% or at least 90% identical to SEQ ID NO: 24. In some embodiments, the V H The FR3 region of the region has the sequence of SEQ ID NO:24.

[0083] In some embodiments, the V of a CXCL16 antibody described herein H The FR4 region of the region is at least 80% or at least 90% identical to SEQ ID NO: 25. In some embodiments, the V H The FR4 region of the region has the sequence of SEQ ID NO:25.

[0084] In some embodiments, the V of a CXCL16 antibody described herein L The FR1 region of the region is at least 80% or at least 90% identical to SEQ ID NO: 26. In some embodiments, the V L The FR1 region of the region has the sequence of SEQ ID NO:26.

[0085] In some embodiments, the V of a CXCL16 antibody described herein L The FR2 region of the region is at least 80% or at least 90% identical to SEQ ID NO: 27. In some embodiments, the V L The FR2 region of the region has the sequence of SEQ ID NO:27.

[0086] In some embodiments, the V of a CXCL16 antibody described herein L The FR3 region of the region is at least 80% or at least 90% identical to SEQ ID NO: 28. In some embodiments, the V L The FR3 region of the region has the sequence of SEQ ID NO:28.

[0087] In some embodiments, the V of a CXCL16 antibody described herein L The FR4 region of the region is at least 80% or at least 90% identical to SEQ ID NO: 29. In some embodiments, the V L The FR4 region of the region has the sequence of SEQ ID NO:29.

[0088] In some embodiments, the CXCL16 antibody is any one of HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5. In some embodiments, the CXCL16 antibody is HC5LC1.

[0089] [Table 1]

[0090] [Table 2]

[0091] [Table 3]

[0092] Note: The name of each CXCL16 antibody in this disclosure consists of two parts: the first part represents the heavy chain and the second part represents the light chain. As an example, the antibody HC5LC1 comprises the heavy chain HC5 (comprising SEQ ID NO: 42) and the light chain LC1 (comprising SEQ ID NO: 43), etc. See Table 4.

[0093] [Table 4]

[0094] [Table 5]

[0095] For the purposes of this disclosure, HC-FR1 refers to the framework 1 (FR1) region of the heavy chain variable region; HC-FR2 refers to the FR2 region of the heavy chain variable region; HC-FR3 refers to the FR3 region of the heavy chain variable region; LC-FR1 refers to the framework 1 (FR1) region of the light chain variable region; LC-FR2 refers to the FR2 region of the light chain variable region; and LC-FR3 refers to the FR3 region of the light chain variable region.

[0096] variant In some embodiments, variants of any of the CXCL16 antibodies disclosed herein can be generated by introducing mutations into the heavy and / or light chain sequences. In some embodiments, mutations are introduced into one or more CDRs of the CXCL16 antibodies disclosed herein, such as antibodies HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5. In some embodiments, mutations are introduced into framework regions. In some embodiments, the CXCL16 antibodies provided herein comprise a VH region of any one of SEQ ID NOS: 1-5 and / or a VL region of any one of SEQ ID NOS: 6-10, or a VH region having at least 80% identity to any one of SEQ ID NOS: 1-5 and a VL region having at least 80% identity to any one of SEQ ID NOS: 6-10, wherein mutations relative to the corresponding VH or VL region are present only in the framework regions.

[0097] The antibodies disclosed herein specifically bind to tumor cells. In some embodiments, the antibodies are added to cancer cell lines, and binding is analyzed using BioLight Interferometry (ForteBio). HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5 all showed strong binding to human CXCL16. See Table 6 and Example 1.

[0098] In some embodiments, the CXCL16 antibody provided herein comprises an HCDR1 of SEQ ID NO: 12, an HCDR2 of SEQ ID NO: 13, an HCDR3 of SEQ ID NO: 14, an LCDR1 of SEQ ID NO: 15, an LCDR2 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO: 17; the FW region within the VH region is at least 80% identical to the FW region present in the VH region of any one of SEQ ID NOs: 1-5, and the FW region within the VL region is at least 80% identical to the FW region present in the VL region of any one of SEQ ID NOs: 6-10.

[0099] Tumor binding activity The CXCL16 antibodies described herein may bind to tumor cells as assessed by assays well known in the art. Non-limiting examples of suitable assays include surface plasmon resonance analysis using a biosensor system such as the BIACORE® system, or flow cytometry assays, or biolight interferometry assays, which are further described in the Examples section.

[0100] In some embodiments, binding assays to assess variant activity are performed on ex vivo tumor tissue or tumor cells, e.g., tumor cells grown in vivo as tumor xenografts in syngeneic (immune-compatible) mice and harvested and processed within 24-48 hours. Binding can be assessed by a number of means, including flow cytometry.

[0101] In some embodiments, antibody binding to tumor cells is assessed by immunofluorescence performed on fresh frozen or fixed human tumor samples using standard immunostaining procedures.

[0102] The antibodies disclosed herein specifically bind to tumor cells. In some embodiments, the antibodies are added to cancer cell lines, and binding is analyzed using biolight interferometry assay. HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5 exhibit strong binding to human CXCL16. See, for example, Table 6 and Example 1.

[0103] Tumor-inhibiting activity The in vivo tumor-inhibitory activity of the CXCL16 antibodies disclosed herein can be assessed by using several assays, including, but not limited to, monitoring tumor growth and animal survival. In some embodiments, the CXCL16 antibodies described herein exhibit tumor-inhibitory effects, including a reduction in tumor growth rate, size, tumor invasion, and / or metastasis. In one illustrative example, a CXCL16 antibody inhibits tumor growth in mice bearing tumors derived from MDA-MB-231 cells (see Example 2). Such antibodies exhibit tumor-targeting effects in vivo, for example, when administered to a subject bearing a tumor that expresses or overexpresses CXCL16.

[0104] In some embodiments, the CXCL16 antibodies disclosed herein inhibit cancer cell chemotaxis and migration. Cancer cell chemotaxis and migration can be assessed using methods well known in the art. These assays are typically designed because CXCR6-expressing cells tend to migrate toward medium containing the CXCL16 cytokine. One exemplary assay is a transwell migration assay. Briefly, a vessel is divided into an upper and lower chamber, CXCR6-expressing cells are seeded into the upper chamber, and CXCL16 is added to the medium contained in the lower chamber. The upper chamber is separated from the lower chamber by a polycarbonate membrane that allows cells to pass through. After a certain period of time, the CXCR6-expressing cells migrate into the medium in the lower chamber containing CXCL16, and the cells in the lower chamber are counted, representing the number of cells that migrated to the lower chamber. Treating CXCR6-expressing cells with the CXCL16 antibodies disclosed herein reduces the number of cells migrating to the lower chamber, indicating that the CXCL16 antibodies can inhibit chemotaxis. An exemplary assay is disclosed in Example 2. The results are shown in Figure 8B, which indicates that various CXCL16 antibodies, including HC5LC1, HC5LC2, and HC5LC5, inhibit chemotaxis and migration.

[0105] In some embodiments, the CXCL16 antibodies disclosed herein inhibit bone metastasis of cancer cells. The activity of the antibodies in inhibiting bone metastasis is assessed by methods known in the art. One exemplary method uses a two-chamber system that mimics the bone metastasis niche. Briefly, the upper chamber, a mesh insert, is suspended within the lower chamber. The lower chamber contains a co-culture of cancer cells / whole bone marrow cells and a CXCL16-enriched solution, mimicking the microenvironment of the bone metastasis niche. The upper chamber contains pre-osteoclasts from bone marrow (e.g., human CD11b+ bone marrow cells). Typically, pre-osteoclasts differentiate into osteoclasts, which support tumor formation in the bone metastasis niche. Therefore, inhibiting the migration of osteoclast precursors can effectively block tumor progression in the bone metastasis niche, i.e., suppress bone metastasis. After contacting the cells in the upper chamber with the CXCL16 antibody, the cells retained in the upper chamber (a.k.a., the mesh of the insert) are counted; the number of these cells positively correlates with the ability of the antibody to inhibit cell recruitment to the metastatic niche.

[0106] Antibody format In a further aspect of the present invention, the CXCL16 antibody of the present disclosure can be an antibody fragment, e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a substantially full-length antibody, e.g., an IgG antibody, or other antibody class or isotype as defined herein. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells.

[0107] In some embodiments, the CXCL16 antibody of the present disclosure administered to a patient is an IgG of the IgG1 subclass. In some embodiments, such an antibody is an IgG of the IgG2, IgG3, or IgG4 subclass. In some embodiments, such an antibody is an IgM. In some embodiments, such an antibody has a lambda light chain constant region. In some embodiments, such an antibody has a kappa light chain constant region.

[0108] In some embodiments, the CXCL16 antibodies of the present disclosure are in a monovalent format. In some embodiments, the tumor-targeting antibody is in the format of a fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.

[0109] In some embodiments, the CXCL16 antibodies disclosed herein, including antibody fragments of the present disclosure, comprise an Fc region that exhibits effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). In some embodiments, the Fc region may be modified to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or ADCC. Thus, the Fc region may contain additional mutations to increase or decrease effector function, i.e., the ability to induce a specific biological function upon binding to an Fc receptor expressed on an immune cell. Immune cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and cytotoxic T cells.

[0110] In some embodiments, the Fc regions described herein may contain additional modifications that modulate effector function. Examples of Fc region amino acid mutations that modulate effector function include, but are not limited to, one or more substitutions at positions 228, 233, 234, 235, 236, 237, 238, 239, 243, 265, 269, 270, 297, 298, 318, 326, 327, 329, 330, 331, 332, 333, and 334 (EU numbering scheme) of the Fc region.

[0111] Exemplary substitutions that reduce effector function include the following: position 329 can be mutated to replace the proline with glycine or arginine, or with an amino acid residue large enough to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and tryptophan residues Trp 87 and Trp 110 of FcγRIII. Additional exemplary substitutions that reduce effector function include S228P, E233P, L235E, N297A, N297D, and P331S. Multiple substitutions may be made to reduce effector function, for example, L234A and L235A in the human IgG1 Fc region; L234A, L235A, and P329G in the human IgG1 Fc region; S228P and L235E in the human IgG4 Fc region; L234A and G237A in the human IgG1 Fc region; L234A, L235A, and G237A in the human IgG1 Fc region; V234A and G237A in the human IgG2 Fc region; L235A, G237A, and E318A in the human IgG4 Fc region; and S228P and L236E in the human IgG4 Fc region. Examples of substitutions that increase effector function include, for example, E333A, K326W / E333S, S239D / I332E / G236A, S239D / A330L / I332E, G236A / S239D / A330L / I332E, F243L, G236A, and S298A / E333A / K334A. In some embodiments, the Fc mutation comprises P329G, L234A, L235A, or a combination thereof. Descriptions of amino acid mutations in the Fc region that can increase or decrease effector function can be found, for example, in Wang et al., Protein Cell. 9(1):63-73, 2018; Saunders, Front Immunol. Jun 7, eCollection, 2019; Kellner et al., Transfus Med Hemother. 44(5):327-336, 2017; and Lo et al., J Biol Chem. 292(9):3900-3908, 2017.

[0112] In some embodiments, the Fc region may have one or more amino acid substitutions that modulate ADCC, for example, substitutions at positions 298, 333, and / or 334 of the Fc region according to the EU numbering scheme. Specifically, S298A, E333A, and K334A may be introduced into the Fc region to increase the affinity of the Fc region for FcγRIIIa and decrease the affinity of the Fc region for FcγRIIa and FcγRIIb.

[0113] The Fc region may also contain additional mutations to increase serum half-life. Such mutations in the Fc region may improve the pharmacokinetics of the antibody through enhanced binding to the neonatal Fc receptor (FcRn). Examples of Fc region substitutions that increase the serum half-life of the antibody include, for example, M252Y / S254T / T256E, T250Q / M428L, N434A, N434H, T307A / E380A / N434A, M428L / N434S, M252Y / M428L, D259I / V308F, N434S, V308W, V308Y, and V308F. Descriptions of amino acid mutations in the Fc region that can increase the serum half-life of antibodies can be found, for example, in Dumet et al., MAbs. 26:1-10, 2019; Booth et al., MAbs. 10(7):1098-1110, 2018; and Dall'Acqua et al., J Biol Chem. 281(33):23514-24, 2006.

[0114] Additionally, in some embodiments, antibodies of the present disclosure may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter one or more functional properties of the antibody, e.g., produced in a cell line and / or under cell culture conditions to alter its glycosylation (e.g., hypofucosylation, afucosylation, or increased sialylation). For example, an antibody may be linked to one of a variety of polymers, e.g., polyethylene glycol. In some embodiments, an antibody may contain mutations to facilitate linkage to chemical moieties and / or to alter residues subject to post-translational modifications, e.g., glycosylation.

[0115] In some embodiments, the CXCL16 antibodies described herein comprise an Fc region with altered glycosylation that enhances the antibody's ability to recruit NK cells and / or increase ADCC. In some embodiments, the Fc region comprises glycans that do not contain fucose (i.e., the Fc region is afucosylated). Afucosylated antibodies can be produced using a cell line expressing a heterologous enzyme that depletes the intracellular fucose pool (e.g., GLYMAXX® from ProBioGen AG, Berlin, Germany). Nonfucosylated antibodies can also be produced using a host cell line in which the endogenous α-1,6-fucosyltransferase (FUT8) gene has been deleted. See Satoh, M. et al., "Non-fucosylated therapeutic antibodies as next-generation therapeutic antibodies," Expert Opinion on Biological Therapy, 6:11, 1161-1173, DOI:10.1517 / 14712598.6.11.1161.

[0116] In some embodiments, CXCL16 antibodies are constructed as multivalent antibodies. In some embodiments, CXCL16 antibodies are constructed as tetravalent molecules containing four CXCL16 binding arms per molecule. Such constructs exhibit increased ADCC activity and increased binding to tumor cells as measured by flow cytometry.

[0117] In some embodiments, the CXCL16 antibodies of the present disclosure are used in a bispecific or multispecific format, e.g., a trispecific format. For example, in some embodiments, the antibodies may be incorporated into bispecific or multispecific antibodies that contain additional binding domains that bind to the same or different antigens.

[0118] There are various formats that can be used in bispecific or multispecific antibodies. The formats can vary, such as the number of binding arms, the format of each binding arm (e.g., Fab, scFv, scFab, or VH only), the number of antigen-binding domains present on the binding arms, the connectivity and binding structure of each arm relative to each other, the presence or absence of an Fc domain, the Ig class (e.g., IgG or IgM), the Fc subclass (e.g., hIgG1, hIgG2, or hIgG4), and mutations to the Fc (e.g., mutations to reduce or enhance effector function or extend serum half-life). For examples of bispecific and multispecific formats, see also Figure 1 in Speiss et al., Alternative Molecular Formats and Therapeutic Applications for Bispecific Antibodies, Mol Immunol, 67, 95-106 (2015).

[0119] CXCL16 antibody conjugates / co-stimulatory agents In a further embodiment, the CXCL16 antibody of the present invention can be conjugated or linked to a therapeutic moiety, an imaging / detection moiety, or an enzyme. For example, a tumor-targeting antibody can be conjugated to a detectable marker, a cytotoxic agent, an immunomodulatory agent, an imaging agent, a therapeutic agent, an oligonucleotide, or an enzyme. Methods for conjugating or linking an antibody to a desired molecule are well known in the art. The moiety can be linked to the antibody by a covalent or non-covalent bond.

[0120] In some embodiments, the antibody is conjugated directly or via a cleavable or non-cleavable linker to a cytotoxic moiety or other moiety that affects critical cellular processes required for survival (the "payload"). In some embodiments, the payload is a microtubule inhibitor that induces apoptosis in cells undergoing mitosis, e.g., by causing cell cycle arrest at G2 / M. Non-limiting examples of microtubule inhibitors that can be used include maytansine derivatives (DM1 / DM4), or auristatins (MMAE / MMAF) and their variants, e.g., monomethyl auristatin D, PF-06380101, duostatin 5, AS269, Tap18Hr1, AGD-0182, HPA-auristatin F. In some embodiments, the payload is a tubulin-targeting agent, e.g., hemiasterin, tubulysin, or eribulin. In some embodiments, the payload is a DNA damaging payload, including enediynes (calicheamicins), duocarmycin derivatives, pyrrolobenzodiazepine dimers (PBD dimers), and indolinobenzodiazepine pseudodimers.

[0121] In some embodiments, the antibody is directed against an antibody that inhibits, for example, auristatin, ricin A chain, doxorubicin, daunorubicin, maytansinoid taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, methotrexate, actinomycin, diphtheria toxin, Pseudomonas exotoxin A, Pseudomonas exotoxin 40, abrin, abrin A chain, modectin A chain, alphasarcin, The antibody may be conjugated to a cytotoxic drug, including, but not limited to, gelonin, mitogen, restrictocin, cobra venom factor, ribonuclease, modified Shiga toxin, phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycin, dolastatin, cc1065, or cisplatin. In some embodiments, the antibody may be linked to a drug such as an enzyme inhibitor, a growth inhibitor, a lytic agent, a DNA or RNA synthesis inhibitor, a membrane permeability modifier, a DNA metabolite, a dichloroethyl sulfide derivative, a protein production inhibitor, a ribosome inhibitor, or an apoptosis inducer. In some embodiments, the antibody is conjugated to a drug such as a topoisomerase inhibitor, e.g., a topoisomerase I inhibitor. Topoisomerase I inhibitors include, but are not limited to, quinoline alkaloids (SN-38, DXd).

[0122] In some embodiments, the CXCL16 antibodies described herein are linked to a molecule that facilitates transport of the antibody across a biological membrane, e.g., by increasing membrane permeability and facilitating translocation of proteins across the membrane. Thus, for example, the antibody may be linked to a cell-penetrating agent, such as a cell-penetrating peptide. Examples of cell-penetrating peptides include TAT, penetrating polyarginine molecules, Kunitz domain-derived peptides, e.g., angiopep-2, SynB, buforin, transportan, amphipathic peptides, and the like. In some embodiments, the antibody may be conjugated to a cationic molecule, such as a polyamine. In some embodiments, the antibody may be conjugated to an agent that facilitates transport across the blood-brain barrier, e.g., transcytosis. Thus, for example, the antibody may be conjugated to an agent that binds to an internalized endothelial cell receptor, e.g., the CXCR6 receptor, insulin receptor, insulin-like growth factor receptor, low-density lipoprotein receptor, and the like. In some embodiments, the antibody may be conjugated to a toxin, e.g., Shiga toxin, that facilitates entry of the antibody into the cytoplasm. In some embodiments, the CXCL16 antibodies described herein may be conjugated to an engineered toxin (ETB) to facilitate internalization of the antibody into cells.

[0123] In some embodiments, the CXCL16 antibodies described herein are conjugated or administered with a polypeptide immunomodulator, e.g., an adjuvant. Examples of immunomodulators include cytokines (e.g., transforming growth factor beta (TGFβ)), growth factors, lymphotoxins, tumor necrosis factors (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-15, IL-15 / IL-15Rα, e.g., Sushi domain complex, IL-18, and IL-21), colony-stimulating factors (e.g., granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF)), interferons (e.g., interferons), and the like. Examples of antibodies include, but are not limited to, feron-α, -β, or -γ, erythropoietin and thrombopoietin, or combinations thereof. In some embodiments, the antibody is linked to or administered with an adjuvant, a compound that stimulates the innate immune system, such as a toll-like receptor (TLR) agonist, a C-type lectin receptor (CLR) agonist, a retinoic acid-inducible gene I-like receptor (RLR) agonist, a polysaccharide such as saponin, chitin, or chitosan, a β-glucan, ISCOM, QS-21, or a stimulator of interferon genes (STING) agonist, or another immune enhancing agent.

[0124] In some embodiments, the CXCL16 antibodies described herein are conjugated to or administered together with an IL-15 receptor agonist, such as an IL-15 fusion construct, an IL-15:IL-15Rα fusion construct, or a single-chain IL-15:IL-15Rα(sushi) fusion construct. In one embodiment, the tumor-targeting antibody conjugated to an IL-15 receptor agonist is a bispecific or multispecific antibody. In some embodiments, the antibody is a bispecific or multispecific antibody comprising an antigen-binding domain described herein that further comprises an IL-15 receptor agonist.

[0125] In one embodiment, a CXCL16 antibody described herein is administered with a single-chain IL-15:IL-15Rα(sushi) fusion construct. In some embodiments, a CXCL16 antibody is administered with a polymer-conjugated IL-15 construct, such as NKTR-255.

[0126] For example, a subject may be administered an IL-15:IL-15Rα single chain construct comprising a therapeutically effective amount in the range of less than 0.01 mg / kg body weight to about 25 mg / kg body weight or 0.1-10 mg / kg, or 1 mg-2 g per patient or about 50 mg-1000 mg per patient.

[0127] In one embodiment, a single-chain IL-15 fusion construct comprises IL-15 linked to IL-15Rα(sushi) using a polypeptide linker. In one embodiment, the single-chain IL-15 fusion construct is linked to another protein, such as Fc, via a polypeptide linker for extended half-life. See, for example, Figure 9B of WO2018071919A1 (corresponding to U.S. Pat. No. 10,550,185). In one embodiment, IL-15 is linked or fused to the N-terminus of the heavy chain of Fc, and IL-15Rα(sushi) is linked or fused to the N-terminus of the other Fc heavy chain using heavy chain heterodimerization technology to form the desired hybrid Fc. See, for example, Figure 9A of WO2018071919A1.

[0128] In some embodiments, the antibody may be linked to a radionuclide, iron-related compound, dye, fluorescent agent, or imaging agent. In some embodiments, the antibody may be linked to an agent such as, but not limited to, a metal; a metal chelator; a lanthanide; a lanthanide chelator; a radioactive metal; a radioactive metal chelator; a positron-emitting nucleus; a microbubble (for ultrasound); a liposome; a molecule microencapsulated in a liposome or nanosphere; a single-crystalline iron oxide nanocompound; a magnetic resonance imaging contrast agent; a light absorbing, reflecting, and / or scattering agent; a colloidal particle; or a fluorophore, such as a near-infrared fluorophore.

[0129] In one embodiment of any of the above constructs, the CXCL16 antibody is any one of HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5. In one embodiment of any of the above constructs, the tumor target binding domain comprises the VH and VL sequences of HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5.

[0130] Antibody generation The disclosed antibodies are generally produced using vectors and recombinant methods well known in the art (see, e.g., Sambrook & Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Ausubel, Current Protocols in Molecular Biology). Reagents, cloning vectors, and kits for genetic manipulation are available from commercial vendors. Thus, in a further aspect of the present invention, provided herein is a V-type antibody or V-type antibody of any of the tumor-targeting antibodies described herein. H Area and / or V L isolated nucleic acids encoding the V region, or fragments thereof; vectors containing such nucleic acids, and host cells into which the nucleic acids have been introduced that are used to replicate the antibody-encoding nucleic acid and / or to express the antibody. Such nucleic acids may be used to encode the V region, or fragments thereof, of a tumor-targeting antibody (e.g., the light and / or heavy chains of the antibody). L and / or V H In some embodiments, the host cell may encode an amino acid sequence comprising: (1) V L Polynucleotides encoding the amino acid sequences and V H a vector containing a polynucleotide encoding an amino acid sequence, or (2) V L a first vector containing a polynucleotide encoding an amino acid sequence and a V H A second vector comprising a polynucleotide encoding the amino acid sequence is included.

[0131] In a further aspect, the invention provides methods of making the CXCL16 antibodies described herein. In some embodiments, the method comprises culturing a host cell described in the preceding paragraph under conditions suitable for expression of the antibody. In some embodiments, the antibody is then recovered from the host cell (or host cell culture medium).

[0132] Suitable vectors containing a polynucleotide encoding an antibody or fragment thereof of the present disclosure include cloning vectors and expression vectors. While the cloning vector of choice may vary depending on the host cell used, useful cloning vectors are generally capable of autonomous replication, may have a single target for a specific restriction endonuclease, and / or may carry a marker gene that can be used to select clones containing the vector. Examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1 plasmid, pCR1, RP4, phage DNA, and shuttle vectors. These and many other cloning vectors, such as pFUSE, pTRIOZ, pETEv2, TGEX-HC-hG1, TGEX-LC-hk, pOpti VEC, pCDNA3.3, pTRIOz, pFUSECHig, pFUSE-CLig, or pOptiVEC, are available from commercial vendors.

[0133] An expression vector is generally a replicable polynucleotide construct containing a nucleic acid of the present disclosure. Expression vectors can be replicable in host cells as episomes or as part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids and viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, and other vectors.

[0134] Suitable host cells for expressing the antibodies described herein include both prokaryotic and eukaryotic cells. For example, if glycosylation and Fc effector function are not required, CXCL16 antibodies can be produced in bacteria. After expression, the antibody can be isolated as a soluble fraction from the bacterial cell paste and further purified. Alternatively, the host cell can be a eukaryotic host cell, including eukaryotic microorganisms such as filamentous fungi or yeast, fungal or yeast strains in which the glycosylation pathway has been "humanized" to produce antibodies with partially or fully human glycosylation patterns, vertebrate, invertebrate, and plant cells. Examples of invertebrate cells include insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells. Plant cell cultures can also be used as host cells.

[0135] In some embodiments, vertebrate host cells are used to produce the antibodies of the present disclosure, for example, mammalian cell lines such as the monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells described by Graham et al., J. Gen Virol. 36:59, 1977); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described by Mather, Biol. Reprod. 23:243-251, 1980); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, e.g., as described by Mather et al., Annals NYAcad. Sci. 383:44-68, 1982; MRC 5 cells; and FS4 cells can be used to express tumor-targeting antibodies. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. Host cells of the present disclosure also include, but are not limited to, isolated cells, in vitro cultured cells, and ex vivo cultured cells. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268, 2003.

[0136] In some embodiments, the CXCL16 antibody of the present invention is produced by a CHO cell line, such as a CHO-K1 cell line. One or more expression plasmids encoding heavy and light chain sequences can be introduced. For example, in one embodiment, an expression plasmid encoding the heavy chain and an expression plasmid encoding the light chain are transfected into host cells as linearized plasmids at a 1:1 ratio in a CHO-K1 host cell line using a reagent such as Freestyle Max reagent. Fluorescence-activated cell sorting (FACS) combined with single-cell imaging can be used as a cloning method to obtain a production cell line.

[0137] Host cells transfected with an expression vector encoding a CXCL16 antibody or fragment thereof of the present disclosure can be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptide can be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide can be retained in the cytoplasm or membrane fraction, and the cells can be harvested, lysed, and the polypeptide isolated by any desired method.

[0138] In some embodiments, antibodies of the present disclosure may be produced by in vitro synthesis (see, for example, the Sutro Biopharma biochemical protein synthesis platform).

[0139] In some embodiments, provided herein are methods for making variants of the CXCL16 antibodies disclosed herein. H The constructs encoding variants of CDR3 can be modified, and the V encoded by the modified constructs H The region is a V L V region or variant region paired with the V described herein H In the context of the field, the compounds can be tested for binding activity to target cells (e.g., breast cancer cells) and / or in vivo tumor targeting activity. LThe constructs encoding variants of CDR3 can be modified, and the V encoded by the modified constructs can be L The regions can be tested for efficacy in binding to target cells or other tumor cells and / or for in vivo tumor targeting activity. Such analyses can also be performed on other CDR or framework regions to select antibodies with the desired activity.

[0140] Cancer treatment In a further embodiment, the CXCL16 antibodies provided herein, or variants thereof described herein, can be used as therapeutic agents to treat cancer.

[0141] In some embodiments, the present disclosure provides methods for identifying subjects who are candidates for treatment with a CXCL16 antibody having tumor-targeting effects. Thus, in one embodiment, the present disclosure provides a method for identifying patients who can benefit from treatment with a CXCL16 antibody of the present disclosure. In one embodiment, the patient has a CXCL16-overexpressing cancer, i.e., a cancer that expresses CXCL16 at levels at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, or 100% higher than normal tissue. In some embodiments, the cancer sample is derived from a primary tumor. In alternative embodiments, the cancer sample is a metastatic lesion. Binding of an antibody to cancer cells via a binding interaction with CXCL16 can be measured using any assay, such as BioLight Interferometry (ForteBio), immunohistochemistry, or flow cytometry. In some embodiments, binding of an antibody to at least 0.2%, 0.5%, or 1%, or at least 5% or 10%, or at least 20%, 30%, or 50% of tumor cells in a sample can be used as a selection criterion to determine patients to be treated with CXCL16, as described herein.

[0142] The CXCL16 antibodies disclosed herein can be used to treat cancer. Tumors generally refer to abnormal growths caused by autonomous excessive proliferation of bodily tissues, and tumors can be divided into benign and malignant tumors. Malignant tumors grow much faster than benign tumors, invade surrounding tissues, and metastasize, threatening life. Such malignant tumors are generally referred to as "cancer." Therefore, the term "cancer" refers to diseases related to the control of cell death or diseases caused by excessive cell proliferation when the normal balance of apoptosis is disrupted. In some cases, these abnormally hyperproliferative cells infiltrate surrounding tissues and organs, forming masses, and this infiltration can cause structural destruction or deformation; these conditions are collectively referred to as cancer.

[0143] In some embodiments, cancers that may benefit from treatment with the CXCL16 antibodies disclosed herein include cervical cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, colon cancer, colorectal cancer, bone cancer, skin cancer, head cancer, cervical cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, liver cancer, brain tumor, bladder cancer, blood cancer, gastric cancer, perianal cancer, breast cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary CNS lymphoma, spinal cord tumors, and brain. In some embodiments, the cancer is glioma or pituitary adenoma.

[0144] In some embodiments, the thyroid cancer that can be treated with the CXCL16 antibodies disclosed herein is anaplastic thyroid cancer. Anaplastic thyroid cancer, also known as histopathogenic thyroid cancer, has the poorest prognosis among thyroid cancers. Distant metastasis to the lungs or bones is often found early on, and if confirmed, is considered stage IV. The average survival time is approximately 3 to 6 months, with a survival rate approaching 0%.

[0145] In some embodiments, the breast cancer that can be treated with the CXCL16 antibodies disclosed herein is triple-negative breast cancer. Triple-negative breast cancer is known to lack estrogen and progesterone receptors (ER- / PR-), and the HER2 gene is not expressed. Therefore, TNBC is resistant to estrogen receptor modulators (tamoxifen) and HER2 inhibitors (trastuzumab). Triple-negative breast cancer accounts for approximately 12-17% of all breast cancer patients in the United States and approximately 15.9% of all breast cancer patients in South Korea. The 5-year survival rate for triple-negative breast cancer patients has been reported to be approximately 77%, which is lower than the approximately 93% survival rate for patients with other types of breast cancer. These cancer patients may benefit from treatment with the CXCL16 antibodies disclosed herein.

[0146] Combination therapy In some embodiments, the CXCL16 antibodies disclosed herein may be administered with one or more additional therapeutic agents, also referred to herein as combination agents. In some embodiments, the CXCL16 antibodies disclosed herein may be administered in combination with one or more targeted anti-cancer agents. In some embodiments, the targeted anti-cancer agents are therapeutic antibodies that target tumor cell antigens. Non-limiting examples of targeted anti-cancer agents include cetuximab, trastuzumab, ibritumomab, rituximab, brentuximab, alemtuzumab, imatinib, nilotinib, radotinib, gefitinib, erlotinib, afatinib, olmutinib, osimertinib, ceritinib, lapatinib, ruxolitinib, tofacitinib, vemuratinib, sunitinib, axitinib, vandetanib, dasatinib, crizotinib, zopanib, regorafenib, bevacizumab, paclitaxel, gemcitabine, docetaxel, axitinib, nintedanib, and lenvatinib.

[0147] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of, but not limited to, a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

[0148] In some embodiments, the co-agent is a PD-L1 inhibitor. Non-limiting examples of PD-L1 inhibitors include atezolizumab, avelumab, duvalumab, embafolimab, cosibelimab, AUNP12, CA-170, BMS-986189, nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, and may be one or more selected from the group consisting of tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514.

[0149] In some embodiments, the co-agent is a CTLA-4 inhibitor. Non-limiting examples of CTLA-4 inhibitors include ipilimumab and tremelimumab.

[0150] In some embodiments, the CXCL16 antibody and the combination agent may be administered simultaneously, separately, or sequentially.

[0151] In some embodiments, the CXCL16 antibody inhibits succinate metabolism.

[0152] In some embodiments, the targeted anti-cancer drug may be administered orally, hi some embodiments, the immune checkpoint inhibitor may be administered by injection.

[0153] In some embodiments, the CXCL16 antibody and the combination agent may each be provided in a single dosage form or in a single dose.

[0154] In some embodiments, the pharmaceutical composition may be for reducing tumor size or inhibiting tumor metastasis.

[0155] The pharmaceutical compositions disclosed herein may also include pharmaceutically acceptable salts, the term "pharmaceutically acceptable salts" as used herein including salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.

[0156] Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess of an aqueous solution of the acid and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. They can also be prepared by heating equimolar amounts of the compound and an acid or alcohol in water, followed by evaporating the mixture to dryness, or by filtering the precipitated salt with suction.

[0157] Salts derived from suitable bases include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.Alkali metal salts or alkaline earth metal salts can be obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate.In this case, it is pharmaceutically appropriate to prepare sodium, potassium, or calcium salts as metal salts, and the corresponding silver salts can be obtained by reacting alkali metal or alkaline earth metal salts with a suitable silver salt (for example, silver nitrate).

[0158] The content of the CXCL16 antibody and / or one or more concomitant agents (e.g., targeted anticancer drugs, immune checkpoint inhibitors) in the pharmaceutical composition can be appropriately adjusted depending on the symptoms of the disease, the progression of the symptoms, the patient's condition, etc., for example, from 0.0001 to 0.0001% by weight relative to the total weight of the composition, which may be 99.9% by weight or from 0.001 to 50% by weight, but is not limited thereto. The content is based on the dry weight excluding the solvent.

[0159] The content of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the severity of the disease and / or the purpose. In some embodiments, the effective dose is 0.01 μg to 10,000 mg, preferably 0.1 μg to 1,000 mg, per administration. It may be administered several times daily. However, the dosage of the pharmaceutical composition is determined by taking into account various factors, such as the formulation method, administration route, number of treatments, and the patient's age, weight, health condition, sex, severity of the disease, diet, and excretion rate, to determine the effective dosage for the patient. Therefore, taking this into consideration, those skilled in the art will be able to determine the appropriate effective dose of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its formulation, administration route, and administration method, as long as the effects of the present invention are demonstrated.

[0160] The pharmaceutical composition of the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipients may be, for example, at least one selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, wetting agents, film coating materials, and release-controlling additives.

[0161] The pharmaceutical compositions of the present invention can be prepared according to conventional methods, and can be formulated into external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, elixirs, emulsions, suspensions, spirits, lozenges, flavors, and lemonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusions, plasters, lotions, pastes, sprays, inhalants, patches, sterile injections, or aerosols. External preparations can be creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or poultices.

[0162] Carriers, excipients, and diluents that may be included in pharmaceutical compositions of the present invention may include lactose, glucose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0163] In the case of a formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc.

[0164] Additives for the tablets, powders, granules, capsules, pills, and lozenges of the present invention include corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, dimannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, crystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, cellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC Hydroxypropyl methyl excipients such as 2910, propylene glycol, casein, calcium lactate, Primogel; gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, carboxymethylcellulose calcium, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, crystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch powder, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. Disintegrants such as chill cellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resins, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelling starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, dissorbitol solution, light anhydrous silicic acid;Lubricants that may be used include calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, limestone, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light silicic anhydride.

[0165] Examples of additives that can be used for the liquid formulation of the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (Twinesters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, and sodium carboxymethylcellulose.

[0166] The syrup according to the present invention may contain sucrose solution, other sugars or sweeteners, and may also contain flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners and the like, as required.

[0167] Purified water may be used in the emulsion of the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0168] The suspending agent of the present invention may be suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, and HPMC 2910. If necessary, surfactants, preservatives, stabilizers, coloring agents, and flavoring agents may also be used.

[0169] The injection solution according to the present invention may contain a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ring Gel injection, glucose injection, glucose + sodium chloride injection, PEG, lactate Ring Gel injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizer such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethyl acetamide, butazolidine, propylene glycol, Tween, nidontinamide, hexamine, and dimethylacetamide; a weak acid and its salt (acetic acid and sodium acetate), a weak base and its salt (ammonia and ammonium acetate), and a weak base and its salt (ammonia and ammonium acetate). buffers such as ethanol, organic compounds, proteins, albumin, peptone, and gums; isotonicity agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfating agents such as 0.1% sodium bisulfide, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium SiMC, sodium alginate, Tween 80, and aluminum monostearate.

[0170] The suppositories of the present invention may contain cocoa butter, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subalanine, cottonseed oil, peanut oil, palm oil, cocoa butter plus cholesterol, lecithin, lanet wax, glyceryl monostearate, Tween or Span, Imhausen, Monolene (propylene glycol monostearate), glycerin, Adeps. Solidus, Butyramutego-G), Sebes Pharma 16, Hexalide Base 95, Cotmar, Hydroxote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrohydrocort 25, Hydrocort 711, Hydropostal, Massa Estralium, A, AS, B, C, D, E, I, T, Massa-MF, Maspol, Maspol-15, Neopostal-N, Paramound-B, Sposhiro (OSI, OSIX, A, B, C, D, H, L), IV suppositories (AB, B, A, BC, BBG, E, BGF, C, D, 299), Sapostal (N, Es), Wecoby (W, R, S, M, Fs), Tester Triglyceride Base (TG-95, MA, 57), etc.

[0171] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such preparations contain at least one excipient in the extract, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may also be used.

[0172] Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavorings, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0173] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective amount is determined by the type, severity, drug activity and type of the patient's disease; factors including drug sensitivity, administration time, administration route and excretion rate, treatment duration, concomitant medication, and other factors well known in the medical field can be determined. In exemplary embodiments, the CXCL16 antibody is administered by intraperitoneal injection at 100 mcg / 20 g / day twice a week; or by intravenous injection at 50 mcg / 20 g / day three times a week; the PD-L1 peptide is administered by intraperitoneal injection at 0.1 mg / 20 g / day five times a week; lenvatinib is administered orally at 30 m / kg / day; and paclitaxel is administered by intraperitoneal injection at 10 mg / kg twice a week, or by intraperitoneal injection at 10 mg / kg / day once a week.

[0174] The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, either sequentially or simultaneously with conventional therapeutic agents.

[0175] The pharmaceutical composition and other therapeutic agents may be administered once or multiple times. Those skilled in the art can determine the minimum amount or the amount that will provide the maximum effect without side effects, taking into account all of the above factors.

[0176] The pharmaceutical composition of the present invention can be administered to an individual via various routes. Any mode of administration can be envisaged, such as oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, or vaginal administration. It can also be administered by internal insertion, ocular administration, aural administration, nasal administration, inhalation, spray into the mouth or nose, dermal administration, transdermal administration, etc.

[0177] The pharmaceutical composition of the present invention is determined depending on the type of drug as an active ingredient as well as various related factors such as the disease to be treated, the route of administration, the age, sex, weight, and severity of the disease of the patient.

[0178] In some embodiments, the present disclosure provides a CXCL16 antibody; and one or more combination agents selected from the group consisting of targeted anti-cancer agents and immune checkpoint inhibitors as active ingredients, wherein the CXCL16 antibody; and the combination agent(s) are administered simultaneously, separately, or sequentially, which provides a pharmaceutical combination formulation for the prevention or treatment of cancer.

[0179] The CXCL16 antibody, targeted anticancer agent, and immune checkpoint inhibitor, which are components of the pharmaceutical combination formulation of the present invention, can be used as they are or in the form of a salt, preferably a pharmaceutically acceptable salt.

[0180] The pharmaceutical combination formulation of the present invention may contain a CXCL16 antibody as a component, depending on the administration method and route. In some embodiments, a combination of one or more drugs selected from the group consisting of targeted anticancer drugs and immune checkpoint inhibitors may also be simultaneously contained in a single formulation together with the CXCL16 antibody. In some embodiments, the combination drug and the CXCL16 antibody may be separately formulated and contained in a single package according to daily or once-daily dosage units. Specific methods for formulating the pharmaceutical combinations disclosed herein are known or apparent to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (19th ed., Mack Publishing Company, Easton, PA, 1995). The CXCL16 antibody and one or more combination drugs selected from the group consisting of targeted anticancer drugs and immune checkpoint inhibitors, which are components of the pharmaceutical combination formulation of the present invention, may be administered simultaneously, separately, or in a predetermined order. As used herein, the term "co-administration" means that, in the case of oral administration, the CXCL16 antibody and the combination are taken together or substantially simultaneously (e.g., within 15 minutes of each other) so that the two components are present in the stomach at the same time. When administered simultaneously, the combination may be formulated to be contained simultaneously in one formulation. When administered orally, the combination may be preferably formulated so that the daily amount is contained in a single dose; however, the combination may also be formulated so that the combination is administered in divided doses, such as two, three, or four times a day.

[0181] Illustrative Embodiments Embodiment 1: A pharmaceutical composition for preventing or treating cancer, comprising a CXCL16 antibody as an active ingredient.

[0182] Embodiment 2: A method of preventing or treating cancer in a subject, comprising administering the pharmaceutical composition of Embodiment 1 and one or more concomitant agents selected from the group consisting of chemotherapeutic agents, targeted anti-cancer agents, and immune checkpoint inhibitors.

[0183] Embodiment 3: The method of embodiment 2, wherein the targeted anticancer agent is at least one selected from the group consisting of cetuximab, trastuzumab, ibritumomab, rituximab, brentuximab, alemtuzumab, imatinib, nilotinib, radotinib, gefitinib, erlotinib, afatinib, olmutinib, osimertinib, ceritinib, lapatinib, ruxolitinib, tofacitinib, vemuratinib, sunitinib, axitinib, vandetanib, dasatinib, crizotinib, pazopanib, regorafenib, bevacizumab, paclitaxel, gemcitabine, docetaxel, axitinib, nintedanib, and lenvatinib.

[0184] Embodiment 4: The method of embodiment 2, wherein the immune checkpoint inhibitor is one selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

[0185] Embodiment 5: The method of embodiment 4, wherein the PD-L1 inhibitor is one of atezolizumab, avelumab, duvalumab, embafolimab, cosibelimab, AUNP12, CA-170, or BMS-986189.

[0186] Embodiment 6: The method of embodiment 4, wherein the PD-1 inhibitor is nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab (tislelizumab), toripalimab, dostarlimab, INCMGA00012, AMP-224, or AMP-514.

[0187] Embodiment 7: The method of embodiment 4, wherein the CTLA-4 inhibitor is ipilimumab or tremelimumab.

[0188] Embodiment 8: The method of embodiment 2, wherein the CXCL16 antibody and the combination agent are administered simultaneously, separately, or sequentially.

[0189] Embodiment 9: The method or pharmaceutical composition of embodiment 2, wherein the CXCL16 antibody inhibits succinate metabolism.

[0190] Embodiment 10: The method of embodiment 2, wherein the cancer is cervical cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, colon cancer, colorectal cancer, bone cancer, skin cancer, head cancer, cervical cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, liver cancer, brain tumor, bladder cancer, blood cancer, stomach cancer, perianal cancer, breast cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis cancer, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, and pituitary tumor.

[0191] Embodiment 11: The method of embodiment 2, wherein the targeted anticancer agent is administered orally.

[0192] Embodiment 12: The method of embodiment 2, wherein the CXCL16 antibody and the combination agent are each provided in a single dosage form or in a single dose.

[0193] Embodiment 13: The method of embodiment 1 or 2, wherein the pharmaceutical composition reduces tumor size or inhibits tumor metastasis.

[0194] Embodiment 14: A pharmaceutical combination formulation for the prevention or treatment of cancer, comprising as active ingredients a CXCL16 antibody and one or more concomitant agents selected from the group consisting of chemotherapeutic agents, targeted anticancer agents, and immune checkpoint inhibitors, wherein the CXCL16 antibody and the concomitant agents are administered simultaneously, separately, or sequentially.

[0195] Embodiment 15: A pharmaceutical composition for inhibiting cancer metastasis, wherein the pharmaceutical composition comprises a CXCL16 antibody as an active ingredient.

[0196] Embodiment 16: A pharmaceutical composition comprising, as active ingredients, a CXCL16 antibody for inhibiting cancer metastasis and one or more concomitant agents selected from the group consisting of chemotherapeutic agents, targeted anticancer agents, and immune checkpoint inhibitors.

[0197] Embodiment 17: A composition for diagnosing cancer, comprising a CXCL16 antibody as an active ingredient.

[0198] Embodiment 18: A kit for diagnosing cancer, comprising the composition of embodiment 17.

[0199] All documents mentioned in this specification are incorporated herein by reference as if the contents were set forth herein. When introducing elements of the invention or preferred embodiments thereof, the articles "a," "an," "the," and "said" refer to one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. Although the present invention has been described with reference to specific embodiments, this should not be construed as limiting the details of these embodiments.

[0200] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are provided merely to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0201] Informal sequence listing: SEQ ID NO: 1: Heavy chain variable region of HC1 QVQLVESGGGVVQPGGSLRLSCAASGFTFSNAVMNWVRQAPGKGLEWVARIRTKPNNYATFYADSVKGRFTFSRDNSKTMLYLQMNSLRAEDTAVYYCTAVTTTEGIYWGQGTLVTVSS

[0202] SEQ ID NO: 2: Heavy chain variable region of HC2 AVQLVESGGGLVQPGGSLKISCAASGFTFSNAVMNWVRQASGKGLEWVGRIRTKPNNYATFYADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAMYYCTAWTTEGIYWGQGTLVTVSS

[0203] SEQ ID NO: 3: Heavy chain variable region of HC3 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNAVMNWVRQAPGKGLEWVGRIRTKPNNYATFYADSVKGRFTFSRDDSKNTAYLQMNILKTEDTAMYYCTAWTTEGIYWGQGTTVTVSS

[0204] SEQ ID NO: 4: Heavy chain variable region of HC4 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAVMNWVRQAPGKGLEWVARIRTKPNNYATFYADSVKGRFTFSRDDSKNTLYLQMNSLKTEDTAVYYCTAWTTEGIYWGQGTTVTVSS

[0205] SEQ ID NO: 5: Heavy chain variable region of HC5 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNAVMNWVRQASGKGLEWVARIRTKPNNYATFYADSVKGRFTFSRDDSKNTAYLQMNSLKTEDTAMYYCTAWTTEGIYWGQGTLVTVSS

[0206] SEQ ID NO: 6: Light chain variable region of LC1 DIVMTQSPDSLAVSAGETVTINCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYDTPWTFGGGTKVEIK

[0207] SEQ ID NO: 7: Light chain variable region of LC2 DIVMTQSPSSLAVSLGETATINCKSSQSLLYSGNQKNYLAWYQQKPGQPPKLLIYWASTRQSGVPDRFTGSGAGTDFTLTISSVQAEDLAIYYCQQYYDTPWTFGGGTKVEIK

[0208] SEQ ID NO: 8: Light chain variable region of LC3 DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDFATYYCQQYYDTPWTFGGGTKVEIK

[0209] SEQ ID NO: 9: Light chain variable region of LC4 DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPSRFSGSGSGTDFTLTISSLQPEDLAIYYCQQYYDTPWTFGGGTKLELK

[0210] SEQ ID NO: 10: Light chain variable region of LC5 DIVMTQSPSSLAVSAGERATINCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPDRFIGSGSGTFTLTISSLQAEDVAIYYCQQYYDTPWTFGGGTKLELK

[0211] SEQ ID NO: 11: Human CXCL16 MGRDLRPGSRVLLLLLLLLLVYLTQPGNGNEGSVTGSCYCGKRISSDSPPSVQFMNRLRKHLRAYHRCLYYTRFQLLSWSVCGGNKDPWVQELMSCLDLKECGHAYSGIVAHQKHLLPTSPPISQAS EGASSDIHTPAQMLLSTLQSTQRPTLPVGSLSSDKELTRPNETTIHTAGHSLAAGPEAGENQKQPEKNAGPTARTSATVPVLCLLAIIFILTAALSYVLCKRRRGQSPQSSPDLPVHYIPVAPDSNT

[0212] SEQ ID NO: 12: HCDR1 GFTFSNAVMN

[0213] SEQ ID NO: 13: HCDR2 RIRTKPNNYATFYADSVKG

[0214] SEQ ID NO: 14: HCDR3 TAWTTEGIY

[0215] SEQ ID NO: 15: LCDR1 KSSQSLLYSGNQKNYLA

[0216] SEQ ID NO: 16: LCDR2 WASTRQS

[0217] SEQ ID NO: 17: LCDR3 QQYYDTPWT

[0218] SEQ ID NO: 18: Heavy chain signal peptide MGWTLVFLFLLSVTAGVHS

[0219] SEQ ID NO: 19: Light chain signal peptide MVSSAQFLGLLLLCFQGTRC

[0220] SEQ ID NO: 20: CXCR6 MAEHDYHEDYGFSSFNDSSQEEHQDFLQFSKVFLPCMYLVVFVCGLVGNSLVLVISIFYHKLQSLTDVFLVNLPLADLVFVCTLPFWAYAGIHEWVFGQVMCKSLLGIYTINFYTSMLILTCITVDRFIVVVKATKAYNQQAKRMTWGKVTSLLIWVISLLVSLPQIIYGN VFNLDKLICGYHDEAISTVVLATQMTLGFFLPLLTMIVCYSVIIKTLLHAGGFQKHRSLKIIFLVMAVFLLTQMPFNLMKFIRSTHWEYYAMTSFHYTIMVTEAIAYLRACLNPVLYAFVSLKFRKNFWKLVKDIGCLPYLGVSHQWKSSEDNSKTFSASHNVEATSMFQL

[0221] SEQ ID NO: 21: HC-FR1 X1VQLVESGGGX2VX3PX4X5SLX6X7SCAAS; where X1 can be A or Q or E; X2 can be L or V; X3 can be Q or K; X4 can be K or G; X5 can be E or G; X6 can be K or R; and X7 can be I or L.

[0222] SEQ ID NO: 22: HC-FR2 WVRQAX1GKGLEWVX2; where X1 can be P or S; X2 can be A or G.

[0223] SEQ ID NO: 23: HC-FR3 FTX1SRDX2SKX3X4X5YLQMX6X7LX8X9EDTAX 10 YYC; where X1 can be F or I; X2 can be D or N; X3 can be S, T, or N; X4 can be M or T; X5 can be V, L, or A; X6 can be D or N; X7 can be N, S, or I; X8 can be K or R; X9 can be T or A; X 10 can be M or V.

[0224] SEQ ID NO: 24: HC-FR4 WGQGTX1VTVSS; where X1 can be L or T

[0225] SEQ ID NO: 25: LC-FR1 DIX1MTQSPX2SLX3X4SX5GX6X7X8TIX9C; where X1 can be V, Q; X2 can be S; D; X3 can be A, S; X4 can be V, A; X5 can be A, L, V; X6 can be E, D; X7 can be T, R; X8 can be V, A; X9 can be N, T.

[0226] SEQ ID NO: 26: LC-FR2 WYQQKPGQX1PKLLIY; X1 can be S or P.

[0227] SEQ ID NO: 27: LC-FR3 GVPX1RFX2GSGX3GTDFTLTISSX4QX5EDX6AX7YYC; X1 can be D, S; X2 can be I, S, or T; X3 can be S or A; X4 can be V or L; X5 can be A, P; X6 can be L, V, or F; and X7 can be I, V, or T.

[0228] SEQ ID NO: 28: LC-FR4 GGGTKX1EX2K; where X1 can be L or V; and X2 can be L or I.

[0229] SEQ ID NO: 29: Chimeric antibody CLS-A1HC0LC0 heavy chain sequence AVQLVESGGGLVQPKESLKISCAASGFTFSNAVMNWVRQAPGKGLEWVARIRTKPNNYATFYADSVKGRFTFSRDDSKSMVYLQMDNLKTEDTAMYYCTAWTTEGIYWGQGTLVTVS

[0230] SEQ ID NO: 30: Chimeric antibody CLS-A1HC0LC0 light chain sequence DIVMTQSPSSLAVSAGETVTINCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPDRFIGSGSGTFTLTISSVQAEDLAIYYCQQYYDTPWTFGGGTKLELK

[0231] SEQ ID NO: 31: Mouse CXCL16 sequence MRRGFGPLSLAFFLFLLALLTLPGDGNQGSVAGSCSCDRTISSGTQIPQGTLDHIRKYLKAFHRCPFFIRFQLQSKSVCGGSQDQWVRELVDCFERKECGTGHGKSFHHQKHLPQASTQTPEA AEGTPSDTSTPAHSQSTQHSTLPSGALSLNKEHTQPWEMTTLPSGYGLEARPEAEANEKQQDDRQQEAPGAGASTPAWVPVLSLLAIVFFLTAAMAYVLCNRRATQQNSAGLQLWYTPVEPRP

[0232] SEQ ID NO: 32: Amino acid sequence of the heavy chain of chimeric antibody (HC0) ACCMGWTLVFLFLLSVTAGVHSAVQLVESGGGLVQPKESLKISCAASGFTFSNAVMNWVRQAPGKGLEWVARIRTKPNNYATFYADSVKGRFTFSRDDSKSMVYLQMDNLKTEDT AMYYCTAWTTEGIYWGQGTLVTVSSAETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVTVPSSTWSSQAVTCNVAHPASST KVDKKIVPRECNPCGCTGSEVSSVFIFPPKTKDVLTITLTPKVTCVVVDISQNDPEVRFSWFIDDVEVHTAQTHAPEKQSNSTLRSVSELPIVHRDWLNGKTFKCKVNSGAFPAP IEKSISKPEGTPRGPQVYTMAPPKEEMTQSQVSITCMVKGFYPPDIYTEWKMNGQPQENYKNTPPTMDTDGSYFLYSKLNVKKETWQQGNTFTCSLVLHEGLHNHHTEKSLSHSPGK

[0233] SEQ ID NO: 33: Optimized coding sequence of the heavy chain of chimeric antibody (HC0) (for Chinese hamster (Cricetulus griseus) (CHO))

[0234] SEQ ID NO: 34: Amino acid sequence of the light chain of the chimeric antibody (LC0) ACCMVSSAQFLGLLLLCFQGTRCDIVMTQSPSSLAVSAGETVTINCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPDRFIGSGSGTDFTLTISSVQAEDLAIYYCQQYY DTPWTFGGGTKLELKRADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDISVKWKIDGTERRDGVLDSVTDQDSKDSTYSMSSTLSLTKADYESHNLYTCEVVHKTSSSPVVKSFNRNEC

[0235] SEQ ID NO: 35: Optimized coding sequence of the light chain of chimeric antibody_LC (LC0) (for Chinese hamster (Cricetulus griseus) (CHO)) ACCATGGTGAGCAGCGCTCAGTTCCTGGGCCTGCTGCTGCTGTGCTTCCAAGGCACAAGATGCGACATCGTGATGACACAGAGCCCTAGCAGCCTGGCCGTGAGCGCCGGCGAGACCGTGACCATCAACTGCAAGAGCTCTCAGAGCCTGCTGTACAGCGGCAATCAGAAGAACTACCTG GCCTGGTATCAGCAGAAGCCTGGACAGAGCCCTAAGCTGCTGATCTACTGGGCTAGCACAAGACAGAGCGGCGTGCCTGACAGATTCATCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCGTGCAAGCCGAGGACCTGGCCATCTACTACTGTCAGCAGTACTACGACA CCCCTTGGACCTTCGGCGGCGGCACCAAGCTGGAGCTGAAGAGAGCCGACGCCGCCCCTACCGTGAGCATCTTCCCTCCTAGCACCGAGCAGCTGGCCACCGGCGGCGCTAGCGTGGTGTGCCTGATGAACAACTTCTACCCTAGAGACATCAGCGTGAAGTGGAAGATCGACGGCACCGA GAGAAGAGACGGCGTGCTGGACAGCGTGACCGACCAAGACAGCAAGGACAGCACCTACAGCATGAGCAGCACCCTGAGCCTGACCAAGGCCGACTACGAGAGCCACAACCTGTACACCTGCGAGGTGGTGCACAAGACAAGCAGCAGCCCTGTGGTGAAGAGCTTCAACAGAAACGAGTGC

[0236] SEQ ID NO: 36: The VH domain of the chimeric antibody HC0LC0 had the following sequence AVQLVESGGGLVQGPKESLKISCAAS GFTFSNAVMN WVRGAPGKGLEWVA RIRTKPNNYATFYADSVKG RFTFSRDDSKSMVYLQMDNLKTEDTAMYYC TAWTTEGIY WGQGTLVTVSS (SEQ ID NO: 36) (CDR residues are underlined).

[0237] SEQ ID NO: 37: The closest human germline gene V region is Homo sapiens IGHV3-73*01 EVQLVESGGGLVQPGGSLKLSCAAS GFTFSGSAMH WVRQGASGKGLEWVG RIRSKANSYATAYAASVKG RFTISRDDSKNTAYLQMNSLKTEDTAVYYC TR

[0238] SEQ ID NO: 38: Heavy chain HC1 QVQLVESGGGVVQPGGSLRLSCAASGFTFSNAVMNWVRQAPGKGLEWVARIRTKPNNYATFYADSVKGRFTFSRDNSKTMLYLQMNSLRAEDTAVYYCTAWTTTEGIYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0239] SEQ ID NO: 39: Heavy chain HC2 AVQLVESGGGLVQPGGSLKISCAASGFTFSNAVMNWVRQASGKGLEWVGRIRTKPNNYATFYADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAMYYCTAWTTEGIYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0240] SEQ ID NO: 40: Heavy chain HC3 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNAVMNWVRQAPGKGLEWVGRIRTKPNNYATFYADSVKGRFTFSRDDSKNTAYLQMNILKTEDTAMYYCTAWTTEGIYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0241] SEQ ID NO: 41: Heavy chain HC4 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAVMNWVRQAPGKGLEWVARIRTKPNNYATFYADSVKGRFTFSRDDSKNTLYLQMNSLKTEDTAVYYCTAWTTEGIYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0242] SEQ ID NO: 42: Heavy chain HC5 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNAVMNWVRQASGKGLEWVARIRTKPNNYATFYADSVKGRFTFSRDDSKNTAYLQMNSLKTEDTAMYYCTAWTTEGIYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0243] SEQ ID NO: 43: Light chain LC1 DIVMTQSPDSLAVSAGETVTINCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYDTPWTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0244] SEQ ID NO: 44: Light chain LC2 DIVMTQSPSSLAVSLGETATINCKSSQSLLYSGNQKNYLAWYQQKPGQPPKLLIYWASTRQSGVPDRFTGSGAGTDFTLTISSVQAEDLAIYYCQQYYDTPWTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0245] SEQ ID NO: 45: Light chain LC3 DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDFATYYCQQYYDTPWTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0246] SEQ ID NO: 46: Light chain LC4 DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPSRFSGSGSGTDFTLTISSLQPEDLAIYYCQQYYDTPWTFGGGTKL ELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0247] SEQ ID NO: 47: Light chain LC5 DIVMTQSPSSLAVSAGERATINCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPDRFIGSGSGTFTLTISSLQAEDVAIYYCQQYYDTPWTFGGGTKL ELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0248] SEQ ID NO: 48: Human IgG1 isotype constant domain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0249] SEQ ID NO: 49: Human IgK isotype constant domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [Example]

[0250] Example Example 1. Binding of human CXCL16 to CXCL16 The binding properties of 25 CXCL16 antibodies were analyzed using Biolight interferometry (ForteBio). All antibodies were diluted in freshly prepared running buffer. Using the capture method, antibodies were immobilized on the surface of a series of biosensors at 0.3 μg / mL. Recombinant human CXCL16 (rhCXCL16) protein (100 nM) was passed over the surface, and the binding reaction was allowed to proceed for 10 minutes. Binding data of the antibody-antigen interaction were collected on the biosensors at 25°C. The running buffer was then allowed to flow for 10 minutes to dissociate the antibodies. The binding constant (k a ) and dissociation constant (k d ) and the equilibrium binding constant (K D Of the 25 antibodies produced, five showed significant binding affinity (K D <1×10 -8 M). R 2 : A value indicating how well the fitted data correlates with the experimental data; X2: A measure of the error between the experimental data and the fitted line.

[0251] To further characterize the binding affinity of the six selected CXCL16 antibodies, a protein-based ELISA was performed. Recombinant human CXCL16 antigen (976-CX, R&D Systems, USA) was immobilized on a 96-well plate and incubated at room temperature for 2 hours. Nonspecific binding sites were blocked by overnight incubation with 1% BSA in PBS at 4°C. The plate was then washed with PBS. Various concentrations (0.001–10 nM) of humanized CXCL16 antibodies were incubated with the immobilized antigen for 2 hours at room temperature. After incubation, the plate was washed and incubated with an HRP-conjugated anti-human IgG antibody (W4031, Promega, USA) for 0.5 hours and developed using substrate solution (DY008B, R&D Systems). The results are shown in Figure 1A, indicating that the binding affinity of the humanized antibodies HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5 was significantly higher than that of the control IgG antibody.

[0252] To evaluate cross-reactivity with mouse CXCL16 antigen, the same ELISA experiment was performed using recombinant mouse CXCL16 antigen (503-CX, R&D Systems). Mouse CXCL16 antigen was immobilized on a 96-well plate by incubation at room temperature for 2 hours. Nonspecific binding sites were blocked by overnight incubation with 1% BSA in PBS at 4°C. After coating, the plate was washed with PBS. Various concentrations (0.001–10 nM) of humanized CXCL16 antibodies were incubated with the immobilized antigen for 2 hours at room temperature. After binding, the plate was washed and incubated with an HRP-conjugated anti-human IgG antibody (W4031, Promega, USA) for 0.5 hours and developed using substrate solution (DY008B, R&D Systems). The results are shown in Figure 1B. The binding affinity of the humanized antibody was similar to that of the IgG antibody. This result indicates that the humanized CXCL16 antibody specifically binds to human CXCL16. See Table 6.

[0253] [Table 6]

[0254] Note: HC0LC0 is a chimeric antibody that binds to human CXCL16; see the related disclosure in Example 7. ND indicates that no signal was detectable. "Res@3.7nM" means resolution at 3.7nM.

[0255] Example 2 Functional Assays a.Inhibition of cancer cell chemotaxis and migration

[0256] CXCR6 is the receptor for the chemokine CXCL16, and CXCR6-bearing cells migrate in response to CXCL16. Therefore, to evaluate the activity of CXCL16 antibodies in inhibiting cell migration induced by the interaction of CXCL16 with CXCR6, a transwell migration assay was performed. Polycarbonate membranes with 8 μm pore size (Corning, NY, USA) were precoated with gelatin and placed in 24-well plates. The membrane separated each well into an upper and lower chamber. CXCR6-overexpressing CHO-K1 cells were seeded in the upper chamber. The lower chamber was filled with medium containing 100 ng / mL rhCXCL16. After 4–5 h, the lower chamber was stained with 1% crystal violet solution, and the number of migrated cells in the lower chamber was counted. The results show that the humanized CXCL16 antibodies HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5 inhibited the migration of CXCR6-overexpressing CHO-K1 cells from the upper to the lower chamber. The results also show that treatment with rhCXCL16 increased the migration of CXCR6-overexpressing CHO-K1 cells to the lower chamber, while treatment with the humanized CXCL16 antibody reduced migration. See Figure 2A.

[0257] To assess whether blocking CXCL16 could lead to the inhibition of cancer cell migration, a transwell migration assay was performed. For the assay, 8-μm pore polycarbonate membranes (Corning, NY, USA) were used. The membranes were precoated with gelatin and placed in a 24-well plate. The membranes separated each well into an upper and lower chamber. CXCL16 antibody (10–100 μg / mL) was preincubated in the upper and lower chambers for 0.5 h. Thyroid cancer cells BHP10-3M (1 × 10 5 / 100 μL) and co-culture conditioned medium (0.5× co-culture-CM), respectively.

[0258] Co-culture CM is the conditioned medium obtained from the co-culture of BHP10-3M cells and THP-1 cells. BHP10-3M cells were cultured at 3 × 10 6 After 24 hours of culture, 6 × 10 THP-1 cells were seeded onto a 100 mm culture dish. 6 Additional cells were seeded. Subsequently, 10 μL of 5 mM PMA stock solution (V1171, Promega) was added to 10 mL of RPMI medium to prepare 5 μM PMA, which induced macrophage differentiation. After 24 h of incubation, conditioned medium (coculture-CM) obtained from the coculture of thyroid cancer cells and macrophages was collected and found to be rich in CXCL16. After incubating thyroid cancer cells in the upper chamber and coculture-CM in the lower chamber for 6 h, the lower chamber was stained with 1% crystal violet solution and the number of migrated cells was counted. Coculture-CM increased BHP10-3M cell migration to the lower chamber, while humanized CXCL16 antibodies HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5 reduced migration. See Figure 2B.

[0259] To assess whether blocking CXCL16 leads to the inhibition of cancer cell migration, a transwell migration assay was performed. For the assay, 8-μm pore polycarbonate membranes (Corning, NY, USA) were used. The membranes were precoated with gelatin, and inserts were placed in 24-well plates. CXCL16 antibody (10–100 μg / mL) was preincubated in the upper and lower chambers for 30 minutes. Breast cancer cells MDA-MB-231 (1 × 10) were then added to the upper and lower chambers. 5 / 100 μL) and co-culture conditioned medium (0.5× co-culture-CM), respectively.

[0260] Co-culture CM is the conditioned medium obtained from the co-culture of MDA-MB-231 and THP-1 cells. BHP10-3M cells were cultured at 3 × 10 in 10 ml of RPMI 1640 medium. 6 After 24 hours of culture, 6 × 10 THP-1 cells were seeded onto a 100 mm culture dish. 6 Additional cells were seeded. Subsequently, 10 μL of 5 mM PMA stock solution (V1171, Promega) was added to 10 mL of RPMI medium to prepare 5 μM PMA, which induced macrophage differentiation. After 24 hours of incubation, conditioned medium (coculture-CM) obtained from the coculture of thyroid cancer cells and macrophages was collected and found to be rich in CXCL16. After incubating breast cancer cells in the upper chamber and coculture-CM in the lower chamber for 6 hours, the lower chamber was stained with 1% crystal violet solution and the number of migrated cells was counted. Coculture-CM increased MDA-MB-231 cell migration to the lower chamber, while humanized CXCL16 antibody reduced it. See Figure 2C.

[0261] To assess whether blocking CXCL16 could lead to the inhibition of monocyte / macrophage migration, a transwell migration assay was performed. For the assay, 5 μm pore polycarbonate membranes (Corning, NY, USA) were used. The membranes were precoated with gelatin, and inserts were placed in 24-well plates. Humanized CXCL16 antibody (10–100 μg / mL) was preincubated in the upper and lower chambers for 30 minutes. Monocytic THP-1 cells and coculture-conditioned medium were seeded into the upper and lower chambers. After 4 hours, the lower chamber was stained with 1% crystal violet solution, and the number of migrated cells was counted. Coculture-conditioned medium increased THP-1 cell migration into the lower chamber, while the humanized CXCL16 antibody reduced it. These results suggest that the humanized CXCL16 antibody inhibits cell migration induced by the interaction between CXCL16 and CXCR6. Humanized CXCL16 antibodies inhibit the migration of cancer cells, including thyroid and breast cancer cells. Inhibition of cancer cell migration by humanized CXCL16 antibodies indicated an anti-cancer effect. See Figure 2D.

[0262] b. Activation of the intracellular Akt signaling pathway, and CXCL16-Akt signaling mediates cell migration CXCL16-CXCR6 interaction activates the intracellular Akt signaling pathway and promotes cell migration. This experiment was designed to evaluate the ability of CXCL16 antibodies to block Akt signaling induced by CXCL16-CXCR6 interaction. Cancer cells were treated with 100 ng / mL rhCXCL16 (976-CX, R&D Systems) for 30 minutes in the presence of 10 μg / mL humanized CXCL16 antibodies (HC4LC1, HC5LC1, HC5LC2, HC5LC3, or HC5LC5) or a control antibody (MAB976, R&D Systems, Minneapolis, MN). Cell lysates were collected. Anti-pAkt (dilution 1:1000, #9102; Cell Signaling), anti-Akt (dilution 1:1000, #4370; Cell Signaling), and anti-β-actin antibodies (dilution 1:10,000, STJ91464, St John's Laboratory, UK) were used. The results show that rhCXCL16 activates Akt phosphorylation in various cancer cells, including thyroid cancer cells (Figure 3B), breast cancer cells (Figure 3C), and prostate cancer cells (Figure 3D), while the CXCL16 antibodies HC4LC1, HC5LC1, HC5LC2, HC5LC3, and HC5LC5 inhibit Akt phosphorylation.

[0263] c. Inhibition of cell migration in an in vitro model of bone metastasis A two-chamber system resembling the bone metastatic niche was constructed. The lower chamber contained conditioned medium from a co-culture (CM) of cancer cells and whole bone marrow cells enriched in CXCL16 (referred to as co-culture-CM). CM from cancer cell culture alone, referred to as sCXCL16-only CM, served as a control. SCXCL16-only CM contained negligible concentrations of CXCL16. The upper chamber (using an insert) housed cells that could be recruited to the metastatic chamber. Human CD11b+ myeloid cells were treated with a CXCL16 antibody for 1 hour, and the treated cells were added to the upper chamber. After 4 hours, the insert (also known as the upper chamber) was harvested, and the cells within the insert were stained to assess the ability of the CXCL16 antibody to inhibit cell recruitment to the metastatic niche. As shown in Figures 8A and 8B, co-culture-CM (high concentration of sCXCL16) exhibited enhanced cell migration potential compared to the sCXCL16-only CM group.

[0264] d. Inhibition of tumor growth in orthotopic xenograft models, alone or in combination with paclitaxel (PTX) This example demonstrates that humanized CXCL16, alone or in combination with paclitaxel (PTX), inhibits tumor growth in an orthotopic xenograft model. To generate a mouse breast cancer model, the triple-negative breast cancer cell line MDA-MB-231 (6.5 × 10 cells) was cultured in a 200-well plate. 5T cell-deficient BALB / c nude mice were transplanted with IgG antibody (MAB006, R&D Systems) into the backs of mice lacking T cells. Starting 5 days after cell transplantation, each experimental group received anticancer drugs. The "IgG control" group received an IgG antibody (MAB006, R&D Systems) administered intravenously at 50 mcg / 20 g / day, three times a week. The "PTX" group received paclitaxel (S1150, Selleckchem) administered intraperitoneally at 10 mg / kg / mouse once a week. The "anti-CXCL16" group received an anti-CXCL16 antibody (MAB503, R&D Systems, Minneapolis, MN) administered intravenously at 50 mcg / 20 g / day, three times a week. The "PTX + anti-CXCL16" group received both paclitaxel and anti-CXCL16 antibody. Tumor size was measured on days 7, 11, 13, 15, 18, and 20. Tumor size was measured with a caliper, and tumor volume was calculated using the following formula: volume = 1 / 2 × a × b2, where a = the longest diameter of the tumor and b = the shortest diameter of the tumor. Mice were sacrificed on day 20, tumors were removed, and tumor weight was measured. The results show that the murine CXCL16 antibody significantly reduced tumor volume and weight in the mouse tumor model. The reduction in tumor volume and weight in the CXCL16 group was comparable to that in the PTX group. This suggests that the humanized CXCL16 antibody may inhibit tumor growth and have anti-cancer effects in vivo. See Figure 4A and Figure 4B.

[0265] Example 3. Effect of combined use with targeted anticancer drugs on thyroid cancer model mice 2×10 6 The anaplastic thyroid cancer cell line FRO was injected into the dorsal region of T cell-deficient nu / nu mice. Starting on day 5, when tumors formed, each experimental group was administered an anticancer drug. The control group received no anticancer drug. Experimental Group 1 received the anti-VEGF-targeted anticancer drug lenvatinib (LENVIMA®) orally at 30 mg / kg / day, five times a week. Experimental Group 2 received lenvatinib and an anti-CXCL16 therapeutic antibody intraperitoneally at 100 mcg / 20 g / day, twice a week. Tumor size was measured with calipers, and tumor volume was calculated using the following formula: volume = 1 / 2 × a × b², where a = the longest diameter of the tumor and b = the shortest diameter of the tumor.

[0266] As shown in Figure 5, in Phase I (up to day 22), when the therapeutic effect of lenvatinib was maintained, the tumor growth rates in Experimental Group 1 and Experimental Group 2 were similar. However, as the therapeutic effect of lenvatinib decreased, the rate became the same as that of the control group. In Phase II (after day 22), when tumor growth began to resume, the tumor growth rate in Experimental Group 2, which was administered with a CXCL16 therapeutic antibody (anti-CXCL16 antibody), was significantly reduced compared to Experimental Group 1, indicating a synergistic effect of the combined treatment. Example 4. Effect of combination therapy with targeted anticancer drugs and / or PD-L1 inhibitors on thyroid cancer mouse models

[0267] 5×10 6 The anaplastic thyroid carcinoma cell line TBP3743 was injected into the dorsal region of C57Bl / 6 mice. Starting on day 5, when tumors formed, each experimental group received an anticancer drug. The control group received no anticancer drug. Experimental Group 1 received oral administration of the targeted anticancer drug lenvatinib at 30 mg / kg five times per week. Experimental Group 2 received oral administration of lenvatinib and injections of an anti-mouse CXCL16 antibody at 100 mcg / 20 g / day twice per week. Experimental Group 3 received oral administration of lenvatinib and a peptide PD-L1 inhibitor (as described in KR-20190072466-A, the disclosure of which is incorporated herein by reference in its entirety) five times per week. Experimental Group 4 received oral administration of lenvatinib, five weekly injections of the PD-L1 inhibitor (peptide), and two weekly injections of an anti-CXCL16 therapeutic antibody.

[0268] From day 15 of the experiment, the combination therapy group showed better therapeutic effects than lenvatinib alone (experimental group 1), lenvatinib plus anti-CXCL16 therapeutic antibody (experimental group 2), and lenvatinib plus PD-L1 inhibitor (experimental group 3). By day 19 of the experiment, it was confirmed that the triple combination therapy group of lenvatinib, PD-L1 inhibitor, and anti-CXCL16 therapeutic antibody (experimental group 4) showed the best therapeutic effect. See Figure 6.

[0269] Example 5. Combination effects of targeted anticancer drugs and / or PD-L1 inhibitors on breast cancer mouse models 2×10 64T1 breast cancer cell lines were injected into the dorsal region of C3H mice, and anticancer drugs were administered to each experimental group starting on day 5 after tumor formation. The control group received no anticancer drug, while the experimental group received the targeted anticancer drug paclitaxel (Taxol) intraperitoneally at 10 mg / kg / day twice weekly. Experimental group 2 received paclitaxel and an anti-CXCL16 therapeutic antibody intraperitoneally at 100 mcg / 20 g / day twice weekly. Experimental group 3 received paclitaxel and a PD-L1 inhibitor (peptide) intraperitoneally at 0.1 mg / 20 g / day five times weekly. Experimental group 4 received paclitaxel, a PD-L1 inhibitor (peptide) five times weekly, and an anti-CXCL16 therapeutic antibody twice weekly.

[0270] As shown in Figure 7, experimental group 1 (taxol alone) and experimental groups 2 and 3 (two-drug combination therapy) showed no obvious therapeutic effect, but on days 17 and 19 of the experiment, the triple combination therapy of paclitaxel + PD-L1 inhibitor + anti-CXCL16 therapeutic antibody (experimental group 4) showed the best therapeutic effect.

[0271] Therefore, the CXCL16 antibody exhibits synergistic effects when combined with a targeted anticancer drug and / or a PD-L1 inhibitor, and it is expected that the CXCL16 antibody of the present invention, a targeted anticancer drug and / or an immune checkpoint inhibitor can be used in combination to prevent or treat cancer.

[0272] Example 6. CXCL16 antibodies can inhibit bone metastasis in a mouse model 1. Higher CXCL16 concentrations in bone marrow serum of zoledronic acid (ZA)-resistant bone metastases Anaplastic thyroid cancer cell line FRO (2 × 10 5) cells / 10 μL PBS) were implanted into the right tibia of 6-week-old female BALB / c nude mice. Mice were divided into a control group and a zoledronic acid (ZA)-treated group. Zoledronic acid is a drug known to be effective in treating bone metastases. The ZA-treated group received intravenous injections of 4 μg zoledronic acid (ZA) per mouse once a week starting on day 3. Bioluminescence imaging (BLI) was performed weekly. The therapeutic response to ZA varied as observed by BLI 4–5 weeks after cell implantation. Bone tumors were observed in 7 of 16 ZA-treated mice. The 9 mice that did not have bone tumors were defined as the "ZA-nonresistant (ZA_nonR)" group, and the 7 mice that had bone tumors were designated as the "ZA-resistant (ZA-R)" group. The ZA_nonR group showed negligible BLI signals in the tibia until week 7, while the ZA-R group showed tumor growth from weeks 4–5 to 7.

[0273] Five weeks after cell transplantation, mice were sacrificed during the early stage of ZA-R. Bone marrow serum was collected and CXCL16 concentrations were measured using ELISA. The results show that CXCL16 concentrations in ZA-R were significantly higher than those in the ZA-nonR and control groups. CXCL16 concentrations in ZA-nonR were lower than those in the control group. See Figures 9B-9C. The results indicate that ZA resistance in bone metastases is associated with high CXCL16 concentrations. 2. Anti-CXCL16 antibody reduced tumor growth in bone metastases

[0274] Anaplastic thyroid cancer cell line FRO (2 × 10 5) cells / 10 μL PBS) were implanted into the right tibia of 6-week-old female BALB / c nude mice. On day 3, the mice were divided into a control group, a CXCL16 antibody (aCXCL16) group, and a zoledronic acid (ZA) group. The CXCL16 group received an intraperitoneal injection of 25 μg / mouse of anti-CXCL16 antibody (rat IgG against human CXCL16 protein, with CDRs of SEQ ID NOs: 12-17) once a week. The ZA-treated group received an intravenous injection of 4 μg / mouse of zoledronic acid (ZA) once a week. Bioluminescence imaging (BLI) was performed weekly. As shown in Figures 10B and 10C, on day 21, the BLI signal in the CXCL16 group was significantly lower than that in the control group but comparable to that in the ZA group. These results indicated that the anti-CXCL16 antibody reduced tumor growth in bone metastases. The results also suggest that CXCL16 antibodies inhibit the progression of bone metastases to a similar extent as ZA, and that CXCL16 antibodies have potential therapeutic efficacy in inhibiting bone metastases.

[0275] 3. aCXCL16 treatment reduced tumor growth in ZA-resistant bone metastases Anaplastic thyroid cancer cell line FRO (2 × 10 5 ) cells / 10 μL PBS) were implanted into the right tibia of 6-week-old female BALB / c nude mice. On day 3, ZA was intravenously injected at 4 μg / mouse once a week. On day 28, ZA-resistant mice, mice that had bone tumors despite ZA treatment, were selected. They were divided into ZA and ZA-aCXCL16 groups. The ZA group was injected with ZA alone, while the ZA + aCXCL16 group was injected with both ZA and an anti-human CXCL16 antibody (rat IgG with CDRs of SEQ ID NOS: 12-17). The anti-CXCL16 antibody was intraperitoneally injected at 25 μg / mouse once a week for 2 weeks from day 28 to day 42. Bioluminescence imaging (BLI) was performed weekly. As shown in Figures 11A-11C, on day 42, the BLI signal in the ZA + aCXCL16 group was significantly lower than that in the ZA group. These results indicated that anti-CXCL16 antibodies reduced the growth of ZA-resistant bone tumors.

[0276] Example 7. Generation of humanized CXCL16 antibodies 1. Generation of Chimeric Antibody HC0LC0 An anti-human CXCL16 antibody produced in rats was obtained. The antibody was sequenced using mass spectrometry and determined to have the CDR sequences disclosed in SEQ ID NOS: 12-17. A variant sequence of the rat antibody was designed by replacing the constant region sequence of the rat antibody with the constant region sequence of a human IgG1 antibody, as further described below. The chimeric antibody is designated HC0LC0.

[0277] DNA sequences (SEQ ID NO: 33 and SEQ ID NO: 35) encoding the variant antibody heavy and light chains (SEQ ID NO: 32 and SEQ ID NO: 34, respectively) were synthesized and cloned into the mammalian transient expression plasmid pETEv2 (TGEX-HC-hG1, TGEX-LC-hk, pOpti VEC, pCDNA3.3, pTRIOz, pFUSECHig, and pFUSE-CLig may also be used). Mouse antibody signal peptides MGWTLVFLFLLSVTAGVHS (SEQ ID NO: 18) and MVSSAQFLGLLLLCFQGTRC (SEQ ID NO: 19), respectively, were used to express the chimeric antibody heavy and light chains, as these peptides can result in higher levels of expression in CHO cells.

[0278] The variant antibodies were expressed using a CHO cell-based transient expression system, and the resulting antibody-containing cell culture supernatant was collected by centrifugation and filtration. These variant antibodies were purified from the cell culture supernatant by affinity chromatography (using a state-of-the-art AKTA chromatography device). The purified antibodies were buffer-exchanged into phosphate-buffered saline. Finally, the chimeric antibody HC0LC0 was purified.

[0279] 2. Humanization of chimeric antibody HC0LC0 To optimally preserve the CDR loop structure, the chimeric antibody HC0LC0 variable domain was sequenced and the CDRs identified using a combination of the IMGT and Kabat antibody numbering systems. The IMGT and Kabat antibody numbering systems are well known, see, for example, Lefranc, M.-P. et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27, 55-77 (2003) PMID: 12477501 LIGM: 268; and Dunbar J and Deane CM. ANARCI: Antigen receptor numbering and receptor classification. Bioinformatics (2016).

[0280] The VH domain of the chimeric antibody HC0LC0 had the following sequence, excluding the signal peptide sequence:

[0281] AVQLVESGGGLVQGPKESLKISCAAS GFTFSNAVMN WVRGAPGKGLEWVA RIRTKPNNYATFYADSVKG RFTFSRDDSKSMVYLQMDNLKTEDTAMYYC TAWTTEGIY WGQGTLVTVSS (SEQ ID NO: 36) (CDR residues are underlined).

[0282] The closest human germline gene V region is Homo sapiens IGHV3-73*01:

[0283] EVQLVESGGGLVQPGGSLKLSCAAS GFTFSGSAMH WVRQGASGKGLEWVG RIRSKANSYATAYAASVKG RFTISRDDSKNTAYLQMNSLKTEDTAVYYC TR (SEQ ID NO: 37) (CDR residues are underlined)

[0284] For comparison with rodent VH domains, a database of human IgG sequences was searched using the BLAST search algorithm, and candidate human variable domains were selected from the top 200 BLAST results. Three human variable domains were selected as acceptor frameworks based on a combination of framework homology, preservation of key framework residues, and canonical loop structures. The CDRs of the VH of HC0LC0 were then grafted onto these acceptor frameworks to create humanized VH variants. In some cases, back mutations were added to make the antibody more similar to the parent antibody (rat CXCL16 antibody) while maintaining close similarity to the germline sequence. CDR residues are underlined.

[0285] >VH1 (SEQ ID NO: 1) QVQLVESGGGVVQPGGSLRLSCAAS GFTFSNAVMN WVRQAPGKGLEWVA RIRTKPNNYATFYADSVKG RFTFSRDNSKTMLYLQMNSLRAEDTAVYYC TAWTTEGIY WGQGTLVTVSS

[0286] >VH2 (SEQ ID NO: 2) AVQLVESGGGLVQPGGSLKISCAAS GFTFSNAVMN WVRQASGKGLEWVG RIRTKPNNYATFYADSVKG RFTISRDDSKNTAYLQMNSLKTEDTAMYYC TAWTTEGIY WGQGTLVTVSS

[0287] >VH3 (SEQ ID NO: 3) EVQLVESGGGLVQPGGSLKLSCAAS GFTFSNAVMN WVRQAPGKGLEWVG RIRTKPNNYATFYADSVKG RFTFSRDDSKNTAYLQMNILKTEDTAMYYC TAWTTEGIY WGQGTTVTVSS

[0288] >VH4 (SEQ ID NO: 4)

[0289] EVQLVESGGGLVKPGGSLRLSCAAS GFTFSNAVMN WVRQAPGKGLEWVA RIRTKPNNYATFYADSVKG RFTFSRDDSKNTLYLQMNSLKTEDTAVYYC TAWTTEGIY WGQGTTVTVSS

[0290] >VH5 (SEQ ID NO: 5)

[0291] EVQLVESGGGLVQPGGSLKLSCAAS GFTFSNAVMN WVRQASGKGLEWVA RIRTKPNNYATFYADSVKG RFTFSRDDSKNTAYLQMNSLKTEDTAMYYC TAWTTEGIY WGQGTLVTVSS

[0292] Similarly, the variable domain of the light chain of chimeric antibody HC0LC0 was determined using a combination of IMGT and Kabat antibody numbering systems for optimal retention of CDR-loop conformations.

[0293] The VL domain of the chimeric antibody HC0LC0 had the following sequence, excluding the signal peptide sequence:

[0294] DIVMTQSPSSLAVSAGETVTINC KSSQSLLYSGNQKNYLA WYQQKPGQSPKLLIY WASTRQS GVPDRFIGSGSGTDFLTISSVQAEDLAIYYC QQYYDTPWT FGGGTKLELK

[0295] CDR residues highlighted in yellow were identified using the IMGT numbering system, and CDR residues highlighted in red were identified using the Kabat numbering system.

[0296] The closest human germline gene V region is Homo sapiens IGKV4-1*01:

[0297] DIVMTQSPDSLAVSLGERATINC KSSQSVLYSSNNKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFLTISSLQAEDCVAVYYC QQYYSTP

[0298] For comparison with the rat VL domain, a database of human IgK sequences was searched using the BLAST search algorithm, and candidate human variable domains were selected from the top 200 BLAST results. These candidates were narrowed down to two based on a combination of framework homology, preservation of key framework residues, and canonical loop structure. Grafting the VL CDRs of HC0LC0 onto these acceptor frameworks resulted in humanized variants. VH1-5 contains back mutations to make the antibody more similar to the parent antibody (rat CXCL16 antibody) while maintaining close similarity to the human germline sequence.

[0299] >VL1 (SEQ ID NO: 6) DIVMTQSPDSLAVSAGETVTINC KSSQSLLYSGNQKNYL AWYQQKPGQSPKLLIY WASTRQS GVPDRFSGSGSGTDFTLTISSVQAEDVAVYYC QQYYDTPWT FGGGTKVEIK

[0300] >VL2 (SEQ ID NO: 7)

[0301] DIVMTQSPSSLAVSLGETATINC KSSQSLLYSGNQKNYLA WYQQKPGQPPKLLIY WASTRQS GVPDRFTGSGAGTDFTLTISSVQAEDLAIYYC QQYYDTPWT FGGGTKVEIK

[0302] >VL3 (SEQ ID NO: 8)

[0303] DIQMTQSPSSLSASVGDRVTITC KSSQSLLYSGNQKNYLA WYQQKPGQSPKLLIY WASTRQS GVPDRFSGSGSGTDFLTISSLQAEDFATYYC QQYYDTPWT FGGGTKVEIK

[0304] >VL4 (SEQ ID NO: 9) DIQMTQSPSSLSASVGDRVTITC KSSQSLLYSGNQKNYLA WYQQKPGQSPKLLIY WASTRQS GVPSRFSGSGSGTDFLTISSLQPEDLAIYYC QQYYDTPWT FGGGTKLELK

[0305] >VL5 (SEQ ID NO: 10) DIVMTQSPSSLAVSAGERATINC KSSQSLLYSGNQKNYLA WYQQKPGQSPKLLIY WASTRQS GVPDRFIGSGSGTDFLTISSLQAEDVAIYYC QQYYDTPWT FGGGTKLELK

[0306] 3. Humanization verification Humanized variants were checked to confirm that they were humanized according to the WHO definition of a humanized antibody. See Ehrenmann F., Kaas Q. and Lefranc M.-P. 2010 IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF Nucleic Acids Res. 38, D301-307. When analyzed as a whole, the variable domains of the humanized chains have V-region amino acid sequences that are closer to those of humans than to those of other species (assessed using the IMMUNOGENETICS INFORMATION SYSTEM® (IMGT®) DomainGapAlign tool). See Table 7.

[0307] [Table 7]

[0308] 4. T Cell Epitope Screening Presentation of peptide sequences in the groove of MHC class II molecules triggers CD4+ T cell activation and immunogenic responses. To reduce this response, therapeutic proteins can be designed to avoid incorporating "T cell epitopes" that can activate T cells by reducing their binding affinity to MHC class II molecules.

[0309] The VH and VL sequences of the original rat antibody and the humanized variant sequences were screened for MHC II-binding peptides, and an in silico algorithm was used to confirm that the humanization process removed peptide sequences with high affinity. The following eight alleles represent over 99% of the world's population and are the standard allele set used to predict MHC class II epitopes: DRB1*01:01; DRB1*03:01; DRB1*04:01; DRB1*07:01; DRB1*08:02; DRB1*11:01; DRB1*13:02; DRB1*15:01. See Wang et al., Peptide binding predictions for HLA DR,DP and DQ molecules.BMC Bioinformatics.11:568; Gonzalez-Galarza FF, Nucleic Acid Research, 39(2011), D913-D919; Greenbaum J, et al., Immunogenetics 63(6):325-35.

[0310] The above description of the present invention is for illustrative purposes only, and those skilled in the art will recognize that the present invention may be easily modified into other specific forms without changing the technical spirit or essential characteristics of the present invention. Therefore, the above-described embodiments are intended to be illustrative in all respects and not restrictive.

Claims

1. An anti-CXCL16 antibody or antigen-binding fragment thereof that binds to CXCL16, The anti-CXCL16 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 12, an HCDR2 of SEQ ID NO: 13, and an HCDR3 of SEQ ID NO: 14, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 15, an LCDR2 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO:

17. An anti-CXCL16 antibody or an antigen-binding fragment thereof.

2. It includes any one selected from the group consisting of the following (i) to (v): (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6; (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6; (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10 The anti-CXCL16 antibody or antigen-binding fragment thereof according to claim 1.

3. Competes with CXCR6 (SEQ ID NO: 21) for binding to CXCL16 The anti-CXCL16 antibody or antigen-binding fragment thereof according to claim 1.

4. The antigen-binding fragment is selected from the group consisting of Fv, Fab, Fab', F(ab')2, scFv, diabody, and combinations thereof. The anti-CXCL16 antibody or antigen-binding fragment thereof according to claim 1.

5. The anti-CXCL16 antibody comprises an Fc region. The anti-CXCL16 antibody or antigen-binding fragment thereof according to claim 1.

6. The Fc region contains mutations to increase serum half-life. The anti-CXCL16 antibody or antigen-binding fragment thereof according to claim 5.

7. the mutations are selected from the group consisting of M252Y / S254T / T256E, T250Q / M428L, N434A, N434H, T307A / E380A / N434A, M428L / N434S, M252Y / M428L, D259I / V308F, N434S, V308W, V308Y, and V308F. The anti-CXCL16 antibody or antigen-binding fragment thereof according to claim 6.

8. It is a humanized antibody, a chimeric antibody, a multispecific antibody, or a bispecific antibody. The anti-CXCL16 antibody or antigen-binding fragment thereof according to claim 1.

9. An immune complex comprising the anti-CXCL16 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and a cytotoxic agent.

10. An isolated nucleic acid molecule encoding an anti-CXCL16 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

11. An expression vector comprising the isolated nucleic acid molecule of claim 10.

12. A host cell transformed with the expression vector of claim 11.

13. A pharmaceutical composition comprising the anti-CXCL16 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition for the prevention or treatment of cancer, comprising the anti-CXCL16 antibody or its antigen-binding fragment according to any one of claims 1 to 8.

15. The pharmaceutical composition induces an immune response. The pharmaceutical composition of claim 14.

16. The cancer is a cancer caused by increased CXCL16 expression. The pharmaceutical composition of claim 14.

17. the cancer is breast cancer, thyroid cancer, cervical cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, colon cancer, colorectal cancer, bone cancer, skin cancer, head cancer, cervical cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, liver cancer, brain tumor, bladder cancer, blood cancer, stomach cancer, perianal cancer, breast cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis cancer, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, or brain stem glioma The pharmaceutical composition of claim 14.

18. The cancer is thyroid cancer or breast cancer.

18. The pharmaceutical composition of claim 17.

19. The breast cancer is triple-negative breast cancer.

19. The pharmaceutical composition of claim 18.

20. Administered intravenously The pharmaceutical composition of claim 14.

21. With chemotherapy or radiation therapy The pharmaceutical composition of claim 14.

22. The chemotherapy is administration of paclitaxel.

22. The pharmaceutical composition of claim 21.

23. Further includes agents that target immunological checkpoint antigens The pharmaceutical composition of claim 14.

24. The drug is an anti-PD-1 antibody.

24. The pharmaceutical composition of claim 23.

25. A pharmaceutical composition for inhibiting tumor metastasis, comprising the anti-CXCL16 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

26. The tumor metastasis is bone metastasis. A pharmaceutical composition for inhibiting tumor metastasis according to claim 25.

27. 1. A method of using an anti-CXCL16 antibody or antigen-binding fragment thereof as a selection criterion for determining subjects who are candidates for treatment with an anti-CXCL16 antibody or antigen-binding fragment thereof, comprising:

9. The method of claim 8, further comprising measuring the level of CXCL16 expression in a biological sample isolated from a subject using the anti-CXCL16 antibody or antigen-binding fragment thereof according to claim 1. A method for using an anti-CXCL16 antibody or an antigen-binding fragment thereof.

28. 1. A method for producing an anti-CXCL16 antibody or antigen-binding fragment thereof, comprising: Culturing the host cell of claim 12. A method for producing an anti-CXCL16 antibody or an antigen-binding fragment thereof, comprising:

29. A pharmaceutical composition comprising the immunoconjugate of claim 9 and a pharmaceutically acceptable carrier.

30. A pharmaceutical composition for the prevention or treatment of cancer, comprising the immune complex described in claim 9.

31. A pharmaceutical composition that inhibits tumor metastasis, comprising the immune complex described in claim 9.

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