阅读说明：本技术 抗人vsig4抗体及其应用 (Anti-human VSIG4 antibodies and uses thereof ) 是由 权炳世 金惠贞 黄善姬 李重远 李承炫 任宣佑 崔镇敬 孙贤台 朴赫俊 于 2019-09-30 设计创作，主要内容包括：提供了结合VSIG4的抗体及其抗原结合片段。涉及抗体的各种体外和体内方法和组合物。方法包括使用结合VISG4的抗体或抗原结合片段预防和/或治疗性处理癌症。(Antibodies and antigen-binding fragments thereof that bind VSIG4 are provided. Various in vitro and in vivo methods and compositions involving antibodies. The methods comprise prophylactic and/or therapeutic treatment of cancer using antibodies or antigen-binding fragments that bind to VISG 4.)

1. An isolated humanized antibody or antigen binding fragment thereof, comprising:

a. heavy chain CDR1 comprising amino acid sequence SEQ ID NO. 17, heavy chain CDR2 comprising amino acid sequence SEQ ID NO. 18, heavy chain CDR3 sequence comprising amino acid sequence SEQ ID NO. 19; and

b. light chain CDR1 comprising amino acid sequence SEQ ID NO. 20, light chain CDR2 comprising amino acid sequence SEQ ID NO. 21, light chain CDR3 comprising amino acid sequence SEQ ID NO. 22 or SEQ ID NO. 23.

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises any one of:

a. a heavy chain variable domain comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SEQ ID NO 2, 6, 14 or 16;

b. a light chain variable domain comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SEQ ID NO 4, 8, 10 or 12; or

c. A heavy chain variable domain comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SEQ ID NO 2, 6, 14 or 16 and a light chain variable domain comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SEQ ID NO 4, 8, 10 or 12.

3. The antibody or antigen-binding fragment of any one of claims 1-2, wherein the antibody or antigen-binding fragment comprises any one of:

a. a heavy chain variable domain comprising the amino acid sequence SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 14 or SEQ ID NO 16;

b. a light chain variable domain comprising the amino acid sequence SEQ ID NO 4, SEQ ID NO 8, SEQ ID NO 10 or SEQ ID NO 12; or

c. A heavy chain variable domain comprising the amino acid sequence SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 14 or SEQ ID NO 16 and a light chain variable domain comprising the amino acid sequence SEQ ID NO 4, SEQ ID NO 8, SEQ ID NO 10 or SEQ ID NO 12.

4. The antibody or antigen-binding fragment of claim 1, wherein the light chain CDR3 of the antibody comprises the amino acid sequence of SEQ ID No. 23.

5. The antibody or antigen binding fragment of claim 2, wherein the antibody or antigen binding fragment comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO 2 and a light chain variable domain comprising the amino acid sequence SEQ ID NO 4.

6. The antibody or antigen binding fragment of claim 2, wherein the antibody or antigen binding fragment comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 8.

7. The antibody or antigen binding fragment of claim 2, wherein the antibody or antigen binding fragment comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 10.

8. The antibody or antigen binding fragment of claim 2, wherein the antibody or antigen binding fragment comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 12.

9. The antibody or antigen binding fragment of claim 2, wherein the antibody or antigen binding fragment comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO. 14 and a light chain variable domain comprising the amino acid sequence SEQ ID NO. 10.

10. The antibody or antigen binding fragment of claim 2, wherein the antibody or antigen binding fragment comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO 16 and a light chain variable domain comprising the amino acid sequence SEQ ID NO 12.

11. The antibody or antigen-binding fragment of any one of claims 1-10, wherein the antibody or antigen-binding fragment has a binding affinity (K) for human V-Set and immunoglobulin domain 4(VSIG4) -containing molecules_D) Is 1x10^-7-1x10^-9。

12. The antibody or antigen-binding fragment of any one of claims 1-11, wherein the antibody or antigen-binding fragment has a binding affinity (K) for VSIG4 molecule_D) Is about 7.156x10^-8-about 7.636x10^-9。

13. The antibody or antigen-binding fragment of any one of claims 1-11, wherein the antibody or antigen-binding fragment has a binding affinity (K) for VSIG4 molecule_D) Is about 7.156x10^-8About 7.636x10^-9About 7.952x10^-9About 8.226x10^-9Or about 8.688x10^-9。

14. A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1-13.

15. A recombinant vector comprising the nucleic acid molecule of claim 14.

16. The recombinant vector of claim 15, wherein the nucleic acid molecule of claim 14 is operably linked to a promoter.

17. The recombinant vector of claim 15 or 16, wherein said vector comprises two separate vectors, each comprising nucleic acid sequences corresponding to the heavy and light chains of said antibody or antigen-binding fragment.

18. A host cell comprising the nucleic acid molecule of claim 14 or the recombinant vector of any one of claims 15-17.

19. The host cell of claim 18, wherein the host cell is a mammalian cell, a yeast cell, or a bacterial cell.

20. The host cell of claim 19, which is a cell selected from the group consisting of: escherichia coli, Pichia pastoris, Sf9, COS, HEK293, Expi293, CHO-S, CHO-DG44, CHO-K1 and mammalian lymphocytes.

21. The host cell of claim 20, wherein the host cell is an Expi293 cell.

22. A pharmaceutical composition comprising:

the antibody or antigen-binding fragment of any one of claims 1-13, the nucleic acid molecule of claim 14, the recombinant vector of any one of claims 15-17, or the host cell of any one of claims 18-21; and a pharmaceutically acceptable carrier.

23. A method of treating a subject in need thereof, the method comprising the steps of:

a. administering to said subject a composition comprising or delivering the antibody or antigen-binding fragment of any one of claims 1-13, the nucleic acid molecule of claim 14, the nucleic acid molecule of claim

15-17 or the host cell of any one of claims 18-21, thereby treating a disease or disorder.

24. The method of claim 23, wherein the subject has or is at risk of developing cancer.

25. The method of claim 24, wherein the cancer is selected from: bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

26. The method of any one of claims 23-25, wherein the subject has received or will receive one or more additional anti-cancer treatments such that the subject receives both treatments, the additional anti-cancer treatments selected from: ionizing radiation, chemotherapeutic agents, antibody substances and cell-based therapies.

27. The method of claim 26, wherein the one or more additional anti-cancer treatments comprise an immune checkpoint inhibitor, IL-12, GM-CSF, an anti-CD 4 substance, cisplatin, fluorouracil, doxorubicin, irinotecan, paclitaxel, an indoleamine 2, 3-dioxygenase-1 (IDOl) inhibitor, or cyclophosphamide.

28. A method for increasing cytokine or chemokine secretion in M2 macrophages, comprising:

a. contacting the M2 macrophage with the antibody or antigen-binding fragment of any one of claims 1-13.

29. Induced CD8⁺A method of T cell proliferation, the method comprising:

a. contacting the M2 macrophage with the antibody or antigen-binding fragment of any one of claims 1-13; and

b. co-incubation of the M2 macrophages with CD8⁺T cells.

30. A method of converting M2 macrophages to M1 macrophages, the method comprising: contacting the M2 macrophage with the antibody or antigen-binding fragment of any one of claims 1-13.

Technical Field

The present disclosure relates to antibodies And antibody binding fragments that bind to V-Set And Immunoglobulin Domain 4-Containing (V-Set And Immunoglobulin Domain binding 4, VSIG4)

Background

Cancer remains one of the leading causes of death in the world to date. Recent statistics have reported that 13% of the global population dies from cancer. According to the estimate of the international agency for research on cancer (IARC), 1410 ten thousand new cancer cases and 820 ten thousand cancer deaths were found worldwide in 2012. By 2030, the global burden is expected to increase to 2170 new cancer cases and 1300 cancer deaths due to population growth and aging and exposure to risk factors such as smoking, unhealthy diet and lack of physical activity. In addition, the pain and medical expense of cancer treatment leads to a reduction in the quality of life of cancer patients and their families. It is clear that cancer is a disease for which better treatment is urgently sought.

Macrophages are multifunctional antigen presenting cells that play a central role in our immune system and are involved in cancer biology. In the case of cancer, tumor-associated macrophages (TAMs) infiltrate malignant tumor tissue and are known to be biologically associated with cancer, and affect tumor progression. TAMs can be described as being divided into two categories: m1 and M2. M1 macrophages were observed to have pro-inflammatory and cytotoxic (anti-tumor) functions, while M2 macrophages had anti-inflammatory (pro-tumor) effects and promoted wound healing.

Consistent with these functions, TAMs, especially macrophages with the M2 phenotype, are closely associated with a poor clinical prognosis for many malignancies. Infiltrated TAMs themselves or polarized pathways of TAMs are considered as new therapeutic targets for the treatment of malignancies.

Disclosure of Invention

The present disclosure relates, at least in part, to antibodies and fragments thereof that bind to VSIG4(V-Set and immunoglobulin domain 4-containing; also referred to as CRIg or Z39Ig), and methods of using such antibodies and antigen binding fragments to treat cancer, induce cytokine and/or chemokine secretion in macrophages, and convert M2 macrophages to M1 macrophages.

In one aspect, the invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising: (a) heavy chain CDR1 comprising amino acid sequence SEQ ID NO. 17, heavy chain CDR2 comprising amino acid sequence SEQ ID NO. 18, heavy chain CDR3 sequence comprising amino acid sequence SEQ ID NO. 19; and (b) a light chain CDR1 comprising the amino acid sequence SEQ ID NO:20, a light chain CDR2 comprising the amino acid sequence SEQ ID NO:21, and a light chain CDR3 comprising the amino acid sequence SEQ ID NO:22 or SEQ ID NO: 23.

In some embodiments, an antibody or antigen-binding fragment described herein can include one of the following: (a) a heavy chain variable domain comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SEQ ID NO 2, 6, 14 or 16; (b) a light chain variable domain comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SEQ ID NO 4, 8, 10 or 12; or (c) a heavy chain variable domain comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SEQ ID NO 2, 6, 14 or 16 and a light chain variable domain comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence SEQ ID NO 4, 8, 10 or 12.

In some embodiments, an antibody or antigen-binding fragment described herein can include one of the following: a heavy chain variable domain comprising the amino acid sequence SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 14 or SEQ ID NO 16, a light chain variable domain comprising the amino acid sequence SEQ ID NO 4, SEQ ID NO 8, EQ ID NO 10 or SEQ ID NO 12, or a heavy chain variable domain comprising the amino acid sequence SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 14 or SEQ ID NO 16 and a light chain variable domain comprising the amino acid sequence SEQ ID NO 4, SEQ ID NO 8, SEQ ID NO 10 or SEQ ID NO 12.

In some embodiments, an antibody or antigen-binding fragment thereof described herein can include light chain CDR3 comprising amino acid sequence SEQ ID No. 23.

In some embodiments, an antibody or antigen-binding fragment thereof described herein can comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID No. 2 and a light chain variable domain comprising the amino acid sequence of SEQ ID No. 4. In some embodiments, an antibody or antigen-binding fragment thereof described herein can comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID No. 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID No. 8. In some embodiments, an antibody or antigen-binding fragment thereof may comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID No. 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID No. 10. In some embodiments, an antibody or antigen-binding fragment thereof may comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID No. 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID No. 12. In some embodiments, the antibodies or antigen binding fragments thereof described herein can comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID No. 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID No. 10. In some embodiments, the antibodies or antigen binding fragments thereof described herein can comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID No. 16 and a light chain variable domain comprising the amino acid sequence of SEQ ID No. 12.

In some embodiments, the antibodies or antigen binding fragments described herein have a binding affinity (K) for human V-Set and immunoglobulin domain 4(VSIG4) -containing molecules_D) Is 1x10^-7-1x10^-9. In some embodiments, the binding affinity (K) of an antibody or antigen binding fragment described herein to a VSIG4 molecule_D) Is about 7.156x10^-8-about 7.636x10^-9. In some embodiments, the binding affinity (K) of an antibody or antigen binding fragment described herein to a VSIG4 molecule_D) Is about 7.156x10^-8About 7.636x10^-9About 7.952x10^-9About 8.226x10^-9Or about 8.688x10^-9。

In another aspect, the invention relates to a nucleic acid molecule encoding any one of the antibodies or antigen-binding fragments described herein.

In another aspect, the invention relates to a recombinant vector comprising any one of the nucleic acids described herein. In some embodiments, a recombinant vector described herein comprises a nucleic acid molecule described herein operably linked to a promoter. In some embodiments, the recombinant vector comprises two separate vectors, each comprising nucleic acid sequences corresponding to the heavy and light chains of an antibody or antigen-binding fragment provided herein.

In another aspect, the invention provides a host cell comprising a nucleic acid molecule or recombinant vector described herein. In some embodiments, the host cell is a mammalian cell, a yeast cell, or a bacterial cell. In some embodiments, the host cell is selected from the group consisting of: escherichia coli, Pichia pastoris, Sf9, COS, HEK293, Expi293, CHO-S, CHO-DG44, CHO-K1 and mammalian lymphocytes. In some embodiments, the host cell is an Expi293 cell.

In another aspect, the present invention relates to a pharmaceutical composition comprising: any one of the antibodies or antigen-binding fragments described herein, any one of the nucleic acid molecules described herein, any one of the recombinant vectors described herein, or any one of the host cells described herein; and a pharmaceutically acceptable carrier.

For another example, in another aspect, the invention relates to a method of treating a subject in need thereof, the method comprising the steps of: (a) administering to a subject a composition comprising or delivering any one of the antibodies or antigen-binding fragments described herein, any one of the nucleic acid molecules described herein, any one of the recombinant vectors described herein, or any one of the host cells described herein, thereby treating a disease or disorder. In certain embodiments, the subject has or is at risk of developing cancer. In certain embodiments, the cancer is selected from: bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

In some embodiments, one or more additional anti-cancer treatments selected from the group consisting of ionizing radiation, chemotherapeutic agents, antibody-based drugs, and cell-based therapies have been or will be administered to the subject such that the subject receives both treatments. For example, in some embodiments, the one or more additional anti-cancer treatments comprise: an immune checkpoint inhibitor, IL-12, GM-CSF, an anti-CD 4 agent, cisplatin, fluorouracil, doxorubicin, irinotecan, paclitaxel, an indoleamine 2, 3-dioxygenase 1(IDO1) inhibitor or cyclophosphamide.

In another aspect, the invention also relates to a method of increasing cytokine or chemokine secretion in an M2 macrophage, comprising contacting an M2 macrophage with any one of the antibodies or antigen binding fragments described herein.

In another aspect, the invention relates to inducing CD8⁺A method of T cell proliferation, the method comprising: (a) contacting M2 macrophages with any one of the antibodies or antigen binding fragments described herein; and (b) co-incubation of M2 macrophages with CD8⁺T cells

In another aspect, the invention also relates to a method of converting M2 macrophages to M1 macrophages, the method comprising contacting M2 macrophages with any one of the antibodies or antigen binding fragments described herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Drawings

FIG. 1 is a graph illustrating how administration of anti-VSIG 4 antibodies to macrophages results in conversion of M2 macrophages to M1 macrophages, resulting in CD8⁺Schematic representation of the mechanism of T cell proliferation and subsequent cancer inhibition.

Figure 2A shows a sequence alignment of VSIG4 with various human B7 protein families.

Figure 2B is a phylogenetic tree showing the evolutionary relationships between VSIG4 and various human B7 protein families.

Fig. 3A and 3B show the correlation of VSIG4 mRNA expression with various genes in tumor tissue.

Figure 4A is an HPLC diagram showing the protein profile of EU103.2 antibody.

Fig. 4B is surface plasmon resonance data for EU103.2 antibody binding to VSIG 4.

Figure 5 shows light microscopy images of M1 and M2 macrophages (top left and top right panels, respectively), and FACS analysis data showing VSIG4 expression in M1 and M2 macrophages.

Figure 6 is a panel of graphs showing the induction of pro-inflammatory cytokines and chemokines by EU103.2 in M1 and M2 macrophages.

Figure 7 is a set of FACS data showing reduced CD163 expression in EU103.2 treated M2 macrophages.

FIG. 8 is a set of FACS data (first six columns) and quantification of these data (last column) showing CD8 when⁺CD8 in T cells co-cultured with EU 103.2-treated M2 macrophages⁺Induction of T cell proliferation.

Figure 9 is a set of FACS data showing VSIG4 expression in macrophages isolated from the peritoneal fluid of ovarian cancer patients.

FIG. 10 is a set of FACS data showing CD8 when co-cultured with EU 103.2-treated macrophages isolated from ovarian cancer patients⁺Induction of T cell proliferation.

FIG. 11 is a graph showing CD8 when co-cultured with anti-EU 103.2 treated macrophages isolated from ovarian cancer patients⁺Induction of T cell proliferation.

Fig. 12 is a set of microscope images of M1 and M2 macrophages showing the morphological differences after conversion of M2 macrophages to M1 macrophages by EU103.2 antibody.

FIG. 13 is a set of FACS data showing the blockade of CD8⁺Interaction between cells and VSIG4 enhanced CD8⁺Proliferation of T cells.

FIGS. 14A and 14B are a set of graphs showing enhancement of CD8 by preventing inhibition of THP-1 cells⁺And (5) cell proliferation.

Figures 15A-15C are a set of graphs showing the anti-tumor activity of anti-VSIG 4 antibodies in three different mouse tumor models.

Figure 16 is a set of graphs showing the anti-tumor activity of anti-VSIG 4 antibodies in a VSIG4 knockout mouse model.

Figure 17A is a set of FACS data showing the activation state of MDSCs and T cells in TDLN in the absence of VSIG4 signaling.

Figure 17B is a set of graphs showing the activation state of MDSCs and T cells in TDLN in the absence of VSIG4 signaling.

Figure 17C is a set of graphs showing the activation state of MDSCs and T cells in TDLN in the absence of VSIG4 signaling.

Figure 17D is a set of graphs showing the activation state of MDSCs and T cells in TDLN in the absence of VSIG4 signaling.

Figure 18A is a graph showing inhibition of tumor growth in the absence of VSIG4 signaling.

Figure 18B is a set of histological slides showing inhibition of tumor growth in the absence of VSIG4 signaling.

Figure 19 is a graph showing the anti-tumor activity of anti-VSIG 4 antibodies in a humanized mouse model.

FIG. 20 is a graph illustrating how administration of EU103.2 antibody to macrophages results in the conversion of M2 macrophages to M1 macrophages, resulting in CD8⁺Schematic representation of the mechanism of T cell proliferation.

Fig. 21A and 21B are schematic diagrams showing expression vectors for cloning and expressing humanized anti-VSIG 4 antibodies.

Fig. 22 is an HPLC chart showing the protein profile of a1 antibody (EU103_ T01.01).

Fig. 23 is an HPLC chart showing the protein profile of a2 antibody (EU103_ T01.02).

Fig. 24 is an HPLC chart showing the protein profile of the a1.3 antibody (EU103_ T01.01S).

Fig. 25 is an HPLC chart showing the protein profile of the a2.3 antibody (EU103_ T01.02S).

Fig. 26A is a set of FACS data showing reduced CD163 expression in M2 macrophages treated with a1 or a2 antibodies.

Fig. 26B is a graph showing reduction of CD163 expression in M2 macrophages treated with a1 or a2 antibody.

Fig. 27 is a set of graphs showing the reduction of M2-type cytokines and chemokines in M2 cells treated with a1 or a2 antibodies.

Figure 28 is a set of FACS data showing increased CD86 expression and decreased CD163 expression in M2 macrophages treated with a1 or a2 antibodies.

Fig. 29 is a set of graphs showing the increase in type M1 cytokines/chemokines in M2 macrophages treated with either a1 or a2 antibodies.

Fig. 30 is a graph showing data from a chemotaxis assay measuring the chemotactic ability of macrophages after conversion of M2 to M1 macrophages by a2 antibody.

FIGS. 31A-31C show the anti-tumor effect of the A1 and A2 antibodies in a humanized mouse model.

FIGS. 32A-32C show macrophage transformation in vivo with the A2 antibody.

Figures 33A and 33B show macrophage transformation of a2 antibody in vivo by analyzing the effect of M2 macrophage transformation to M1 macrophage transformation on tumor growth.

FIGS. 34A-34E show the anti-tumor effect of the A2 antibody in the humanized mouse model.

FIGS. 35A-35D show the anti-tumor effect of A2 and A2.3 antibodies in a humanized mouse model.

FIGS. 36A and 36B are data sets from co-culture experiments showing that co-culturing A1, A1.3, A2, and A2.3 antibodies and M2 macrophages showed CD8⁺T cell proliferation is increased.

FIG. 37 is a graph showing data from co-culture experiments showing that co-culturing A2 and A2.3 antibodies and M2 macrophages showed CD8⁺T cell proliferation is increased.

FIGS. 38A and 38B are a set of graphs showing the observation of passage through A2 or A2 using HeLa-h VSIG4 cells expressing hVSIG43 CD8 of antibody⁺T cells proliferate.

Fig. 39 is a graph showing the signaling pathway for observing macrophage transformation by a2 antibody using a human phosphor-kinase array.

Figure 40 shows the sequence alignment of the heavy and light chains of the humanized EU103.3, A1, A2, a1.3 and a2.3 antibodies.

FIG. 41 shows the generation and characterization of VSIG 4K/O mice.

Fig. 42 is data of gene array analysis, which shows changes in gene expression after conversion of M2 macrophages to M1 macrophages by the a2 antibody.

Detailed Description

The present invention is based, at least in part, on the discovery that: inhibition of VSIG4 and CD8 using certain anti-VSIG 4 antibodies⁺T cell interaction leads to CD8⁺T cells proliferate and can thus lead to cancer suppression.

As used herein, the term "about," when used in reference to a numerical value, refers to a value that is similar in context to the value. In general, one skilled in the art familiar with relevant content will understand the degree of difference encompassed by "about" in respect of the relevant content. For example, in some embodiments, the term "about" means a value within a range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the referenced value.

Herein, "administering" generally refers to administering a composition to a subject or system to effect delivery of a substance, i.e., the composition or contained in the composition. One of ordinary skill in the art will know of various routes for administration to a subject (e.g., a human) where appropriate. For example, in some embodiments, administration can be ocular, oral, parenteral, topical, and the like. In certain embodiments, administration can be bronchial (e.g., via bronchial perfusion), buccal, dermal (which can be or include, for example, one or more of topical to dermal, intradermal (intradermal), transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, buccal, rectal, subcutaneous, sublingual, topical, tracheal (e.g., via intratracheal perfusion), vaginal, vitreous, and the like. In some embodiments, administration comprises only one dose. In some embodiments, administration may include administering a fixed number of doses. In some embodiments, administration may include intermittent administration (e.g., multiple doses separated in time) and/or periodic administration (e.g., each dose is spaced equally apart). In some embodiments, administration may include continuous administration (e.g., infusion) for at least a selected period of time.

As used herein, the term "affinity" generally refers to a measure of how tightly a particular ligand binds its ligand (partner). Affinity can be determined by different methods. In some embodiments, affinity is determined using a quantitative assay. In some embodiments, the binding partner concentration may be fixed in excess of the ligand concentration, thereby mimicking physiological conditions. Alternatively or additionally, in some embodiments, the concentration of the binding partner and/or the concentration of the ligand may be varied. In some embodiments, affinity can be compared to a reference under comparable conditions (e.g., concentration).

As used herein, the term "affinity maturation" refers to a process in which an antibody evolves (typically by mutating one or more amino acid residues) from a reference antibody (also referred to herein as a template or parent antibody) such that the activity towards a target antigen is increased over the activity of the corresponding form of the reference antibody towards the same target antigen. Thus, the evolved antibody is optimized compared to a reference antibody or a template antibody. As used herein, the term "affinity matured antibody" generally refers to an antibody that has increased activity against a target antigen relative to a reference antibody. In some embodiments, the affinity matured antibody exhibits increased binding to the target antigen as compared to a reference or parent antibody. Typically, affinity matured antibodies bind to the same epitope as a reference antibody.

As used herein, the term "antibody" refers to a polypeptide comprising sufficient binding activity to a particular targetTypical immunoglobulin sequence elements that antigen specifically binds. As known in the art, a naturally occurring intact antibody is a tetramer of about 150kD, comprising two identical heavy chain polypeptides (each about 50kD) and two identical light chain polypeptides (each about 25kD) associated with each other in a so-called "Y-shaped" structure. Each heavy chain comprises at least four domains (each about 110 amino acids in length) -an amino-terminal Variable (VH) domain (located at the two top ends of the Y structure), followed by three constant domains: CH1, CH2 and carboxy-terminal CH3 (at the dry bottom of Y). A short region called a "switch" connects the heavy chain variable region to the constant region. The "hinge" connects the CH2 and CH3 domains to the rest of the antibody. In a complete antibody, two disulfide bonds within the hinge region link two heavy chain polypeptides to each other. Each light chain comprises two domains, an amino-terminal Variable (VL) domain followed by a carboxy-terminal Constant (CL) domain, which are separated from each other by another "switch fork". A complete antibody tetramer comprises two heavy chain-light chain dimers, wherein the heavy and light chains are linked to each other by a disulfide bond; two more disulfide bonds link the heavy chain hinge regions to each other, thereby linking the dimers to each other to form the tetramer. Naturally occurring antibodies are also glycosylated, typically being glycosylated in the CH2 domain. Each domain in a native antibody has a structure characterized as an "immunoglobulin fold" that is formed by two beta sheets (e.g., 3-, 4-, or 5-strand sheets) stacked within a flattened antiparallel beta barrel. Each variable domain comprises three hypervariable loops called "complementarity determining regions" CDR1, CDR2, and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR 4). When a natural antibody is folded, the FR regions form the structural framework of the beta sheet providing domains, and the CDR loop regions of the heavy and light chains converge with one another in three dimensions, thereby forming a hypervariable antigen-binding site at the top of the Y structure. The Fc region of a natural antibody binds to elements of the complement system and also binds to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity and/or other binding properties of an Fc region for an Fc receptor can be modulated by glycosylation or other modifications. In certain embodiments, generated and/or caused in accordance with the present inventionAntibodies useful comprise glycosylated Fc domains, including Fc domains having a modified or engineered glycosylation. For the purposes of the present invention, in certain embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences found in a native antibody, whether such polypeptide is naturally-produced (e.g., by an organism's reaction to an antigen) or recombinantly engineered, chemically synthesized or produced by other artificial systems or methods, may be referred to and/or used as an "antibody". In certain embodiments, the antibody is a polyclonal antibody; in certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the constant region sequence of the antibody has mouse, rabbit, primate, or human antibody properties. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art. Furthermore, the term "antibody" herein refers in appropriate embodiments to any construct or form known or available in the art that utilizes structural and functional characteristics of antibodies in other manifestations (unless otherwise indicated or clear from context). For example, as an embodiment, the antibody used in the present invention may be in a form selected from the group consisting of, but not limited to: intact IgA, IgG, IgE or IgM antibodies; bi-or multispecific antibodies (e.g. antibodiesEtc.); antibody fragments, such as Fab fragments, Fab ' fragments, F (ab ') 2 fragments, Fd ' fragments, Fd fragments, and isolated CDRs or sets of CDRs; a single-chain Fvs; a polypeptide-Fc fusion; single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof); a camel antibody; masking antibodies (e.g. antibodies) (ii) a Small modular immunopharmaceuticals (' SMIPs)^TM”) (ii) a Single chain or tandem bifunctional antibodiesHumabody antibodies, VHH;a minibody;ankyrin repeat proteins or DART; a TCR-like antibody; MicroProteins；andin certain embodiments, the antibody may lack covalent modifications in the native form (e.g., tipping glycans). In certain embodiments, the antibody can comprise a covalent modification (e.g., a tipping glycan, a payload [ e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.)]Or other ancillary groups [ e.g. polyethylene glycol, etc. ]]。

Herein, "antibody fragment" refers to a portion of an antibody or antibody class as described herein, typically refers to a portion that includes an antigen binding portion or a variable region thereof. Antibody fragments can be made in any manner. For example, in certain embodiments, antibody fragments can be produced by enzymatically or chemically fragmenting an intact antibody or antibody class. Alternatively, in certain embodiments, the antibody fragment may be produced using recombinant techniques (i.e., by expression of an engineered nucleic acid sequence). In certain embodiments, the antibody fragment may be produced synthetically, in whole or in part. In certain embodiments, antibody fragments (particularly antigen-binding antibody fragments) can be at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or longer in length, in certain embodiments at least about 200 amino acids.

As used herein, the term "association" generally refers to a non-covalent association between two or more entities. "direct" binding includes physical contact between entities or moieties; indirect binding includes physical interaction that occurs through physical contact with one or more intermediate entities. Binding between two or more entities can generally be assessed in a variety of situations-including studies of interacting entities or moieties alone or in a more complex system context (e.g., covalently or otherwise associated with a carrier and/or within a biological system or cell).

As used herein, the terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" generally refer to cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, and which therefore exhibit an abnormal growth phenotype characterized by significant uncontrolled cell proliferation. In some embodiments, a tumor can be or comprise precancerous (e.g., benign), malignant, pre-metastatic, and/or non-metastatic cells. This document specifically suggests certain cancers that may be particularly relevant. In some embodiments, the related cancer may be characterized as a solid tumor. In some embodiments, the associated cancer may be characterized as a hematological tumor. In general, examples of different types of cancer known in the art include, for example: cancers of the hematopoietic system including leukemia, lymphoma (hodgkin and non-hodgkin), myeloma, and myeloproliferative diseases; sarcomas, melanomas, adenomas, solid tissue cancers, squamous cell carcinoma of the mouth, throat and lung, liver cancer, genitourinary system cancers such as prostate, cervical, bladder, uterine, endometrial and renal cell cancers, bone, pancreatic, skin or intraocular melanoma, cancers of the endocrine system, thyroid, parathyroid, head and neck, breast, gastrointestinal and nervous system, benign lesions such as papillomas, and the like.

As used herein, the term "CDR" refers to complementarity determining regions within an antibody variable region. The variable regions of the heavy and light chains each have three CDRs, which are CDR1, CDR2, and CDR3 of each variable region. "set of CDRs" or "set of CDRs" refers to a set of three or six CDRs that are either capable of binding to CDRs within a single variable region of an antigen or are capable of binding to CDRs of interrelated heavy and light chain variable regions of an antigen. Certain systems for defining CDR boundaries have been established in the art (e.g., Kabat, Chothia, etc.); those skilled in the art are aware of the differences between these systems and are able to understand the boundaries of the CDRs to the extent necessary to understand and practice the claimed invention.

As used herein, the term "chemotherapeutic agent" has its art-recognized meaning and refers to one or more pro-apoptotic, cytostatic, and/or cytotoxic substances, including, for example, specifically for and/or recommended for use in the treatment of one or more diseases, disorders, or conditions associated with undesired cellular proliferation. In many embodiments, chemotherapeutic agents can be used to treat cancer. In some embodiments, the chemotherapeutic agent may be or comprise one or more alkylating agents, one or more anthracycline antibiotics, one or more cytoskeletal disrupting agents (e.g., microtubule targeting agents such as taxanes, maytansine and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and/or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based drugs, one or more retinoids, one or more vinca alkaloids, and/or one or more analogs (i.e., also having related antiproliferative activity) of one or more of the following. In certain embodiments, the chemotherapeutic agent may be or comprise one or more of the following: actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and/or analogs thereof (e.g., DM1), dichloromethyldiethylamine (nitrogen mustard, Mechlorethamine), mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine (tiogouanine), topotecan, valrubicin (valbicin), vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. In some embodiments, chemotherapeutic agents may be used for the antibody-drug conjugates. In some embodiments, the chemotherapeutic agent is a chemotherapeutic agent selected from the group consisting of antibody-drug conjugates set forth below: hLL 1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4/D10-doxorubicin, gemtuzumab-ozuramicin (gemtuzumab ozogamicin), bentuximab-vittamicin (brentuximab), tuzumab-tamumab (trastuzumab), tamumab-tamuzumab (tamuzumab), and tamuzumab (tamuzumab-tamuzumab), ) Geobatemomab-vedottin (glembatemomab vedotin), SAR3419, SAR566658, BIIBO15, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, Vorstuzumab-madotin (vortuzumab fodotin), and Rotatuzumab-maytansinoidin-1 (merlostemozumab).

The term "combination therapy" as used herein refers to a situation in which a subject is simultaneously exposed to 2 or more treatment regimens (e.g., 2 or more therapeutic agents). In some embodiments, 2 or more treatment regimens may be administered simultaneously. In some embodiments, the 2 or more treatment regimens are administered sequentially (e.g., the first regimen is administered before the second regimen is administered at any dose). In some embodiments, the 2 or more treatment regimens are administered in overlapping dosing regimens. In some embodiments, administration of the combination therapy comprises administering 1 or more therapeutic agents or therapies to a subject receiving the other agent or therapy (modality).

As used herein, the term "framework" or "framework region" refers to a variable region sequence minus the CDRs. Since the CDR sequences can be determined in many different systems, the framework region sequences are likewise subject to correspondingly different interpretations. The six CDRs divide the framework regions on the heavy and light chains into four subregions on each chain (FR1, FR2, FR3 and FR4), with CDRl between FRl and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR 4. Unless a subregion is specified as FR1, FR2, FR3 or FR4, the framework regions represent, as otherwise stated, the sum of the FRs within a single native immunoglobulin variable region. Herein, a single FR represents one of the four subregions, e.g., FR1 represents the first framework region closest to the amino terminus of the variable region, located 5' to CDR1, while multiple FRs represent two or more subregions constituting the framework region.

As used herein, the term "humanized" is used generally to refer to antibodies (or antibody components) whose amino acid sequence includes V from a reference antibody produced in a non-human species (e.g., a mouse)_HAnd V_LThe region sequences, but also including modifications in these sequences relative to a reference antibody, are intended to make them more "human-like", i.e., more similar to human germline variable sequences. In certain embodiments, a "humanized" antibody (or antibody component) immunospecifically binds to an antigen of interest and has Framework (FR) regions having substantially the same amino acid sequence as human antibody framework regions and Complementarity Determining Regions (CDRs) having substantially the same amino acid sequence as a non-human antibody. Humanized antibodies comprise substantially all of at least one, and typically two, variable domains (Fab, Fab ', F (ab')₂(iii), (FabC), Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor immunoglobulin) and all or substantially all of the framework regions are human immunoglobulin consensus sequence framework regions. In some embodiments, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain and at least the variable domain of the heavy chain. The antibody may further comprise C of the heavy chain constant region_H1. Hinge region、C_H2、C_H3 and optionally C_HZone 4.

As used herein, the term "in vitro" refers to events occurring in an artificial environment (e.g., in a test tube or reaction vessel, in a cell culture, etc.) rather than in a multicellular organism.

As used herein, the term "in vivo" refers to events occurring within multicellular organisms (e.g., humans and non-human animals). In the case of cell-based systems, the term may refer to events occurring within living cells (as opposed to in vitro systems).

As used herein, the term "isolated" refers to a substance and/or entity that: (1) at least some of the components with which it was originally produced (whether in natural and/or experimental settings) have been isolated, and/or (2) have been artificially designed, generated, prepared, and/or manufactured. An isolated substance and/or entity may be one that has been separated by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99% or more of the other components with which it was originally associated. In some embodiments, the isolated material is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. Herein, a substance is considered "pure" if it is substantially free of other components. In some embodiments, a substance may still be considered "isolated" or even "pure" after combination with certain other components, e.g., one or more carriers or excipients (e.g., buffers, solvents, water, etc.), as understood by those skilled in the art. To give just one example: in some embodiments, a native biopolymer, such as a polypeptide or polynucleotide, is considered "isolated" when: a) as far as the origin or source of derivation is concerned, it is not associated in part or in whole with the accompanying component in its natural state; b) it is substantially free of other polypeptides or nucleic acids from the species produced by its natural producer; c) a component of a cell or other expression system that is expressed or otherwise associated with but is not the natural producer of the cell or other expression system. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized by a cell system other than its natural producer is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has undergone one or more purification techniques may be considered "isolated" to the extent that it has been separated from other components with which it is associated a) in nature, and/or b) at the time of its original production.

As used herein, the term "K_D"refers to the dissociation constant for dissociation of a binding substance (e.g., an antibody or binding component thereof) from a complex with its partner (e.g., an epitope bound by the antibody or binding component thereof).

As used herein, the term "macrophage" refers to a cell of the monocyte/macrophage lineage present in the spleen or that has differentiated into tissue macrophages. These cells include Follicular Dendritic Cells (FDCs), dendritic cells, langerhans cells, and other tissue macrophages. Macrophages are phagocytic and antigen-presenting cells that differentiate from monocytes in the circulating peripheral blood. They play an important role in both innate and adaptive immunity by activating T lymphocytes. Macrophages that activate Th 1T lymphocytes provide an inflammatory response and are referred to as M1 macrophages. M1 macrophages, also known as "killer macrophages," inhibit cell proliferation, cause tissue damage, and have antibacterial capabilities. Macrophages that activate Th 2T lymphocytes provide an anti-inflammatory response and are called M2 macrophages. M2 macrophages, also known as "repair macrophages," promote cell proliferation and tissue repair and have anti-inflammatory effects. As used herein, the term "tumor-associated macrophages" (TAMs) generally refers to macrophages present in the microenvironment of a cancer (e.g., a tumor).

As used herein, the term "operatively linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. The control elements "operatively connected" to the functional elements are interrelated in such a way that: expression and/or activity of the functional element is effected under conditions compatible with the control element. In some embodiments, a control element that is "operably linked" is adjacent to (e.g., covalently linked to) an encoding element of interest; in some embodiments, the control element acts in trans or otherwise with the functional element of interest.

As used herein, the term "pharmaceutical composition" refers to a composition in which the active substance is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in a unit dose that is suitable for administration in a treatment regimen and that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when used in a relevant population.

As used herein, the term "polypeptide" generally has the meaning well known in the art herein, i.e., a polymer of at least three amino acids. It will be understood by those of ordinary skill in the art that the term "polypeptide" is to be understood in a broad enough sense to encompass not only polypeptides having the complete sequence herein, but also polypeptides that represent functional fragments of these entire polypeptides (i.e., fragments that retain at least one activity). Also, as will be appreciated by those of ordinary skill in the art, protein sequences typically allow for some substitution without compromising their activity. Thus, herein, the relative term "polypeptide" encompasses any polypeptide as described below: it retains activity and has at least about 30-40% overall sequence identity, typically greater than about 50%, 60%, 70% or 80%, and typically comprises at least one region of much greater identity, typically greater than 90% or even 95%, 96%, 97%, 98% or 99%, typically comprising at least 3-4, and typically up to 20 or more amino acids, within one or more highly conserved regions, as compared to another polypeptide of the same class. The polypeptide may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, the protein may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides that are less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids in length. In certain embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and/or a characteristic portion thereof.

As used herein, the terms "prevent" or "preventing," when used in reference to the occurrence of a disease, disorder, and/or condition, refer to reducing the risk of developing the disease, disorder, and/or condition and/or delaying the onset and/or severity of one or more characteristics or symptoms of the disease, disorder, and/or condition. In some embodiments, prevention is based on a population assessment, i.e., an agent/substance is considered to be capable of "preventing" a disease, disorder or condition if a statistically significant decrease in the occurrence, development, frequency and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder or condition.

The term "recombinant" as used herein means a polypeptide designed, engineered, prepared, expressed, created, manufactured and/or isolated by recombinant means, such as a polypeptide expressed with a recombinant viral vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or otherwise manipulated to express one or more genes or gene components that encode and/or directly express the polypeptide or one or more components, portions, elements, or domains thereof; and/or by any other means involving splicing or linking selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/or otherwise producing a nucleic acid encoding and/or directly expressing a polypeptide or one or more components, portions, elements, or domains thereof. In some embodiments, one or more such selected sequence elements are native. In some embodiments, one or more of the selected sequence elements are designed in silico. In some embodiments, one or more of the selected sequence elements are derived from a mutation of a known sequence element (e.g., in vivo or in vitro), e.g., from a natural or synthetic source, such as in a species of interest (e.g., human, mouse, etc.).

As used herein, the term "specific binding" refers to the ability to distinguish between potential binding partners in the environment in which binding occurs. A binding substance, when interacting with a particular target in the presence of other potential targets, is said to "specifically bind" the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the extent of binding between a binding substance and its partner; in some embodiments, specific binding is assessed by detecting or determining the extent of dissociation of the binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining the ability of a binding agent to compete for selective interaction between its partner and other entities. In some embodiments, specific binding is assessed by performing such detection or assay over a range of concentrations.

As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments, a prenatal form of a human). In some embodiments, the subject has an associated disease, disorder, or condition. In some embodiments, the subject is predisposed to the associated disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject has one or more susceptibility characteristics or risk characteristics of a disease, disorder, or condition. In some embodiments, the subject is a human. In some embodiments, the subject is an individual diagnosed and/or treated or an individual who has been diagnosed and/or treated.

As used herein, the phrase "therapeutic agent" broadly refers to any substance that elicits a desired pharmacological effect upon administration to an organism. In some embodiments, a substance is considered a therapeutic agent if it exhibits a statistically significant effect in the appropriate population. In some embodiments, a suitable population may be a model biological population. In some embodiments, a suitable population may be defined using various criteria, such as a particular age group, gender, genetic background, pre-existing clinical condition, and the like. In some embodiments, a therapeutic agent is a substance that can be used to reduce, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and/or reduce the incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, a "therapeutic agent" is a substance that has been or is presently approved by a governmental agency for human consumption in the marketplace. In some embodiments, a "therapeutic agent" is a substance that is prescribed for human use.

The term "therapeutically effective amount" as used herein refers to an amount sufficient to treat a disease, disorder, and/or condition when administered to a population suffering from or susceptible to such a disease, disorder, and/or condition according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and/or severity of one or more symptoms of a disease, disorder, and/or condition, stabilizes one or more characteristics of the symptoms, and/or delays the onset of the symptoms. One of ordinary skill in the art will appreciate that a "therapeutically effective amount" need not actually achieve therapeutic success in a particular individual. Conversely, a therapeutically effective amount may be that amount which, when administered to a patient in need of such treatment, provides a particular desired pharmacological response in a significant number of subjects. For example, in some embodiments, the term "therapeutically effective amount" refers to an amount that, in the context of innovative therapy, blocks, stabilizes, reduces, or reverses the supportive course of cancer in an individual in need thereof, or that will enhance or increase the inhibitory course of cancer in the individual. In the context of cancer treatment, a "therapeutically effective amount" is an amount that, when administered to an individual diagnosed with cancer, prevents, stabilizes, inhibits or reduces the further development of cancer in the individual. A particularly preferred "therapeutically effective amount" of the compositions described herein reverses (in the therapeutic setting) the development of a malignant tumor, such as pancreatic cancer, or assists in achieving or prolonging remission of the malignant tumor. The therapeutically effective amount administered to an individual to treat the individual's cancer may be the same or different from the therapeutically effective amount used to promote remission or inhibit metastasis. As with most cancer therapies, the treatment methods described herein should not be construed as being limited or otherwise limited to a "cure" of cancer; in contrast, a method of treatment refers to the use of the composition to "treat" cancer, i.e., to produce a desired or beneficial change in the health of an individual with cancer. Such benefits are known to skilled healthcare providers in the oncology arts, including but not limited to patient condition stabilization, tumor reduction (tumor regression), improvement in vital functions (e.g., improvement in function of cancerous tissues or organs), reduction or inhibition of further metastasis, reduction in opportunistic infections, improvement in viability, reduction in pain, improvement in motor function, improvement in cognitive function, improvement in energy (energy, discomfort), improvement in wellness, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. In addition, regression of a particular tumor in an individual (e.g., as a result of a treatment described herein) can also be assessed as follows: cancer cells, such as pancreatic cancer, are sampled from the tumor site (e.g., during treatment) and monitored for levels of metabolic and signaling markers to monitor the status of the cancer cells, thereby molecularly verifying cancer cell regression to a low malignancy phenotype. For example, tumor regression induced using the methods of the invention may be indicated by: a decrease in any of the aforementioned pro-angiogenic markers is found, an increase in the anti-angiogenic marker described herein, a normalization of the metabolic pathway or intercellular or intracellular signaling pathway (exhibiting abnormal activity in the individual diagnosed with cancer) (i.e., a change in state towards a non-cancerous, normal individual). One of ordinary skill in the art will appreciate that in some embodiments, a therapeutically effective amount can be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated for multiple dose and/or administration in multiple doses, e.g., as part of a dosing regimen.

The term "variant" in the context of a molecule (e.g., a nucleic acid, protein, or small molecule) as used herein refers to a molecule that exhibits significant structural identity to, but is structurally distinct from, a reference molecule, e.g., differs in the presence or absence or level of one or more chemical moieties as compared to the reference. In some embodiments, the variant is also functionally distinct from its reference molecule. In general, a particular molecule is considered to be a "variant" of a reference molecule based on the degree of structural identity to the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a distinct molecule that shares one or more of such characteristic structural elements, but differs from a reference molecule in at least one aspect. Polypeptides may have characteristic sequence elements consisting of a plurality of amino acids having specified positions relative to each other in linear or three-dimensional space and/or involved in constituting specific structural motifs and/or biological functions, to name a few; a nucleic acid can have a characteristic sequence element composed of a plurality of nucleotide residues having specified positions relative to each other in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid by one or more differences in amino acid or nucleotide sequence. In some embodiments, the overall sequence identity of a variant polypeptide or nucleic acid is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% to a reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more biological activities with a reference polypeptide or nucleic acid.

The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and other episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Also, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". Recombinant DNA, oligonucleotide synthesis and tissue culture and transformation can be performed using standard techniques (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or according to routine procedures in the art or as described herein. The techniques and methods described above can be generally performed as described in numerous comprehensive and monographic documents as known in the art, and also as cited and discussed in this specification. See, e.g., Sambrook et al, Molecular Cloning: a Laboratory Manual (molecular cloning: A Laboratory Manual) (2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for all purposes.

Macrophages in cancer

Macrophages are phagocytic and antigen-presenting cells that differentiate from monocytes in the circulating peripheral blood. These cells are known to play an important role in both innate and adaptive immunity by activating T lymphocytes. Macrophages that activate Th 1T lymphocytes provide an inflammatory response and are referred to as M1 macrophages. M1 macrophages, also known as "killer macrophages," inhibit cell proliferation, cause tissue damage, and have antibacterial capabilities. Macrophages that activate Th 2T lymphocytes provide an anti-inflammatory response and are called M2 macrophages. M2 macrophages are also known as "repair macrophages," which promote cell proliferation and tissue repair.

Although macrophages are formed by the differentiation of monocytes, monocytes mature to form M1(CD 68)⁺And CD80⁺) Or M2(CD 68)⁺And CD163⁺) Macrophages, depending on the cytokines and growth factors that cause them to differentiate. Lipopolysaccharide (LPS) and interferon gamma (IFN γ) activate monocytes to differentiate into M1 macrophages, which secrete high levels of interleukin 1(IL-1) and interleukin 12(IL-12) and low levels of interleukin 10 (IL-10). Alternatively, interleukin-4 (IL-4), IL-10, interleukin-1 receptor antagonist (IL-1ra) and transponderantsMetagrowth factor beta (TGF β) activates monocytes to differentiate into M2 macrophages that secrete high levels of IL-10, TGF β, and insulin-like growth factor 1(IGF-1), as well as low levels of IL-12.

The role of immunity in carcinogenesis is becoming increasingly important. Since macrophages are known to play an important role in both innate and adaptive immunity, they are considered to be a key component of tumors and their microenvironment. Tumor Associated Macrophages (TAMs) generally refer to macrophages present in the cancer microenvironment. The role of tumor-associated macrophages (TAMs) in tumor growth, invasion and metastasis has been extensively studied and TAMs are known to exhibit a wide range of phenotypes, ranging from an M1-like phenotype in the early stages of selected tumors to an M2-like phenotype in most advanced tumors. As evidence of its role in promoting tumorigenesis, M2 macrophages exhibit a characteristic phenotype: IL-10, IL4, MMP and VEGF expression were elevated, but pro-inflammatory cytokines and cytotoxic iNO and ROI expression were decreased, which was associated with tumoricidal activity. In addition to its intrinsic function in promoting tumorigenesis, TAMs also result in the suppression of anti-tumor immunity by alternating T cell responses and balance in the tumor microenvironment.

In cancer, M2 macrophages can induce angiogenesis in the tumor region in addition to promoting cell proliferation and acting as an anti-inflammatory. Therefore, in this case, it would be useful to inhibit M2 polarization of macrophages and induce M1 polarization of macrophages known to attack tumor cells.

VSIG4

VSIG4(V-set and immunoglobulin domain 4-containing) is a B7 family-related membrane protein, a complement receptor belonging to the immunoglobulin superfamily (CRIg), which is known to down-regulate CD8 by binding to iC3B and C3B⁺T cell proliferation and IL-2 production. Expression of VSIG4 was restricted to tissue macrophages, including peritoneal macrophages and liver-resident Kupffer (Kupffer) cells. Figures 2A and 2B show the relationship (homology and phylogenetic relationship, respectively) of VSIG4 to other B7 protein families (VSIG4 is referred to as EU103 in figures 2A and 2B). Figure 2A shows an amino acid sequence alignment of VSIG4 with various other B7 protein families. FIG. 2B showsThe evolutionary relationship between VSIG4 and other B family proteins.

anti-VSIG 4 antibodies for the treatment of cancer

Tumor-associated macrophages (TAMs) are key cells in the generation of the immunosuppressive Tumor Microenvironment (TME), providing multiple targets for immunotherapy. Macrophages are also known to have high plasticity and can also repolarize and acquire an anti-tumorigenic M1-like phenotype.

It is known that tumor-associated macrophages (TAMs) are characterized by tumor-promoting functions, such as promotion of cancer cell motility, metastasis formation and angiogenesis. TAMs are formed depending on the micro-environmental factors present in developing tumors. TAMs are present in high levels in many cancers, especially in the tumor microenvironment, and often exhibit an immunosuppressive M2-like phenotype that promotes tumor growth and enhances resistance to therapy. TAM also produces immunosuppressive cytokines such as IL-10, TGF β and PGE2, very small amounts of NO or ROIs and low levels of inflammatory cytokines such as IL-12, IL-1 β, TNF α and IL-6. The conversion of macrophages to TAMs results in a reduced ability to present tumor associated antigens and stimulate the anti-tumor function of T and NK cells. Furthermore, TAMs are unable to lyse tumor cells. Thus, targeting TAMs is a novel therapeutic strategy for inhibiting or treating cancer, e.g., by delivering agents to alter the recruitment and distribution of TAMs, depleting existing TAMs or inducing relegation (or transformation) of TAMs from the M2 to M1 phenotype.

The present invention is based on the discovery that: m2 macrophages expressing VSIG4 can be treated with humanized anti-VSIG 4 antibodies to convert (or repolarize) M2 macrophages into tumor-suppressor M1 macrophages, thereby inducing CD8⁺T cell proliferation and proinflammatory cytokine production, leading to tumor suppression. In addition, by targeting (1) the transformation of M2 macrophages to M1 macrophages, and (2) induction of CD8⁺T cell proliferation and pro-inflammatory cytokine production, the use of anti-VSIG 4 antibodies can effectively inhibit cancer, thereby affecting the tumor microenvironment itself. This method of inhibiting cancer using an anti-VSIG 4 antibody is superior to other therapies that induce only T cell proliferation or block only macrophage tumor-accessory activity. See FIG. 1 (schematically showing the effect of anti-VSIG 4 antibody on macrophage functionIts effect on T cell proliferation, and subsequent cancer inhibition).

Humanization of mouse antibodies

Although monoclonal antibodies can be rapidly produced by the mouse immune system for biological studies, in a clinical setting, the use of these murine antibodies may result in a human anti-mouse antibody response (HAMA). Although chimeric antibodies can reduce human anti-IgG responses, the murine variable domains may still possess aggressive (pro-volatile) T cell epitope content, which requires "humanization" of their framework regions.

Classical antibody humanization typically begins with the transfer of all six murine Complementarity Determining Regions (CDRs) to a human antibody framework (Jones et al, Nature 321,522-525 (1986)). These CDR grafted antibodies typically do not retain their original affinity for antigen binding, and in fact affinity is often severely compromised. In addition to the CDRs, certain non-human framework residues must be incorporated into the variable domain to maintain the proper CDR conformation (Chothia et al, Nature 342:877 (1989)). The inclusion of murine residues at key positions in the human framework to restore function is commonly referred to as "back-mutation". Back mutations can support the structural conformation of the grafted CDRs and restore antigen binding and affinity. A variety of framework positions have been identified that are likely to affect affinity, so structural modeling in a stepwise manner to select novel residues can often lead to variants with restored antigen binding. Alternatively, phage antibody libraries targeting these residues can also be used to enhance and accelerate the affinity maturation process (Wu et al, J.mol.biol.294:151-162(1999) and Wu, H., Methods in mol.biol.207:197-212 (2003)).

Antibody affinity maturation

Affinity maturation is the process by which T_FHCell-activated B cells produce antibodies with enhanced affinity for a particular antigen during the course of an immune response. By repeated exposure to the same antigen, the host will produce antibodies of successively higher affinity. The secondary response may elicit antibodies with an affinity several times greater than the primary response. Affinity maturation is an important strategy for antibody optimization to produce safe and effective second generation therapeutics. The classical way is by applyingThe antigen is used to immunize a mouse or transgenic animal expressing human immunoglobulin genes to obtain a therapeutic antibody. Antigen-stimulated immune cells from these animals are transformed into hybridomas and subsequently screened to identify monoclonal antibodies with low nanomolar affinity for the target antigen. In vivo, natural affinity maturation by the immune system occurs through somatic hypermutation and clonal selection, while in vitro, in laboratory affinity maturation, can occur through mutation and selection. Furthermore, in addition to methods of affinity maturation using TFH cell-activated B cells, other methods of affinity maturation are known in the art and are within the scope of the present disclosure.

Examples

The invention is further described in the following examples, which do not limit the scope of the invention as claimed.

Materials and methods

The following methods and materials were used in the experiments described in the examples.

Isolation of CD14 from human PBMC⁺Monocyte cell

Differentiation of macrophages into M1 or M2 macrophages was performed: the macrophages were either isolated from the subjects and incubated for 6 days in 50ng/ml hGM-CSF for conversion to M1 macrophages or 100ng/ml M-CSF for 6 days for conversion to M2 macrophages (FIG. 5).

The conversion of macrophages to M1 or M2 macrophages was confirmed by phenotypic examination (fig. 6 and 7).

Subsequent conversion of M1 or M2 macrophages to M1 macrophages was performed by incubating the macrophages in LPS (100ng/ml) + IFN γ (100ng/ml) or 500ng/ml anti-VSIG 4 antibody (EU103.2) for 24 hours (fig. 12).

M1 or M2 macrophages were converted to M2 macrophages by incubating the macrophages in 20ng/ml IL-4 for 24 hours (FIG. 12).

Humanized anti-VSIG 4 antibody, EU103.2 antibody

The EU103.2 antibody is a humanized anti-VSIG 4 antibody generated from the mouse anti-VSIG 4 antibody mu6H 8. Figures 4A and 4B show biochemical characterization of EU103.2 antibody using size exclusion HPLC (figure 4A) and surface plasmon resonance experiments, showing binding of VSIG4 to EU103.2 antibody (figure 4B). A summary of EU103.2 antibody purification data is shown in table 1 below.

Table 1: summary of EU103.2 antibody size exclusion HPLC data

Humanization of mouse anti-VSIG 4 antibody, EU103.3 antibody

The humanized anti-VSIG 4 antibody hu6H8 (or EU103.3) was generated as described below.

mu6H8 VH humanization

The framework of the VH humanized variants (germline genes: VH2-5/D3-3/JH6c) was generated using the mouse 6H8 antibody and Blast (https:// blast.ncbi. nlm. nih. gov/blast.cglip page ═ Proteins) Kabat numbering for the sorting of the CDRs and back mutations in the framework and sorted mu6H8 VH CDRs, VH2, VH27, VH30, VH93 and VH94 were used to design humanized VH (hu6h8.3 VH).

mu6H8 VL humanization

The framework (germline genes: a17/JK2) Kabat numbering of VL humanized variants was generated using mouse 6H8 antibody and Blast (https:// blast.ncbi. nlm. nih. gov/blast.cgipage ═ Proteins) for CDR classification, and humanized VL was designed using framework and back-mutations of the classified mu6H8 VL CDRs, VH2, VH4, VH36 and VH46 (hu6h8.3 VL).

Cloning and expression of IgG antibodies-EU 103.3 antibody

The heavy chain variable region sequence was modified by FES (L234F, L235E, P331S) mutation to construct a heavy chain pOptivec (Invitrogen) expression plasmid without Fc effector function, as shown in fig. 21A. The light chain variable region sequence was constructed using pcDNA3.3 (Invitrogen) and synthesized using IDT, as shown in FIG. 21B. The gene encoding the Heavy Chain (HC) was flanked by the Nhe1 restriction enzyme EcoR1 to construct pOptivEc (FES) plasmid vector, and the gene encoding the Light Chain (LC) was flanked by BsiW1 restriction enzyme EcoR1 to construct pcDNA3.3 plasmid vector. Cloning was performed by subcloning the mutation site into the hu6H8.3 backbone. Use ofThe resulting insert gene and linearized vector were cloned separately with the HD Cloning Kit (Clontech) and the sequencing primers were identified using the CMV forward, EMCV IRES reverse primers.

VSIG4 knockout mice

VSIG K/O mice were generated by homologous recombination replacing exon 1 with the neomycin resistance gene. A targeting vector is generated for homologous recombination in ES cells. E1 and E1 and E2 indicate exons 1 and 2 of the CRIg gene (fig. 41A). Homologous recombination of the CRIg allele was confirmed by Southern blotting into heterozygous female offspring bred from the ES cell clones 1 and 2(C1, C2) chimeric mice to WT mice (FIG. 41B). The number of leukocytes in peripheral blood of WT and K/O male and female mice was compared. Total blood counts were determined using a hemocytometer. Leukocytes were incubated with fluorochrome-conjugated antibodies specific for various cell surface markers and the number of different leukocyte subsets was determined by flow cytometry. Data represent mean + SD of 5-7 mice (fig. 41C).

A1, A2, A1.3 and A2.3 antibodies

The a1 and a2 antibodies were raised against the affinity matured EU103.3 antibody (see table 2 below), in which the light chain variable region at positions 76, 90 and/or 92(kabat numbering) was mutated as shown in table 2 below.

The a1.3 and a2.3 antibodies were generated from the A1 and A2 antibodies, respectively, to further improve the affinity for VSIG 4.

FIG. 40 provides an amino acid sequence alignment of the heavy and light chains of EU103.3, A1, A2, A1.3 and A2.3, and the consensus amino acid sequences of the heavy and light chains. The amino acid residues that differ between different antibodies are shown in rectangular boxes.

Table 2: cloning of humanized anti-VSIG 4 antibodies screened in affinity maturation experiments

Production of antibodies with high binding affinity

The humanized antibody gene was inserted into a plasmid and expressed in an IgG form using an Expi293 expression system (Invitrogen), and then purified using an AktaPure purifier (GE Healthcare), an AktaPrime purifier (GE Healthcare), and a Mabselect SURE column (GE Healthcare). The purified antibody was run through a desalting column (GE healthcare, Cat. No. 17-1408-01) with PBS buffer changes and antibody concentration was measured with Multiskan GO (Siemer fly (Thermo)).

Table 3: yield of antibodies with high binding affinity

PBMC-derived macrophage differentiation

M1 and M2 macrophages were obtained from PBMCs using the following protocol.

1. Blood mixture (1:1) containing PBS 20ml were overlaid with 10ml of Ficoll-Paque^TMPlus (GE healthcare company, catalog number 17-1440-02)

2. Centrifuge at 400Xg for 35 minutes (2 acceleration, 0 brake)

PBMC were isolated and washed 5 min x2 times with RPMI-1640 medium (WelGene, catalog # LB011-01) at 2000rpm

4. Counting

1-2 ml of a PBS (WelGene, catalog number LB004-02) suspension of MAC buffer (2% FBS (Millipore, catalog number TMS-013-BKR)).

6. Addition of CD 14-Microbeads (20 ul/10)⁷Cells) (Miltenyi Biotech, catalog No. 130-

7. Incubate on ice for 30 min

8. Wash 5 min x2 times by MAC buffer at 2000rpm

9. Counting

10. Loading of cells onto MAC column (America whirly Biotech, catalog No. 130-

11. Positive selection and counting

12. Suspending was carried out in a medium (RPMI-1640+ 10% FBS + penicillin/streptomycin (Gibco, catalog No. 15140-122)) + Glutamax (Gibco, catalog No. 35050-061) + rhM-CSF (Bailejin, catalog No. 574806) at 20-40 ng/ml

13. Will CD14⁺Cells were seeded on 100mm dishes (1X 10)⁶Cells/10 ml/dish) (Seimer Feishell science, Cat # 150466)

14. After every 3 days, the medium was replaced with fresh one

After 15.7-10 days, detecting the differentiation of M0 macrophage by FAC

16. Differentiation from M0 to M1 or M2 macrophages was carried out 2 days with LPS 20ng/ml (Sigma-Aldrich, Cat. L4391) + rhIFN γ 20ng/ml (Leulejin, Cat. 570204) (M1) and rhIL 420 ng/ml (Leulejin, Cat. 574002) + rhIL 1320 ng/ml (Leulejin, Cat. 571102) (M2) medium (RPMI-1640+ 10% FBS + penicillin/streptomycin + Glutamax)

Examination of cell phenotype by FACs after differentiation of M1 or M2

18. For M2 to M1 macrophage transformation, 20ug/ml of antibody or 20ng/ml of LPS +20 ng/ml of rhIFN γ (positive control) was added to fresh medium (RPMI-1640+ 10% FBS + penicillin/streptomycin + Glutamax) for 2 days

Phenotyping of cells after transformation of M2 to M1 was performed by FACs, while cytokine/chemokine examination in cultured suspension was performed by LEGENDplex^TM(Bailejin Co., Cat. No. 740502)

FACS analysis

The following antibodies were used for FAC analysis:

hCD14-BV650(BD Bioscience, Cat. No. 563419)

hCD14-BV421(BD biosciences, Cat. No. 565283)

hIFN γ -PE/Cy7(BD biosciences, catalog No. 557844)

hCD3-BV510(BD biosciences, Cat. No. 563109)

hCD8-V450(BD bioscience, Cat. No. 560347)

hCD68-PE (Bilojin Co., Ltd. (Biolegend, Cat. 333808)

hCD93-PE (Bailejin Co., Cat No. 336108)

HLA-DR-BV421 (Bailejin Co., Cat. No. 307636)

hCD45-PE (Bailejin Co., Cat No. 304008)

hCD64-APC (Bailejin Co., Cat No. 305014)

hCD163-APC/Cy7 (Bailejin, Cat. No. 333622)

hCD86-PerCP/Cy5.5 (Bailejin Co., Cat. No. 305420)

hCD86-BV421(BD biosciences, Cat. No. 562432)

Example 1:VSIG4 expression in macrophages

VSIG4 was expressed in M2 macrophages. Figures 3A and 3B show data relating VSIG4 expression (measured by mRNA) and various genes associated with type 2 macrophages (M2) and tumor associated macrophages. As shown in fig. 3A, VSIG4 expression was negatively correlated to [ CXCL11, CXCL13, ZNMB, IFNAR1, IFNAR2] expression, while it was positively correlated to CCL19, IRF5, and IL1A expression. Figure 3B shows that VSIG4 expression is positively correlated with CD163, CSF1R, MSR1, TGFBR2, STAT6, IL1R1, IL10RA, MS4A4A, CCL2, CCL14, CCL17, and MS4A6A expression.

Figure 5 shows VSIG4 expression in M2 macrophages. The two panels in the upper row of fig. 5 show light microscopy images showing the morphology of M1 and M2 macrophages. The second and third rows show flow cytometry data for lymphocytes stained for CD14 and VSIG4, indicating that M2 cells (positive for CD14 staining) also express VSIG 4.

Example 2: human anti-VSIG 4 antibodies induced cytokine and chemokine secretion in M2 macrophages M1 and M2 macrophages were treated with EU103.2 antibody and measured for proinflammatory cytokine and chemokine secretion. As shown in figure 6, treatment of M2 macrophages with EU103.2 resulted in the induction of cytokines and chemokines IL12, IFN γ, IL10 and IL 23.

Example 3: humanized anti-VSIG 4 antibodies convert M2 macrophages to M1 macrophages

To further test the effect of EU103.2 on macrophages, M2 macrophages were treated with EU103.2 antibody and stained for M2 macrophage marker CD 163. As shown in figure 7, EU103.2 treatment reduced M2 macrophage marker CD163 expression in M2 macrophages, suggesting that blocking VSIG4 with EU103.2 results in the conversion of M2 macrophages to another different cell type.

Then, by co-incubating M2 macrophages and CD8⁺T cells (treated or not with EU103.2 antibody) were tested for the effect of EU103.2 antibody on macrophage-T cell interaction. As shown in FIG. 8, M2 macrophages treated with EU103.2 are CD 8-dependent⁺T cell co-incubation will result in CD8⁺T cell proliferation, which indicates that M2 macrophages will be converted to M1 macrophages when treated with EU103.2 antibody, whereas M1 macrophages induce CD8⁺T cell proliferation, while M2 macrophages inhibit CD8⁺T cells proliferate.

Then, to further investigate the effect of EU103.2 antibody on human macrophages in the context of cancer biology, ascites samples from ovarian cancer patients were collected and VSIG4 expression was first analyzed. Macrophages obtained from the peritoneal fluid of ovarian cancer patients included M2 macrophages co-expressing VSIG4 and CD14, as shown in fig. 9, while induced CD8, as shown in fig. 10 and 11⁺T cells proliferate.

In addition, microscopic images demonstrated that EU103.2 antibody treatment converted M2 macrophages to M1 macrophages, as shown in fig. 12.

As shown in figure 13, VSIG4 was blocked by co-culturing HeLa cells expressing VSIG4 with PBMC, with or without anti-VSIG 4 antibody, VSIG4 signaling on CD8 by macrophages⁺The role in T cell proliferation was further confirmed. This experiment shows blockade of VSIG4 and CD8⁺The interaction between T cells results in CD8⁺Proliferation of T cells is enhanced.

For further examination of VSIG4 at CD8⁺Effect in T cells mononuclear THP-1 cells were co-incubated with T cells at various ratios in the presence of anti-VSIG 4 antibody or control IgG antibody to show that anti-VSIG 4 antibody was able to increase CD8 by 4divisions (4divisions) compared to control IgG⁺T cells.

Example 4: anti-tumor effects of blocking VSIG4 signaling using anti-VSIG 4 antibodies or VSIG4 knockout mouse models

To determine the anti-tumor effect of the anti-VSIG 4 antibody, three mouse tumor models were used: MC38 colon adenocarcinoma mouse tumor model, B16F10 melanoma mouse tumor model, and 3LL lung cancer mouse tumor model (using VSIG4 knockout mice), and compared to wild-type mice, as shown in fig. 15A, 15B, and 15C, respectively. Tumor growth was inhibited compared to wild type mice, particularly for MC38 and 3LL mouse tumor models in VSIG4 knockout mice (see fig. 15A and 15C, respectively), demonstrating that tumor growth was inhibited in the absence of VSIG4 signaling.

Similar tumor growth inhibition was observed in the MC38 mouse tumor model in wild type mice injected with anti-VSIG 4 antibody compared to control IgG injected mice, as shown in figure 16. The extent of tumor growth inhibition by the anti-VSIG 4 antibody was at least, if not significantly greater than that of the VSIG4 knockout mouse, evident in the VSIG4 knockout mouse.

The activation status of lymphocytes from tumor draining lymph nodes was then examined in VSIG4 knock-out mice and compared to wild-type mice. As shown in figures 17A and 17B, comparable CD4 was observed in VSIG4 knockout mice compared to their wild-type counterparts⁺And CD8⁺Lymphocyte levels, and comparable CD62L expression was observed in those lymphocytes expressing CD4 and CD8 between the VSIG4 knockout mouse and the wild type mouse. Furthermore, VSIG4 knockout mice have increased CD8 β compared to wild type mice⁺T cells (as shown in FIG. 17C), but with comparable levels of CD11 β⁺Gr-1 lymphocytes (as shown in the figure)Shown as 17D).

To further assess the role of VSIG4 signaling in tumor growth, a CD38 colon adenocarcinoma mouse tumor model was employed with VSIG4 knockout mice and wild type mice, in which the chemotherapeutic agent, clauran (ctx), was injected intraperitoneally 18 or 23 days after tumor injection into the subject mice, as shown in the upper schematic of fig. 18A. The clauran injection resulted in a greater reduction in tumor volume in the VSIG4 knockout mice compared to wild type mice, and the reduction in tumor size was maintained 40 days after tumor injection. In contrast, in wild type mice, CTX injection resulted in a slight decrease in tumor size, followed by a continued increase in tumor size, as shown in fig. 18A. Mouse images and micrographs of tumor sections of VSIG4 knockout mice and wild type mice at day 24 post tumor injection are shown in fig. 18B. On day 24 by VSIG4^+/+And VSIG4^-/-Tumor sections were collected from C57BL/6 mice and treated with H&E staining paraffin sections of tumor tissue.

The effect of anti-VSIG 4 on tumor inhibition in the humanized mouse model was assessed by injecting 10mg/kg of anti-VSIG 4 antibody on days 19, 22, 25, 28 and 31 following HT29 cancer cell injection in the humanized mice, as shown in fig. 19. Significant inhibition of tumor growth was observed in mice receiving the anti-VSIG 4 antibody compared to mice receiving IgG control injections.

These experiments demonstrate that, as exemplarily shown in fig. 20, VSIG4 signaling modulates the inhibition of T cell proliferation by M2 macrophages, and that blocking VSIG4 signaling results in (1) abrogation of T cell proliferation induced by M2 macrophages, leading to CD8⁺T cell proliferation and tumor suppression; (2) m2 macrophages were converted to M1 macrophages.

Example 5: evaluation of A1, A2, A1.3 and A2.3 antibodies

Antibody clones A1, A2, a1.3 and a2.3 were developed by affinity maturation of the EU103.2 antibody. Protein profiles by size exclusion HPLC are shown in fig. 22, 23, 24 and 25, respectively, and summarized in table 4 below.

Table 4: SECHPC data for A1, A2, A1.3, and A2.3 antibodies

As shown in fig. 26A and 26B, a1 or a2 antibody was used for differentiated M2 macrophages for 2 days, and FACS analysis showed a decrease in CD163 (marker for M2 macrophages) and a significant increase in CD86 (marker for M1 macrophages). LPS/IFN gamma treatment for 2 days was used as a positive control. Treatment with A1 and A2 antibodies showed an increase in the M1/M2 ratio. Specifically, the a2 antibody showed an increased rate of closeness to the positive control group.

Then, as shown in FIG. 27, A1 or A2 antibody was applied to differentiated M2 macrophages for 2 days, repolarized to M1 macrophages, and LEGENDplex was used^TMChanges in cytokine and chemokine production in the culture medium are measured.

This was to confirm that M2 macrophages repolarize to M1 macrophages. LPS/IFN gamma treatment for 2 days was used as a positive control. Both groups A1 and A2 showed an increase in the production of cytokines/chemokines of type M1 (TNF α, IL6, IFN γ, IP-10 and IL12p40) and of cytokines/chemokines of type M2 (IL-10, arginase, TARC, and IL-1RA) compared to M2 macrophages. Specifically, the increase in TNF α and IL6 production was comparable to the positive control group.

Furthermore, as shown in fig. 28, a1 or a2 antibody was used for differentiated M2 macrophages for 2 days, and FACS analysis showed decreased expression of CD163 (a marker of M2 macrophages) and significantly increased expression of CD86 (a marker of M1 macrophages). LPS/IFN gamma treatment for 2 days was used as a positive control.

To further confirm that the a1 and a2 antibodies repolarize M2 macrophages to M1 macrophages, a1 or a2 antibodies were applied to differentiated M2 macrophages at different concentrations (5, 10, and 20ug/ml) for 2 days and cytokines and chemokines produced by the macrophages were evaluated, as shown in fig. 29. Using LEGENDplex^TMChanges in cytokine/chemokine production in the culture medium are measured. LPS/IFN gamma treatment for 2 days was used as a positive control. The A1 and A2 groups showed increased TNF α, IL6 and IP-10 associated with M1 macrophages, and this trend was particularly evident when M2 macrophages were treated with A2 antibody. Whether or not resistingAt any concentration of body, production of arginase associated with M2 macrophages was reduced.

Chemotactic ability of macrophages after conversion from M2 macrophages to M1 macrophages by the a2 antibody was assessed by chemotaxis analysis. Using Transwell 24-well 5 μ M-well chambers (Corning, Cat. No. CLS3421-48EA), the lower chamber was treated with the chemoattractant rhCCL19 (Bailejin, Cat. No. 582104) (chemokine type M1) at a concentration of 100ng/ml (400 μ l in volume), and the upper chamber was treated with re-polarized M1 macrophages at 1.5-5X 10⁵Cells/600 μ l treatment with M2 or A2 administered for 2 days. (treatment with LPS/IFN. gamma. for 2 days was used as a positive control). At 37 ℃ and 5% CO₂After a4 hour incubation period, 100 μ l of cells in the lower chamber were transferred to a 96-well plate. Then, 10. mu.l of a CCK-8 solution (catalog number CK04 of Dojindo chemical Co., Ltd.) was added to each well, and the absorbance (450nm) was measured after 1 hour incubation period. As shown in fig. 30, M1 macrophages from group a2 showed chemotactic capacity, confirming that the a2 antibody converted macrophages from M2 to M1.

Gene array analysis was performed to analyze changes in gene expression after conversion of M2 macrophages to M1 macrophages by a2 antibody, as shown in fig. 42. M2 macrophages were treated with a2 antibody and cells were harvested 2 days later. (Macrogen, Agilent Human GE 8x60K V3) analysis showed that, similar to LPS/IFN γ -treated cells as positive control, expression of the M1 phenotypic marker and the M1 type cytokines/chemokines increased, while expression of the M2 phenotypic marker and the M2 type cytokines/chemokines decreased.

Example 6: anti-tumor effects of A1, A2, A1.3, and A2.3 antibodies

The anti-tumor effects of the a1 and a2 antibodies were evaluated using a humanized mouse model. Human CD34 cells were injected into NBSGW mice, blood samples were then collected and human CD45 cells in PBMCs were measured to observe humanization of the mice within 12 to 14 weeks. HCT-15 colon cancer cells at 1X10⁷Cells/mice were injected into humanized mice, and after 5 days the mice were divided into three groups, each receiving an injection of hIgG (sigma-olidge, cat # I4506), a1 antibody or a2 antibody. Antibodies were injected every three days for a total of 5 injections, as shown in FIG. 31AAs shown. Tumor size was observed and after sacrifice, sera from blood were used to measure IFN γ using ELISA (invitrogen, catalog No. 88-7316-88) and tumor samples were used to analyze infiltrated leukocytes.

As shown in fig. 31B and 31C, no anti-tumor effect of the a1 antibody was observed, but the samples from the a2 antibody group showed smaller tumor size and increased IFN γ, which confirmed the anti-tumor effect of the a2 antibody.

The effect of the a2 antibody in converting M2 macrophages to M1 macrophages was then evaluated in vivo for tumor growth. As shown in fig. 32A, SW480 colon cancer cells were injected into mice (1x 10)⁷Cells/mouse), and once the tumor size grows to a certain size (about 1000mm3), differentiated M2 macrophages (7x 10)⁵Cell/mouse) was injected with hIgG or a2 antibody. Antibodies were injected every 2 days for a total of 5 injections, and after the first injection, blood samples were collected on days 4, 7 and 11 after tumor injection, and blood samples were used to isolate serum or PBMCs for analysis of changes in macrophage phenotype using FACS analysis.

On day 7 after tumor cell injection, changes in the M1 macrophage phenotype were observed from the a2 antibody panel, as shown in fig. 32B. Although CD163(M2 macrophage marker) was unchanged, the expression of CD86 and HLA-DR (M1 macrophage marker) was significantly increased compared to the hIgG group, confirming that the a2 antibody converts M2 to M1 macrophages, as shown in fig. 32C.

The effect of M2 conversion to M1 macrophages on tumor growth was then analyzed.

As schematically shown in FIG. 33A, HCT-15 colon cancer cells (8X 10)⁶Cells/mouse) and different concentrations of M2 macrophages (2.5x 10)⁵/5x10⁵/1x10⁶Cell/mouse) was injected into the mice. After 2 days, hIgG or A2 antibody was injected every 3 days for a total of 5 injections.

As shown in fig. 33B, the a2 antibody reduced or slowed tumor growth in a dose-dependent manner at day 14 post tumor injection compared to hIgG control mice, and this effect persisted at day 25 post tumor injection.

The humanized mouse model was then evaluated for the anti-tumor effect of the a2 antibody using a different mouse tumor model. As schematically shown in fig. 34A, human CD34 cells were injected into NBSGW mice, after which blood samples were collected and human CD45 cells in PBMCs were measured to observe humanization of the mice over a 12-14 week period. SW480 colon cancer cell (1x 10)⁷Cells/mouse) were injected into the mice, and 5 days later the mice were divided into two groups, each group being injected with hIgG or a2 antibody (20 mg/kg). Each group was injected with antibody every three days for a total of 5 injections. Tumor size was observed and after sacrifice, sera from blood were used to measure IFN γ using ELISA and tumor samples were used to analyze infiltrated leukocytes.

As shown in fig. 34B and 34C, the anti-tumor effect of the a1 antibody was not observed, but the samples from the a2 antibody group showed smaller tumor size and an increase in IFN γ was observed, which further confirmed the anti-tumor effect of the a2 antibody.

In conclusion, the group injected with the a2 antibody showed a smaller tumor size and an increase in IFN γ in serum. In addition, as shown in fig. 34D, CD8 was observed⁺T cells, particularly CD8 γ T cells secreting IFN γ, are increased. As shown in fig. 34E, decreased expression of CD93 and CD163(M2 macrophage marker) was observed, while increased expression of CD86(M1 macrophage marker) was observed, while no change in HLA-DR was observed. These data demonstrate that the a2 antibody mediates CD8⁺Cytotoxic activity of T cells.

The anti-tumor effects of the A2 and a2.3 antibodies were then compared in a humanized mouse model. As schematically shown in fig. 35A, human CD34 cells were injected into NBSGW mice, after which blood samples were collected and human CD45 cells in PBMCs were measured to observe humanization of the mice over a 12-14 week period. HCT-15 colon cancer cells at 1X10⁷Cells/mice were injected into humanized mice, and grown to a specific size (about 100 mm) in tumor size³) Thereafter, mice were divided into three groups, each receiving an injection of hIgG, A2 or a2.3 antibody (20 mg/kg). Antibodies were injected every three days for a total of 5 injections. Tumor size was observed and after the first injection, blood samples were collected at D5 and D13, where in serum from bloodInflammatory cytokines were discovered. As shown in fig. 35B, the A2 and a2.3 antibodies reduced tumor size.

Then, four anti-VSIG 4 antibodies A1, a1.3, A2, and a2.3 were evaluated against CD8⁺Effects of T cell proliferation. Addition of A1, a1.3, A2, and a2.3 antibodies to macrophages isolated from a donor to convert M2 macrophages to M1 macrophages and to CD8 isolated from the same donor PBMC⁺T cells were co-cultured for co-culture experiments. CD8⁺T cells were labeled with CFSE (Life Technologies, Cat. No. V12883) and co-cultured with transformed macrophages in a 2:1 ratio in anti-CD 3 coated 96-well plates (BD Biocoat, Cat. No. 354725) (CD 8T: macrophages 2X 10)⁵Cell/well 1X10⁵Cells/well). After 5 days, the harvested cells were stained with hCD8-V450 and analyzed by FACS analysis. CD8 confirmed by observing reduced CFSE levels⁺T cells proliferate. Although M2 macrophages down-regulate CD8⁺T cells proliferate, but co-culture of A1, A1.3, A2, and A2.3 antibodies resulted in the conversion of M2 macrophages to M1 macrophages, and in CD8⁺T cell proliferation increased as shown in fig. 36A and 36B.

Similar experiments comparing the effects of the A2 antibody and the a2.3 antibody were performed using macrophages and T cells isolated from different donors (i.e., different from those in fig. 36) relative to the experiments described above. Application of A2 and A2.3 antibodies to macrophages to convert M2 macrophages to M1 macrophages, and to CD8 isolated from the same donor PBMC⁺T cells were co-cultured for co-culture experiments. CD 8T cells were labeled with CFSE (life technologies, cat # V12883) and co-cultured with transformed macrophages in a 1:1 or 2:1 ratio in anti-CD 3 coated 96-well plates (BD Biocoat, cat # 354725) (CD 8T: macrophage: 2x 10)⁵Cell/well 2X10⁵Cell/well or 2x10⁵Cell/well 1X10⁵Cells/well). After 5 days, the harvested cells were stained with hCD8-V450 and analyzed by FACS analysis. CD8 confirmed by observing a reduction in CFSE levels⁺T cells proliferate. Although M2 macrophages down-regulate CD8⁺The proliferation of the T-cells is carried out,however, addition of A2 and a2.3 antibodies to convert M2 macrophages to M1 macrophages resulted in CD8⁺T cell proliferation is increased.

Then, HeLa cells expressing hVSIG4 (HeLa-hVSIG4 cells) were used to confirm CD8 mediated by VSIG4 signaling against A2 and a2.3 antibodies⁺Effects in the induction of T cell proliferation.

CD8⁺T cells were isolated from PBMCs of healthy donors, labeled with CFSE, and applied to anti-CD 3 coated plates (2X 10)⁵Cells/well). After 1 day, HeLa or HeLa-hVSIG4 cells were added to the wells after 30GY irradiation (1X 10)⁵Or 0.5x10⁵Cells/well). After 5 days, CD8 was analyzed by measuring CFSE levels using FACS analysis⁺T cells proliferate. CD8 was also treated with various concentrations of anti-CD 3 (Gentle Biotech, catalog No. 130-⁺T cells, and after 1 day, HeLa or HeLa-hVSIG4 cells were added to the wells after 30Gy irradiation (X-ray) (1X 10)⁵Cells/well). After 5 days, 100. mu.l of the cultured cells were transferred to separate 96-well plates, and CCK-8(10 ul/well) was added to each well. After 5 hours, absorbance was measured at 450nm to confirm CD8⁺T cells proliferate. As shown in FIGS. 38A and 38B, HeLa-hVSIG4 negatively regulated CD8⁺T cells proliferate, whereas treatment with A2 or a2.3 induced T cell proliferation.

Human phosphokinase arrays were used to examine the signaling pathway whereby the a2 antibody repolarizes macrophages. As shown in FIG. 39, a protome Profiler is used^TMAntibody array (R)&D systems Co Ltd (R)&D Systems), catalog number ARY003B), transformation of M2 macrophages to M1 macrophages by a2 antibody treatment resulted in a significant increase in JNK, MSK1/2 and p38a phosphorylation.

Antibody sequences and binding affinity information

Sequence information and binding affinity information for the various anti-VSIG 4 antibodies described herein are provided in tables 5-12 below.

Table 5: VH and VL sequences of EU103.2 antibody

Table 6: VH and VL sequences of EU103.3 antibody

Table 7: VH and VL sequences of A1 antibody

Table 8: VH and VL sequences of A2 antibody

Table 9: VH and VL sequences of A1.3 antibody

Table 10: VH and VL sequences of A2.3 antibody

Table 11: CDR sequences of EU103.2, EU103.3, A1, A2, A1.3 and A2.3 antibodies

Table 12: binding affinities of EU103.2, EU103.3, A1, A2, A1.3 and A2.3 antibodies to VSIG4 (K)_D)

Other embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.