RU2776638C1 - Antibodies against human vsig4 and application thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and constitutes an isolated humanised anti-VSIG4 antibody or an antigen-binding fragment thereof with a certain structure. Invention also relates to a pharmaceutical composition containing said antibody.

EFFECT: possibility of effectively treating a VSIG4-associated disease.

30 cl, 42 dwg, 12 tbl, 6 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a PCT application claiming priority and benefit under U.S. Provisional Application No. 62/738255 filed September 28, 2018 and U.S. Provisional Application No. 62/776523 filed December 7, 2018. Contents of the above applications incorporated herein by reference in its entirety.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to antibodies and antigen-binding fragments that bind to the V-set and immunoglobulin domain containing protein 4 (VSIG4).

BACKGROUND OF THE INVENTION

Cancer remains one of the leading causes of death in the world. Recent statistics report that 13% of the world's population dies of cancer. The International Agency for Research on Cancer (IARC) estimated that in 2012 there were 14.1 million new cancer cases and 8.2 million cancer deaths worldwide. By 2030, the global burden is expected to increase to 21.7 million new cancer cases and 13 million cancer deaths due to population growth and aging, as well as exposure to risk factors such as smoking, unhealthy diet and physical inactivity. In addition, the suffering and medical costs of cancer treatment result in a reduced quality of life for both cancer patients and their families. Clearly, cancer is primarily a disease for which improved treatments are urgently needed.

Macrophages are polyfunctional antigen-presenting cells that play a central role in our immune system and are associated with cancer biology. In the context of cancer, tumor-associated macrophages (TAMs) infiltrate malignant tumor tissues and, being related to cancer biology, influence tumor progression. OAM is believed to be divided into two categories: M1 and M2. M1 macrophages are known to have pro-inflammatory and cytotoxic (anti-tumor) function, while M2 macrophages are anti-inflammatory (pro-tumor) and promote wound healing. Because of these functions, TAMs, in particular, macrophages of the M2 phenotype, are closely associated with a poor clinical prognosis in many types of malignant tumors. The infiltrating TAMs themselves, or the TAM polarization pathway, are considered new therapeutic targets in cancer therapy.

SUMMARY OF THE INVENTION

The present invention relates, at least in part, to antibodies and fragments thereof that bind to VSIG4 (V-set and immunoglobulin domain containing protein 4; also referred to as CRIg or Z39Ig), as well as methods of using such antibodies and antigen-binding fragments for the treatment of cancer. , inducing the secretion of cytokines and/or chemokines in macrophages and the conversion of M2 macrophages to M1 macrophages.

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

In some embodiments, an antibody or antigen binding fragment described herein may comprise any of: (a) a heavy chain variable domain containing an amino acid sequence of at least 80%, 90%, 95%, 96%, 97% , 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14 or SEQ ID NO: 16; (b) a light chain variable domain containing an amino acid sequence at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of 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 an amino acid sequence at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14 or SEQ ID NO: 16, and a light chain variable domain containing an amino acid sequence at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid the sequences 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 described herein may comprise any of: a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14, or SEQ ID NO: 16, a light chain variable domain containing the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12, or a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14, or SEQ ID NO: 16, and a light chain variable domain containing the amino acid sequence of 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 described herein may comprise a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 23.

In some embodiments, an antibody or antigen-binding fragment described herein may 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, implementation an antibody or antigen-binding fragment described herein 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: 8. In some embodiments, the antibody or antigen-binding fragment 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, the antibody, or antigen binding fragment, may comprise l 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, an antibody or antigen binding fragment described herein may comprise a heavy variable domain. chain containing the amino acid sequence of SEQ ID NO: 14, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, an antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing an amino acid the sequence of SEQ ID NO: 16, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 12.

In some embodiments, an antibody or antigen-binding fragment described herein has a binding affinity (K _D ) for a human V-set molecule and an immunoglobulin domain containing protein 4 (VSIG4) of 1×10 ^-7 -1×10 ^-9 . In some embodiments, an antibody or antigen-binding fragment described herein has a binding affinity (K _D ) for the VSIG4 molecule of about 7.156 x 10 ^-8 to about 7.636 x 10 ^-9 . In some embodiments, an antibody or antigen-binding fragment described herein has a binding affinity (K _D ) for the VSIG4 molecule of about 7.156×10 ^-8 , about 7.636×10 ^-9 , about 7.952×10 ^-9 , about 8.226 ×10 ^-9 or about 8.688×10 ^-9 .

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

In another aspect, the present invention relates to a recombinant vector containing any of the nucleic acid molecules described herein. In some embodiments, the recombinant vector described herein contains a nucleic acid molecule described herein that is operably linked to a promoter. In some embodiments, the implementation of the recombinant vector includes two separate vectors, each of which contains a nucleotide sequence corresponding to the heavy chain and light chain of the antibody, or antigennegative fragment proposed herein.

In another aspect, the present invention provides a host cell comprising the 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 a cell selected from the group consisting of E. coli, P. pastoris , Sf9, COS, HEK293, Expi293, CHO-S, CHO-DG44, CHO-K1, and a mammalian lymphocyte. In some embodiments, the host cell is an Expi293 cell.

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

As a further example, in another aspect, the present invention relates to a method of treating an individual in need thereof, comprising the steps of: (a) administering to the individual a composition that contains or delivers any of the antibodies or antigen-binding fragments described herein, any of nucleic acid molecules described herein, any of the recombinant vectors described herein, or any of the host cells described herein, thereby treating a disease or condition. In some embodiments, the individual is suffering from, or at risk of developing, cancer. In some 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, hematologic 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, the individual has received, or will receive, one or more additional cancer therapies selected from ionizing radiation, a chemotherapeutic agent, an antibody, and cell therapy such that the individual is receiving both therapies. For example, in some embodiments, one or more additional cancer therapies may include the use of an immune checkpoint inhibitor, IL-12, GM-CSF, an anti-CD4 agent, cisplatin, fluorouracil, doxorubicin, irinotecan, paclitaxel, an indolamine-2,3 inhibitor -dioxygenase 1 (IDO1) or cyclophosphamide.

In another aspect, the present invention also relates to a method of increasing the secretion of cytokines or chemokines in M2 macrophages, including contacting M2 macrophages with any of the antibodies or antigen-binding fragments described herein.

In another aspect, the present invention provides a method for inducing CD8 ⁺ T cell proliferation comprising: (a) contacting an M2 macrophage with any of the antibodies or antigen binding fragments described herein; and (b) co-incubation of M2 macrophage with CD8 ⁺ T cells.

In another aspect, the present invention also provides a method for converting an M2 macrophage to an M1 macrophage, comprising contacting the M2 macrophage with any of the antibodies or antigen-binding fragments described herein.

Unless otherwise indicated, all technical and scientific terms used in this document have the meaning that is usually given to them by specialists in the field to which the present invention relates. While methods and materials similar or equivalent to those described herein may 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 in this document are incorporated by reference in their entirety. In the event of a conflict, this specification, including definitions, shall prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

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

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a schematic diagram illustrating the mechanism by which delivery of an anti-VSIG4 antibody to macrophages results in the conversion of M2 macrophages to M1 macrophages, resulting in CD8 ⁺ T cell proliferation and subsequent cancer suppression.

FIGURE 2A shows the alignment of VSIG4 sequences with various human B7 family proteins.

FIGURE 2B presents a phylogenetic tree showing the evolutionary relationship between VSIG4 and various human B7 family proteins.

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

FIGURE 4A is an HPLC graph showing the protein profile of the EU103.2 antibody.

FIGURE 4B presents surface plasmon resonance data showing EU103.2 antibody binding to VSIG4.

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

FIGURE 6 presents a set of graphs showing the induction of pro-inflammatory cytokines and chemokines in M1 and M2 macrophages by EU103.2.

FIGURE 7 presents a FACS analysis data set showing a decrease in CD163 expression in M2 macrophages treated with EU103.2.

FIGURE 8 shows the FACS analysis data set (first six columns) and quantification of these data (last column) which show the induction of CD8 ⁺ T cell proliferation when CD8 ⁺ T cells are co-cultured with M2 macrophages treated with EU103.2.

FIGURE 9 is a FACS analysis dataset demonstrating VSIG4 expression in macrophages isolated from the abdominal fluid of ovarian cancer patients.

FIGURE 10 presents a FACS analysis dataset demonstrating the induction of CD8 ⁺ T cell proliferation when co-cultured with EU103.2 treated macrophages obtained from ovarian cancer patients.

FIGURE 11 is a graph showing the induction of CD8 ⁺ T cell proliferation when co-cultured with EU103.2 antibody-treated macrophages from ovarian cancer patients.

FIGURE 12 presents a set of microscopic images of M1 and M2 macrophages showing morphological differences after conversion of M2 macrophages to M1 macrophages by exposure to the EU103.2 antibody.

FIGURE 13 is a FACS analysis dataset demonstrating that blocking the interaction between CD8 ⁺ cells and VSIG4 results in increased proliferation of CD8 ⁺ T cells.

FIGURES 14A and 14B present a set of graphs showing an increase in CD8 ⁺ cell proliferation as a result of blocking the suppression of THP-1 cells.

FIGURES 15A-15C are sets of graphs showing the antitumor activity of an anti-VSIG4 antibody in three different mouse tumor models.

FIGURE 16 is a set of graphs showing the antitumor activity of an anti-VSIG4 antibody in a VSIG4 knockout mouse model.

FIGURE 17A presents a FACS analysis dataset showing the activation status of T cells and MSCs in the DOLA in the absence of VSIG4 signaling.

FIGURE 17B presents a set of graphs showing the activation status of T-cells and MSCs in DOL in the absence of VSIG4 signaling.

FIGURE 17C is a set of graphs showing the activation status of T cells and MSCs in DOL in the absence of VSIG4 signaling.

FIGURE 17D is a set of graphs showing the activation status of T-cells and MSCs in DOL in the absence of VSIG4 signaling.

FIGURE 18A is a graph showing the suppression of tumor growth in the absence of VSIG4 signaling.

FIGURE 18B is a set of histological sections showing suppression of tumor growth in the absence of VSIG4 signaling.

FIGURE 19 is a graph showing the antitumor activity of an anti-VSIG4 antibody in a humanized mouse model.

FIGURE 20 is a schematic diagram illustrating the mechanism by which delivery of the EU103.2 antibody to macrophages results in the conversion of M2 macrophages to M1 macrophages, resulting in proliferation of CD8 ⁺ T cells.

FIGURES 21A and 21B are schematic representations showing expression vectors used to clone and express humanized anti-VSIG4 antibodies.

FIGURE 22 is an HPLC graph showing the protein profile of the A1 antibody (EU103_T01.01).

FIGURE 23 is an HPLC graph showing the protein profile of antibody A2 (EU103_T01.02).

FIGURE 24 is an HPLC graph showing the protein profile of the A1.3 antibody (EU103_T01.01S).

FIGURE 25 is an HPLC graph showing the protein profile of the antibody A2.3 (EU103_T01.02S).

FIGURE 26A is a FACS analysis data set showing a decrease in CD163 expression in M2 macrophages treated with A1 or A2 antibodies.

FIGURE 26B is a graph showing the reduction in CD163 expression in M2 macrophages treated with A1 or A2 antibodies.

FIGURE 27 is a set of graphs showing the reduction in M2-type cytokines and chemokines in M2 cells treated with A1 or A2 antibodies.

FIGURE 28 is a FACS analysis data set showing a decrease in CD163 expression and an increase in CD86 expression in M2 macrophages treated with A1 or A2 antibodies.

FIGURE 29 presents a set of graphs showing the increase in expression of M1-type cytokines/chemokines in M2 macrophages treated with A1 or A2 antibodies.

FIGURE 30 is a graph showing data from a chemotaxis assay that measures the chemotaxis ability of macrophages after conversion of M2 macrophages to M1 macrophages by A2 antibodies.

FIGURES 31A-31C show the antitumor effect of A1 and A2 antibodies in a humanized mouse model.

FIGURES 32A-32C show macrophage conversion by A2 antibodies in vivo .

FIGURES 33A and 33B show macrophage conversion by A2 antibodies in vivo by analyzing the effect of conversion of M2 macrophages to M1 macrophages on tumor growth.

FIGURES 34A-34E show A2 anti-tumor antibodies in a humanized mouse model.

FIGURES 35A-35D show antitumor antibodies A2 and A2.3 in a humanized mouse model.

FIGURES 36A and 36B present a dataset of co-cultivation analysis showing that co-cultivation of antibodies A1, A1.3, A2 and A2.3 with M2 macrophages increased CD8 ⁺ T cell proliferation.

FIGURE 37 is a graph based on co-culture assay data showing that when A2 and A2.3 antibodies are co-cultured with M2 macrophages, there is an increase in CD8 ⁺ T cell proliferation.

FIGURES 38A and 38B are a set of graphs showing the use of hVSIG4-expressing HeLa-hVSIG4 cells to observe CD8 ⁺ T cell proliferation in response to A2 or A2.3 antibodies.

FIGURE 39 is a graph showing the use of a human phosphokinase panel to analyze the macrophage signaling pathway in response to A2 antibodies.

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

FIGURE 41 shows the creation and characterization of VSIG4 knockout mice.

FIGURE 42 shows gene array analysis data showing the change in gene expression after conversion of M2 macrophages to M1 macrophages by A2 antibodies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery that inhibition of VSIG4 interaction with CD8 ⁺ T cells by certain anti-VSIG4 antibodies leads to CD8 ⁺ T cell proliferation, which can lead to suppression of cancer.

As used herein, the term "about" when used in relation to a value refers to a value that is similar in the context of a reference value. Typically, those skilled in the art familiar with the context will understand the relative degree of variation encompassed by the term "about" in a given context. For example, in some embodiments, the term "about" may include a range of values within 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 reference value.

As used herein, the term "administration" generally means administering a composition to an individual or system to effect delivery of an agent that is in, or included in, the composition. Those skilled in the art are aware of various routes which, under appropriate circumstances, can be used to administer to an individual, eg a human. For example, in some embodiments, administration may be ophthalmic, oral, parenteral, topical, and so on. In some specific embodiments, the implementation may be bronchial (for example, by instillation into the bronchi), buccal, dermal (which may be or include, for example, one or more of topical application to the skin, intradermal, intradermal, transdermal administration, and so on) , enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, to a specific organ (eg, liver), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, intratracheal (eg , by intratracheal instillation), vaginal, into the vitreous body, and so on. In some embodiments, the implementation of the introduction may include the introduction of only one dose. In some embodiments, the implementation of the introduction include the use of a fixed number of doses. In some embodiments, administration may include dosing that is discontinuous (eg, multiple doses separated by time) and/or intermittent (eg, separate doses separated by equal time periods) dosing. In some embodiments, administration may include continuous dosing (eg, perfusion) for at least a selected period of time.

As used herein, the term "affinity" generally refers to a measure of the strength of a particular ligand's binding to its binding partner. Affinity can be measured in various ways. In some embodiments, affinity is measured by quantitative analysis. In some such embodiments, the binding partner concentration may be fixed in excess of the ligand concentration to mimic physiological conditions. Alternatively or additionally, in some embodiments, the concentration of the binding partner and/or the concentration of the ligand can be varied. In some such embodiments, affinity can be compared to a reference under comparable conditions (eg, concentrations).

As used herein, the term "affinity maturation" means a process in which an antibody is altered relative to a reference antibody (also referred to herein as a base or parent antibody), typically by mutating one or more amino acid residues, to acquire higher activity in against the target antigen than the activity of the corresponding form of the reference antibody for the same target antigen. Thus, the modified antibody is optimized in comparison to the reference or base antibody. As used herein, the term "affinity matured antibody" generally means an antibody that has increased activity against a target antigen compared to a reference antibody. In some embodiments, the affinity matured antibody exhibits enhanced binding to the target antigen compared to the reference or parent antibody. Typically, an affinity matured antibody binds to the same epitope as the reference antibody.

As used herein, the term "antibody" means a polypeptide that includes canonical immunoglobulin sequence elements sufficient to provide specific binding to a particular target antigen. As is known in the art, intact antibodies naturally produced are approximately 150 kDa tetrameric molecules composed of two identical heavy chain polypeptides (approximately 50 kDa each) and two identical light chain polypeptides (approximately 25 kDa each) that are linked with each other into a structure that is commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains (each about 110 amino acids long): an amino-terminal variable (VH) domain (located at the ends of a Y-shaped structure), followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 (located at the base of the "Y" stem). A short region, known as the "arrow", connects the variable and constant regions of the heavy chain. The "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect two heavy chain polypeptides to each other in an intact antibody. Each light chain consists of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "arrow". Intact antibody tetramers consist of two heavy chain-light chain dimers in which the heavy and light chains are linked by a single disulfide bond; two other disulfide bonds connect the hinge regions of the heavy chains to each other so that the dimers link together and form a tetramer. Naturally produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure called an "immunoglobulin fold" formed from two beta sheets (eg, 3-, 4-, or 5-strand sheets) packed opposite each other in a compressed anti-parallel beta-stem. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2 and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3 and FR4). When natural antibodies are folded, the FR regions form beta sheets, which provide a structural scaffold for the domains, and the CDR loop regions of both the heavy and light chains converge in three-dimensional space, forming one hypervariable antigen-binding site located at the end of the Y-shaped structure. The Fc region of natural antibodies binds to elements of the complement system as well as 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 characteristics of Fc regions to Fc receptors can be modulated by glycosylation or other modification. In some embodiments, the antibodies made and/or used in accordance with the present invention contain glycosylated Fc domains, including Fc domains modified or recombinantly altered by such glycosylation. For the purposes of the present invention, in specific embodiments, any polypeptide, or complex of polypeptides, containing sufficient sequences of immunoglobulin domains present in natural antibodies can be named, and/or used as, "antibody", whether such a polypeptide is naturally produced (e.g., produced an antigen-responsive organism), or produced by recombinant modification, chemical synthesis, or other artificial system or methodology. In some embodiments, the antibody is polyclonal; in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, and so on, as is known in the art. In addition, as used herein, the term "antibody" in the respective embodiments (unless otherwise indicated or it is not clear from the context) can mean any of the constructs or formats known in the art or developed for using the structural and functional features of antibodies in an alternative representation. . For example, in embodiments, an antibody used in the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi - or multispecific antibodies (eg, Zybodies ^® and so on); antibody fragments such as Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fd' fragments, Fd fragments, as well as selected CDR regions or sets thereof; single chain Fv fragments; polypeptide-Fc fusion proteins; single domain antibodies (eg single domain shark antibodies such as IgNARs or fragments thereof); camelid antibodies; masked antibodies (eg ^Probodies® ); small modular immunopharmaceuticals (“SMIP™”); single-chain or tandem diabodies (TandAb ^® ); ^Humabodies® , VHH; anticalin ^® ; nanotel ^® , minitel; ^BiTE® ; proteins with ankyrin repeats or darpins ^® ; avimers ^® ; DART; TCR-like antibodies; adnectins ^® ; affilins ^® ; transtel ^® ; affitel ^® ; ^TrimerX® ; microproteins; finomers ^® , sentirins ^® and calbitors ^® . In some embodiments, an antibody may lack a covalent modification (eg, glycan attachment) that it would have if produced naturally. In some embodiments, the antibody may have a covalent modification (eg, glycan attachment, payload [eg, detectable moiety, therapeutic moiety, catalytic moiety, etc.] or other pendant group [eg, polyethylene glycol, etc.].

As used herein, the term "antibody fragment" means a portion of an antibody or antibody agent described herein, and generally means a portion that includes an antigen-binding portion or variable region thereof. The antibody fragment can be obtained by any method. For example, in some embodiments, an antibody fragment can be obtained by enzymatic or chemical fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, the antibody fragment may be obtained by recombinant methods (i.e., by expression of a genetically modified nucleotide sequence. In some embodiments, the antibody fragment may be wholly or partially obtained by synthesis. In some embodiments, the antibody fragment (in particular antigen-binding antibody fragment) may be at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more in length, in some embodiments, at least least about 200 amino acids.

Used in this document, the term "binding", as a rule, means non-covalent association between, or among, two or more molecules. "Direct" binding includes physical contact between molecules or fragments; indirect binding involves physical interaction through physical contact with one or more intermediate molecules. Binding between two or more molecules can generally be assessed in any of a variety of contexts - including cases where interacting molecules or fragments are studied in isolation or in the context of more complex systems (e.g., covalently or otherwise bonded to a carrier molecule and/or in 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 such that they have a phenotype aberrant growth, characterized by a significant loss of control of cell proliferation. In some embodiments, the tumor may be, or contain, cells that are precancerous (eg, benign), malignant, pre-metastatic, metastatic, and/or non-metastatic. This document specifically identifies certain types of cancer in which the ideas described in the document may be particularly useful. In some embodiments, the implementation of the relevant cancer can be characterized as a solid tumor. In some embodiments, the implementation of the relevant cancer can be characterized as a hematological tumor. Generally, examples of various cancers known in the art include, for example, hematopoietic cancers, including leukemias, lymphomas (Hodgkin's and non-Hodgkin's), myelomas, and myeloproliferative diseases; sarcomas, melanomas, adenomas, hard tissue carcinomas, squamous cell carcinoma of the mouth, throat, larynx and lung, liver cancer, cancer of the genitourinary system such as prostate cancer, cervical cancer, bladder cancer, cancer of the uterus, endometrial cancer and renal cell carcinoma , bone cancer, pancreatic cancer, skin cancer, skin or intraocular melanoma, endocrine system cancer, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, cancer of the gastrointestinal tract and cancer of the nervous system, benign lesions, such as papillomas, and the like.

As used herein, the term "CDR" refers to a complementarity-determining region within the variable region of an antibody. In each of the heavy chain and light chain variable regions, there are three CDR regions, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. "Set of CDRs" or "set of CDRs" means a group of three or six CDRs that are present in any one variable region capable of antigen binding, or the CDRs of the respective heavy and light chain variable regions capable of antigen binding. In the field, specific systems have been created to define CDR boundaries (eg Kabat, Chothia, etc.); those skilled in the art understand the differences between and among these systems and are able to understand the boundaries of the CDR to the extent necessary to understand and practice the claimed invention.

As used herein, the term "chemotherapeutic agent" has the meaning accepted in the art referring to one or more pro-apoptotic, cytostatic and/or cytotoxic agents, for example, including agents used and/or recommended for use in the treatment of one or more diseases, disorders or conditions associated with unwanted cell proliferation. In many embodiments, chemotherapeutic agents can be used to treat cancer. In some embodiments, the chemotherapeutic agent may be, or contain, one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g., microtubule-targeting agents such as taxanes, maytansine, and their analogs), one or more epothilones, one or more HDAC histone deacetylase inhibitors), one or more topoisomerase inhibitors (e.g., topoisomerase I and/or topoisomerase II inhibitors), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and/or one or more analogs of one or more of the foregoing (i.e., also having appropriate antiproliferative activity). In some specific embodiments, the chemotherapeutic agent may be, or contain, one or more of 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 its analogs (e.g. DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. In some embodiments, a chemotherapeutic agent may be used in the context of an antibody-drug conjugate. In some embodiments, the chemotherapeutic agent is an agent in an antibody drug conjugate selected from the group consisting of: hLL1-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 Ozagomycin, Brentuximab Vedotina, Trastuzumab Emtansine, Inotuzumab Ozagomycin, Glembatumomab Vedotina, 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, vorsetuzumab mafodotina and lorvotuzumab mertanzine.

As used herein, the term "combination therapy" refers to situations in which two or more therapeutic regimens (eg, two or more drugs) are used simultaneously for an individual. In some embodiments, two or more therapeutic regimens may be used simultaneously. In some embodiments, two or more therapeutic regimens may be used sequentially (eg, the first regimen is used before any dose from the second regimen is used). In some embodiments, two or more therapeutic regimens are used in overlapping dosing regimens. In some embodiments, the implementation of the use of combination therapy may include the receipt of one or more drugs, or procedures, an individual receiving another drug(s) or procedure.

The term "framework" or "framework region" as used herein refers to variable region sequences minus CDRs. Since the CDR sequence can be determined using different systems, the framework sequence is also subject to different interpretations. The six CDRs divide the framework regions on the heavy and light chains into four subregions (FR1, FR2, FR3 and FR4) on each chain, with CDR1 located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying sub-regions, FR1, FR2, FR3 or FR4, a framework region, as mentioned elsewhere, is the combined FR regions in the variable region of a single natural immunoglobulin chain. Herein, FR is one of four sub-regions, FR1, for example, is the first framework region closest to the amino terminus of the variable region and located in the 5' direction relative to CDR1, and FR regions are two or more of the sub-regions constituting frame area.

As used herein, the term "humanized" is generally used to refer to antibodies (or antibody components) whose amino acid sequence includes the sequences of the VH and VL regions from a reference antibody produced in a non-human species (e.g., a mouse), but also has modifications in these sequences compared to a reference antibody, designed to make them more "human-like", that is, more similar to human germline variable region sequences. In some embodiments, a “humanized” antibody (or antibody component) is an antibody that binds an antigen of interest with immunological specificity and that has a framework (FR) region containing an amino acid sequence substantially the same as that of a human antibody and defining complementarity. a region (CDR) containing an amino acid sequence substantially the same as that of a non-human antibody. A humanized antibody contains substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') ₂ , FabC, Fv) in which all or substantially all of the CDR regions correspond to those in a non-human immunoglobulin (ie, a donor immunoglobulin), and all, or substantially all, of the framework regions are regions having a human immunoglobulin consensus sequence. In some embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically from a human immunoglobulin constant region. In some embodiments, the humanized antibody contains both a light chain and at least a heavy chain variable domain. The antibody may also contain a CH1 region, a hinge, a CH2, a CH3, and optionally a CH4 heavy chain constant region.

As used herein, the term " in vitro " refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, and so on, and not in a multicellular organism.

As used herein, the term " in vivo " refers to events that occur in a multicellular organism such as a human and a non-human animal. In the context of cellular systems, the term can be used in relation to events that occur in a living cell (as opposed to, for example, in vitro systems).

As used herein, the term "isolated" refers to a substance and/or molecule that has been (1) separated from at least some of the components with which it was associated during initial production (either in nature and/or under experimental conditions). ) and/or (2) designed, produced, obtained and/or manufactured by a human hand. The isolated substances and/or molecules can be separated from 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 more than about 99% of the other components with which they were originally associated. In some embodiments, isolated agents are about 80% pure, about 85% pure, about 90% pure, about 91% pure, about 92% pure, about 93% pure, about 94% pure, about 95% pure, about 96% pure, about 97% pure, about 98% pure. %, about 99% or more than about 99%. In this document, a substance is considered "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, the substance can still be considered "isolated", or even "pure", after combining it with some other components, such as, for example, one or more carriers or excipients (for example , buffer, solvent, water and so on); in such embodiments, percent recovery or purity is calculated without taking into account such carriers or excipients. As one example, in some embodiments, a biological polymer, such as a naturally occurring polypeptide or polynucleotide, is considered "isolated" when a) due to its origin or source of preparation, it is not associated with some or all of the components, which accompany him in his natural state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that naturally produces it; c) it is expressed, or otherwise associated with, components from a cell or other expression system that does not belong to the species that naturally produces it. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system other than that which naturally produces it is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification methods may be considered an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is naturally associated; and/or b) with which it was associated in the original production.

As used herein, the term "K _D " means the dissociation constant of a binding substance (eg, an antibody or its binding component) from a complex with its binding partner (eg, an epitope to which the antibody or its binding component binds).

As used herein, the term "macrophage" means a cell of the monocyte/macrophage lineage that resides in the spleen or has been differentiated into a tissue macrophage. These cells include follicular dendritic cells (FDCs), dendritic cells, Langerhans cells, and other tissue macrophages. Macrophages are phagocytes and antigen presenting cells that differentiate from monocytes in the circulating peripheral blood. They play an important role in both innate and adaptive immunity through the activation of T-lymphocytes. Macrophages that activate Th1 T lymphocytes mediate the inflammatory response and are referred to as M1 macrophages. M1 macrophages, also called killer macrophages, inhibit cell proliferation, cause tissue damage, and are aggressive towards bacteria. Macrophages that activate Th2 T lymphocytes provide an anti-inflammatory response and are referred to as M2 macrophages. M2 macrophages, also called "repair macrophages", stimulate cell proliferation and tissue repair, and are anti-inflammatory. As used herein, the term "tumor-associated macrophages" (TAM) generally refers to macrophages that are present in the microenvironment of a cancer, such as a tumor.

As used herein, the term "operably linked" refers to an arrangement in which the components described are in a relationship that allows them to function in their intended manner. A control element "operably linked" to a functional element is linked in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, "operably linked" control elements are adjacent (eg, covalently linked) to the coding elements of interest; in some embodiments, the control elements operate in a trans position, or other position, with respect to the functional element of interest.

Used in this document, the term "pharmaceutical composition" means a composition in which the active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to an individual, human, or animal. In some embodiments, the active agent is present in a unit dosage amount suitable for administration in a therapeutic regimen that has a statistically significant probability of achieving a predetermined therapeutic effect when administered in an appropriate population.

The term "polypeptide" as used herein generally has the meaning recognized in the art, i.e., means a polymer of at least three amino acids. Those skilled in the art will appreciate that the term "polypeptide" should be generic enough to include not only polypeptides having the complete sequence recited herein, but also polypeptides that are functional fragments (i.e., fragments that retain at least at least one activity) of such complete polypeptides. In addition, those skilled in the art will appreciate that protein sequences generally allow for some substitution without disruption of activity. Thus, any polypeptide that retains activity and has at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and in addition typically has at least one area with a higher degree of identity, often more than 90% or even 95%, 96%, 97%, 98% or 99% in one or more highly conservative areas, usually covering at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed by the appropriate term "polypeptide" as used herein. The polypeptides may contain L-amino acids, D-amino acids, or both, and may have any of a variety of amino acid modifications or analogues known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and so on. In some embodiments, proteins may contain natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

As used herein, the term "prevent" or "prevention", when used in relation to the development of a disease, disorder and/or condition, means to reduce the risk of developing the disease, disorder and/or condition, and/or delay the onset and/or reduce the severity of one or more signs or symptoms of a disease, disorder, or condition. In some embodiments, prevention is assessed on a population basis, such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the degree, frequency, and/or intensity of one or more symptoms of the disease, disorder, or condition is observed in the population. affected by a disease, disorder, or condition.

As used herein, the term "recombinant" refers to polypeptides that are designed, constructed, manufactured, expressed, created, produced and/or isolated by recombinant methods, for example, polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial library of human polypeptides; polypeptides derived from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or otherwise altered to express a gene or genes or components of genes that encode and/or directly express a polypeptide or one or more than its components, parts, elements or domains; and/or polypeptides obtained, expressed, created or isolated by any other means, including splicing or ligation of selected elements of the nucleotide sequence with each other, chemical synthesis of selected elements of the sequence and/or otherwise obtaining a nucleic acid that encodes and/or directs the expression of the polypeptide or one or more of its components, parts, elements or domains. In some embodiments, one or more of such selected sequence elements occur naturally. In some embodiments, one or more of such selected sequence elements are designed in silico . In some embodiments, one or more of such selected sequence elements result from mutagenesis (e.g., in vivo or in vitro ) of a known sequence element, e.g., from a natural or synthetic source, such as, for example, in the germline of the parent organism of interest (e.g., human, mouse, etc.).

As used herein, the term "specific binding" means the ability to discriminate between possible binding partners in the environment in which the binding will take place. A binding agent that interacts with one specific target in the presence of other potential targets is referred to as "specifically binding" the target with which it interacts. In some embodiments, specific binding is assessed by detecting, or determining the extent of, an association between a binding agent and its partner; in some embodiments, specific binding is assessed by detecting, or determining the extent, 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 with an alternative interaction between its partner and another molecule. In some embodiments, specific binding is assessed by conducting such a detection or determination over a range of concentrations.

As used herein, the term "individual" refers to an organism, typically a mammal (eg, a human, in some embodiments including a prenatal human). In some embodiments, the implementation of the individual suffers from a corresponding disease, disorder or condition. In some embodiments, the individual is predisposed to the disease, disorder, or condition. In some embodiments, the individual exhibits one or more symptoms or signs of a disease, disorder, or condition. In some embodiments, the individual does not exhibit any symptom or sign of the disease, disorder, or condition. In some embodiments, the implementation of the individual is an individual with one or more features that are characteristic of the predisposition or risk of developing a disease, disorder or condition. In some embodiments, the individual is a patient. In some embodiments, the individual is an individual who is diagnosed and/or has been diagnosed and/or is receiving and/or has received therapy.

As used herein, the term "drug" generally means any agent that produces the desired pharmacological effect when administered to the body. In some embodiments, an agent is considered a drug if it exhibits a statistically significant effect in an appropriate population. In some embodiments, the implementation of the corresponding population may be a population of model organisms. In some embodiments, an appropriate population may be determined based on various criteria such as specific age group, gender, genetic background, pre-existing clinical conditions, and so on. In some embodiments, a drug is a substance that can be used to alleviate, improve, alleviate, inhibit, prevent, delay the onset of development, reduce the severity and/or reduce the incidence of one or more symptoms or signs of a disease, disorder and/or states. In some embodiments, a "drug" is an agent that has been, or must be, approved by a government agency before it can be marketed for administration to humans. In some embodiments, a "drug" is an agent for which humans require a medical prescription to administer.

As used herein, the term "therapeutically effective amount" means an amount that is sufficient when administered to populations of individuals suffering from, or predisposed to a disease, disorder and/or condition, in accordance with a therapeutic dosage regimen for the treatment of a disease, disorder and/ or states. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and/or severity, stabilizes one or more characteristics, and/or delays the onset of one or more symptoms of a disease, disorder, and/or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not actually mean that successful treatment has been achieved for a particular individual. Rather, a therapeutically effective amount may be that amount which provides a particular desired pharmacological response in a significant number of individuals when administered to patients in need of such treatment. For example, in some embodiments, the term "therapeutically effective amount" means an amount that, when administered to an individual in need thereof, in the context of the therapy of the invention, will block, stabilize, alleviate, or reverse a cancer-supporting process occurring in the body of said individual, or will enhance or stimulate a cancer-suppressing process in said individual. In the context of cancer treatment, a "therapeutically effective amount" is an amount that, when administered to an individual diagnosed with cancer, will prevent, stabilize, inhibit, reduce the further development of cancer in the individual. A particularly preferred "therapeutically effective amount" of the composition described herein reverses (in therapeutic treatment) the progression of a cancer, such as pancreatic carcinoma, or helps achieve or prolong remission of a cancer. A therapeutically effective amount administered to an individual to treat that individual's cancer may be the same as, or different from, a therapeutically effective amount administered to induce remission or inhibit metastasis. As is the case with most anti-cancer therapies, the therapeutic methods described herein are not to be construed, limited, or otherwise limited to "cure" cancer; rather, the methods of treatment are directed to using the described compositions to "treat" cancer, that is, to bring about a desired or beneficial change in the health status of an individual suffering from cancer. Such benefits are recognized by those skilled in the art of oncology and include, but are not limited to, stabilization of the patient's condition, reduction in tumor size (tumor regression), improvement in vital functions (e.g., improved function of cancerous tissues or organs), reduction or inhibition of further metastasis, decrease in opportunistic infections, increase survival, decrease pain, improve motor function, improve cognitive function, improve sense of energy (vitality, decrease malaise), improve well-being, restore normal appetite, restore healthy weight gain, and combinations thereof. In addition, the regression of a particular tumor in an individual (for example, as a result of the treatments described herein) can also be assessed by sampling cancer cells from a tumor site, such as pancreatic adenocarcinoma (for example, throughout the course of treatment), and testing for cancer cells at the level of metabolic and signaling markers to monitor the state of cancer cells in order to confirm at the molecular level the regression of cancer cells to a less malignant phenotype. For example, tumor regression induced by the methods of the present invention would be indicated by the detection of a decrease in any of the pro-angiogenic markers described above, an increase in the anti-angiogenic markers described herein, normalization (i.e., a change towards a state characteristic of healthy individuals not suffering from cancer) metabolic pathways, intercellular signaling pathways, or intracellular signaling pathways that exhibit abnormal activity in individuals diagnosed with cancer. Those skilled in the art will appreciate that in some embodiments, a therapeutically effective amount may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and/or administered in multiple doses, eg, as part of a dosing regimen.

As used herein in the context of molecules, such as nucleic acids, proteins, or small molecules, the term "variant" means a molecule that has significant structural identity with a reference molecule, but differs structurally from a reference molecule, such as the presence or absence of, or level, one or more chemical moieties compared to a reference molecule. In some embodiments, the variant also differs functionally from its reference molecule. Generally, the decision as to whether a particular molecule can be considered a "variant" of a reference molecule is based on its degree of structural identity with the reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is another molecule that shares one or more of these characteristic structural elements, but differs in at least one aspect from the reference molecule. As some examples, a polypeptide may have a characteristic element of the sequence, consisting of a plurality of amino acids having certain positions relative to each other in linear or three-dimensional space and/or participating in a specific structural motif and/or biological function; a nucleic acid may have a characteristic element of the sequence, consisting of a plurality of nucleotide residues having certain 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 as a result of one or more amino acid or nucleotide sequence differences. In some embodiments, the variant polypeptide or nucleic acid has a common sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97% or 99%. In some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more biological activities with the reference polypeptide or nucleic acid.

As used herein, the term "vector" means a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. One type of vector is a "plasmid", which is a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is the viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they have been introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of a host cell when introduced into the host cell and thus replicate along with the host genome. In addition, some vectors are capable of direct expression of the genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". Standard techniques can be used for recombinant DNA production, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification methods can be used according to the manufacturer's instructions, or as is commonly done in the art, or as described herein. The above methods and procedures may generally be performed in accordance with conventional methods well known in the art and as described in the various general and more specific literature sources cited and described in this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), the contents of which are incorporated herein by reference for all purposes.

Macrophages in cancer

Macrophages are phagocytes 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 through the activation of T-lymphocytes. Macrophages that activate Th1 T lymphocytes mediate the inflammatory response and are referred to as M1 macrophages. M1 macrophages, also called killer macrophages, inhibit cell proliferation, cause tissue damage, and are aggressive towards bacteria. Macrophages that activate Th2 T lymphocytes provide an anti-inflammatory response and are referred to as M2 macrophages. M2 macrophages, also called "repair macrophages", stimulate cell proliferation and tissue repair, and are anti-inflammatory.

When macrophages are formed by monocyte differentiation, monocytes mature into either M1 (CD68 ⁺ and CD80 ⁺ ) or M2 (CD68 ⁺ and CD163 ⁺ ) macrophages depending on the cytokines and growth factors that cause them to differentiate. Lipopolysaccharide (LPS) and interferon-gamma (IFNγ) 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, an interleukin 1 receptor antagonist (IL-1ra), and transforming growth factor beta (TGFβ) activate monocytes to differentiate into M2 macrophages that secrete high levels of IL-10, TGFβ, and insulin-like growth factor 1 (IGF-1), and low levels of IL-12.

The role of immunity in oncogenesis is becoming increasingly recognized. Since macrophages are known to play important roles in both innate and adaptive immunity, they have been recognized as key components of tumors and their microenvironment. Tumor-associated macrophages (TAMs) are typically macrophages that reside in the microenvironment of a cancerous tumor. The role of tumor-associated macrophages (TAMs) in tumor growth, invasion, and metastasis has been extensively investigated, and TAMs are known to exhibit a wide range of phenotypes, from an M1-like phenotype in the early stages of selected tumors to an M2-like phenotype in the most advanced tumors. As evidence of their role in promoting oncogenesis, M2 macrophages show a characteristic phenotype with increased expression of IL-10, IL4, MMP, and VEGF, but reduced expression of pro-inflammatory cytokines and cytotoxic iNO and ROI, which are involved in tumoricidal activity. In addition to their intrinsic function in promoting tumorigenesis, TAMs are also involved in the suppression of antitumor immunity by altering T-cell responses and balance in the tumor microenvironment.

In addition to stimulating cell proliferation, by playing an anti-inflammatory role, in M2 cancer, macrophages can induce vascularization in the tumor area. Therefore, in such scenarios, it would be beneficial to inhibit M2 polarization of macrophages and induce M1 polarization of macrophages known to attack tumor cells.

VSIG4

VSIG4 (V-set and domain-containing immunoglobulin protein 4) is a B7-family-related membrane protein belonging to the complement receptors of the immunoglobulin superfamily (CRIg) known to negatively regulate CD8 ⁺ T cell proliferation and IL-2 production by binding iC3b and C3b. Expression of VSIG4 is limited to tissue macrophages, including abdominal macrophages and liver-based Kupffer cells. Figures 2A and 2B show the association of VSIG4 (homology and phylogenetic relationships, respectively) with other B7 family proteins (VSIG4 is referred to as EU103 in Figures 2A and 2B). Figure 2A shows the amino acid sequence alignment of VSIG4 with various other B7 family proteins. Figure 2B shows the evolutionary relationship between VSIG4 and other B family proteins.

Anti-VSIG4 antibody for cancer treatment

Tumor-associated macrophages (TAMs) are the key cells that create the immunosuppressive tumor microenvironment (MTM), providing multiple targets for immunotherapy. It is also known that macrophages are very plastic and can easily change polarity and acquire an antitumorogenic M1-like phenotype.

Tumor-associated macrophages (TAMs) are generally known for their protumorogenic functions, such as promoting cancer cell movement, metastasis formation, and angiogenesis. The formation of OAM depends on the microenvironmental factors that are present in the developing tumor. TAMs are abundant in many types of cancers, especially in the tumor microenvironment, and often exhibit an immunosuppressive M2-like phenotype that stimulates tumor growth and contributes to the development of therapy resistance. TAMs also produce immunosuppressive cytokines such as IL-10, TGFβ and PGE2, very small amounts of NO or ROI, and low levels of inflammatory cytokines such as IL-12, IL-1β, TNFα and IL-6. The conversion of macrophages into OAM leads to a decrease in the ability to present tumor-associated antigens and stimulate the antitumor functions of T and NK cells. In addition, OAMs are unable to lyse tumor cells. As a result, targeting of TAMs is a novel therapeutic strategy for suppressing and treating cancer, for example, by delivering agents to either alter the recruitment and distribution of TAMs, deplete existing TAMs, or induce "relearning" (or conversion) of TAMs from an M2 to an M1 phenotype.

The present invention is based on the discovery that VSIG4-expressing M2 macrophages can be treated with humanized anti-VSIG4 antibodies to convert (or reverse) M2 macrophages into tumor-suppressing M1 macrophages, thereby inducing CD8 ⁺ T cell proliferation and production of pro-inflammatory cytokines, leading to tumor suppression. In addition, the use of anti-VSIG4 antibodies can effectively suppress cancer by both (1) converting M2 macrophages to M1 macrophages and (2) inducing CD8+ T cell proliferation and production of pro-inflammatory cytokines, with effects on the tumor microenvironment itself. This anti-VSIG4 antibody approach for suppressing cancer is superior to other therapies that only induce T cell proliferation or only block the tumor-stimulating activity of macrophages. See Figure 1 (schematically depicting the effects of anti-VSIG4 antibodies on macrophage function, their effects on T cell proliferation and subsequent cancer suppression.)

Mouse antibody humanization

Although monoclonal antibodies can be rapidly produced by the immune system of mice for biological research, under clinical conditions, the use of these mouse antibodies can lead to the development of a human response to mouse antibodies (HAMA). While chimeric antibodies may elicit less pronounced anti-IgG responses in humans, murine variable domains may still contain T-cell provoking epitopes, resulting in the need to "humanize" their frameworks.

Classical antibody humanization typically begins by transferring all six murine complementarity determining regions (CDRs) to a human antibody framework (Jones et al., Nature 321, 522-525 (1986)). These CDR-grafted antibodies generally do not retain their original affinity upon antigen binding and, in fact, affinity is often seriously degraded. In addition to the CDRs, some non-human framework residues must also be included in the variable domains to maintain the correct CDR conformation (Chothia et al., Nature 342:877 (1989)). The incorporation of murine residues into key positions of human antibody framework regions to restore function is generally referred to as "backmutations". Back mutations can help maintain the structural conformation of the grafted CDRs and restore antigen binding and affinity. Many of the framework positions that are likely to affect affinity have been identified, so structural modeling to select new residues in a step-by-step fashion can usually result in variants with restored antigen binding capacity. Alternatively, antibody phage 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 TFH-activated B cells produce antibodies with increased affinity for a particular antigen during an immune response. With repeated exposures to the same antigen, the host will produce antibodies with successively increasing affinity. The secondary response can produce antibodies with an affinity that is several times higher than the primary response. Affinity maturation is an important strategy in antibody optimization for the development of safe and effective second generation therapeutics. Typically, therapeutic antibodies are obtained by immunizing mice or transgenic animals expressing human immunoglobulin genes with the desired antigen. Antigen-stimulated immune cells from these animals are transformed into hybridomas and then screened to identify monoclonal antibodies with low nanomolar affinity for their target antigen. In vivo , natural affinity maturation in the immune system occurs as a result of somatic hypermutations and clonal selection, while in vitro , with laboratory affinity maturation, it can be achieved through mutations and selection. In addition, other methods of affinity maturation, in addition to methods using TFH-activated B cells, are known in the art and are within the scope of the present invention.

EXAMPLES

Further, the invention is described using the following examples, which do not limit the scope of the invention described in the claims.

METHODS AND MATERIALS

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

Isolation of CD14 ⁺ monocytes from human PBMCs

Differentiation of macrophages into M1 or M2 macrophages was performed by obtaining PBMCs from individuals and incubating macrophages in 50 ng/ml hGM-CSF solution for 6 days to convert to M1 macrophages or incubating macrophages in 100 ng/ml M-CSF solution for 6 days to convert to M2 macrophages (Figure 5).

The transformation of macrophages into M1 or M2 macrophages was confirmed by checking the phenotype (Figures 6 and 7).

The subsequent transformation of M1 or M2 macrophages into M1 macrophages was performed by incubating macrophages in a mixture of LPS (100 ng/ml) + IFNγ (100 ng/ml) or 500 ng/ml solution of anti-VSIG4 antibody (EU103.2) for 24 hours ( Figure 12).

The transformation of M1 or M2 macrophages into M2 macrophages was performed by incubation of macrophages in 20 ng/ml IL-4 solution for 24 hours (Figure 12).

Humanized anti-VSIG4 antibody - EU103.2 antibody

The EU103.2 antibody is a humanized anti-VSIG4 antibody derived from the mouse anti-VSIG4 antibody mu6H8. Figures 4A and 4B show the biochemical characterization of the EU103.2 antibody by size exclusion HPLC (Figure 4A) and surface plasmon resonance, demonstrating the binding of VSIG4 to the EU103.2 antibody (Figure 4B). A summary of the purification of the EU103.2 antibody is shown in Table 1 below.

TABLE 1: SEC HPLC summary for EU103.2 antibody

Concentration 30 ml total (x5) when transfected with Expi293F EU103.2 1.2 mg/ml × 7 ml
0.37 mg/ml × 2 ml 9.14 mg

Humanization of mouse anti-VSIG4 antibody - EU103.3 antibody

Humanized anti-VSIG4 antibody hu6H8 (or EU103.3) was obtained as described below.

Humanization mu6H8 VH

A scaffold for a humanized VH variant was generated using mouse 6H8 antibody and Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) (germline gene: VH2-5/D3-3/JH6c ). The Kabat numbering system for CDR classification was used, and humanized VHs were designed with scaffold and mu6H8 classified VH CDRs, VH2, VH27, VH30, VH93 and VH94 backmutations. (hu6H8.3VH).

Humanization mu6H8 VL

A scaffold for a humanized VL variant was generated using mouse 6H8 antibody and Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) (germline gene: A17/JK2). The Kabat numbering system was used to classify CDRs and humanized VLs were designed with scaffold and mu6H8 classified VL CDRs, VH2, VH4, VH36 and VH46 backmutations. (hu6H8.3 VL).

Cloning and expression of IgG antibody - EU103.3 antibody

The heavy chain variable region sequence was modified by FES (L234F, L235E, P331S) mutations to construct the heavy chain expression plasmid pOptivec (Invitrogen) without Fc effector functions as shown in Figure 21A. The light chain variable region sequence was constructed using pcDNA3.3 (Invitrogen) and synthesized using IDT as shown in Figure 21B. The gene encoding the heavy chain (HC) was flanked with restriction enzyme sites EcoR1, Nhe1 to construct the plasmid vector pOptivec (FES), and the gene encoding the light chain (LC) was flanked by restriction enzyme sites EcoR1, BsiW1 to construct the plasmid vector pcDNA3.3. Cloning was performed with mutation sites subcloned into the hu6H8.3 framework. Each of the resulting insert genes and linearized vectors was cloned using In-Fusion^® HD cloning kit (Clontech), and sequencing primer was defined with CMV forward, EMCV IRES reverse primer.

VSIG4 knockout mice

VSIG knockout (K/O) mice were generated by homologous recombination replacing exon 1 with the neomycin resistance gene. A targeting vector was prepared for use in homologous recombination in ES cells. E1 and E1 and E2 denote exon 1 and 2 of the CRIg gene (Figure 41A). Homologous recombination of the CRIg allele in heterozygous female offspring from crossing chimeric mice with ES cell clones 1 and 2 (C1, C2) with DT mice was confirmed by Southern blotting (Figure 41B). The number of leukocytes in the peripheral blood of female and male DT and K/O mice was compared. The total number of cells was determined using a hematocytometer. Leukocytes were incubated with fluorochrome-conjugated antibodies specific for several cell surface markers, and the number of different subgroups of leukocytes was determined by flow cytometry. Data represent means+SD for 5-7 mice (Figure 41C).

Antibodies A1, A2, A1.3 and A2.3

Antibodies A1 and A2 were generated by affinity maturation of the EU103.3 antibody (see Table 2 below) with the light chain variable regions at positions 76, 90 and/or 92 (Kabat numbering) mutated as shown in Table 2 below.

Antibodies A1.3 and A2.3 were generated from antibodies A1 and A2, respectively, to further improve affinity for VSIG4.

Figure 40 shows the amino acid sequence alignment for heavy chains and light chains EU103.3, A1, A2, A1.3 and A2.3, along with consensus amino acid sequences for the heavy chain and light chain. Amino acid residues that differ between antibodies are shown in rectangular boxes.

TABLE 2: Humanized anti-VSIG4 antibody clones screened in affinity maturation experiments

Name At Library HC LC Mutation sites (HC)
(Kabat numbering) Mutation sites (LC)
(Kabat numbering) T01.01(A1) L3A DT T01L1 - H90Q, R92G T01.02(A2) L3A DT T01L2 - L76F, H90Q, R92G T01.03 L3B DT T01L3 - L76F, H90Q, R92F T01.04 L3B DT T01L4 - L76F, H90Q, R92L T01.05 H3B T01H1 DT N99H - T01.06 H3B T01H2 DT Q1K, N99S - T01.07 H3B T01H3 DT K98E, N99K - T01.08 L2 DT T01L5 - S52E, M106I T01.09 L2 DT T01L6 - L54R, M106I T01.10 L2 DT T01L7 - S52E, L54R, M106I T01.11 H1 T01H4 DT S32Y - T01.12 H1 T01H5 DT S32F - T01.13 H2A T01H6 DT D50E, F52Y, W53S, D54G, D55E - T01.14 H2A T01H7 DT D50E, W53S - T01.15 L1C DT T01L8 - L76F T01.16 L1C DT T01L9 - M33V, L76F T01.17 L1A DT T01L10 - K27E, L76F T01.18 L1B DT T01L11 - T27E, L76F T01.19 L1B DT T01L12 - T27K, L76F T01.20 H1 T01H8 DT S32W -

Obtaining antibodies with high binding affinity

The humanized antibody gene was inserted into a plasmid and expressed as IgG using the Expi293 Expression System (Invitrogen), then purified using AktaPure (GE healthcare), AktaPrime purifier (GE healthcare), and a MabselectSURE column (GE healthcare cat. no. 11-0034-95 ). Purified antibodies were passed through a desalting column (GE healthcare cat. no. 17-1408-01) with PBS buffer exchange and the antibody concentration was measured using Multiskan GO (Thermo).

TABLE 3: Yields for High Binding Affinity Antibodies

Name At Culture volume (ml) Ab concentration (mg/ml) Yield (mg) T01.01(A1) thirty 0.68 1.020 T01.02(A2) thirty 0.22 0.330 T01.03 thirty 0.87 0.104 T01.04 thirty 0.99 0.119 T01.05 thirty 0.26 0.390 T01.06 thirty 1.2 0.144 T01.07 thirty 0.33 0.495 T01.08 thirty 1.46 0.292 T01.09 thirty 1.73 0.346 T01.10 thirty 2.07 0.414 T01.11 thirty 0.27 0.054 T01.12 thirty 1.26 0.252 T01.13 thirty 0.72 0.144 T01.14 thirty 0.89 0.178 T01.15 thirty 0.21 0.042 T01.16 thirty 1.05 0.21 T01.17 thirty 0.76 0.15 T01.18 thirty 0.33 0.07 T01.19 thirty 0.44 0.09 T01.20 thirty 0.99 0.198

Differentiation of PBMC-derived macrophages

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

1. 20 ml blood/PBS mixture (1:1) overlaid on 10 ml Ficoll-Paque™ Plus (GE Healthcare p/n 17-1440-02)

2. Centrifuge at 400xg for 35 min (acceleration 2, brake 0)

3. PBMC is isolated and washed with RPMI-1640 (WelGene, Cat # LB011-01) at 2000 rpm for 5 min x 2 times

4. Cells are counted

5. MACs buffer (2% EBS (Millipore, cat. no. TMS-013-BKR) in PBS (WelGene, cat. no. LB004-02) 1~2 ml suspension

6. Add CD14 microbeads (20 µl/10 ⁷ cells) (Miltenyi Biotec, cat. no. 130-050-201)

7. Incubate for 30 min on ice

8. Wash MACs with buffer at 2000 rpm for 5 min x 2 times

9. Cell count

10. Load the cells onto a MACs column (Miltenyi Biotec, cat. no. 130-042-401)

11. Perform positive selection and cell count

12. Suspend in culture medium (RPMI-1640+10% EBS+penicillin/streptomycin (Gibco, cat. no. 15140-122) + Glutamax (Gibco, cat. no. 35050-061) + 20~40 ng/ml rhM-CSF ( Biolegend, cat. no. 574806)

13. Seeding CD14 ⁺ cells in a 100 mm culture dish (1 x 10 ⁶ cells/10 ml/dish) (Thermo Scientific, cat. no. 150466)

14. Then change culture medium every 3 days

15. After 7~10 days, check the differentiation of M0 macrophages by FACS

16. Differentiate M0 into M1 or M2 macrophages with LPS 20 ng/ml (Sigma-Aldrich, cat. no. L4391) + rhIFNγ 20 ng/ml (Biolegend, cat. no. 570204) (M1) and rhIL4 20 ng/ml (Biolegend 574002) + rhIL13 20 ng/mL (Biolegend ref 571102) (M2) in culture medium (RPMI-1640 +10% FBS+penicillin/streptomycin+Glutamax) for 2 days

17. After differentiation into M1 or M2, check the cell phenotype by FACS

18. To convert M2 to M1, macrophages add antibodies 20 µg/ml or LPS 20 ng/ml+rhIFNγ 20 ng/ml (positive control) in fresh culture medium (RPMI-1640 +10% FBS+penicillin/streptomycin+Glutamax) for 2 days

19. After converting M2 to M1, macrophages check the cell phenotype by FACS and check the content of cytokines/chemokines in the culture supernatant using LEGENDplex™ (Biolegend, catalog # 740502).

FACS analysis

The following antibodies were used for FACS analysis:

- hCD14-BV650 (BD Bioscience, catalog # 563419)

- hCD14-BV421 (BD Bioscience, catalog # 565283)

- hIFNγ-PE/Cy7 (BD Bioscience, catalog # 557844)

- hCD3-BV510 (BD Bioscience, catalog # 563109)

- hCD8-V450 (BD Bioscience P/N 560347)

- hCD68-PE (Biolegend, cat. no. 333808)

- hCD93-PE (Biolegend, cat. no. 336108)

- HLA-DR-BV421 (Biolegend, cat. no. 307636)

- hCD45-PE (Biolegend, cat. no. 304008)

- hCD64-APC (Biolegend, cat. no. 305014)

- hCD163-APC/Cy7 (Biolegend, cat. no. 333622)

- hCD86-PerCP/Cy5.5 (Biolegend p/n 305420)

- hCD86-BV421 (BD Bioscience, catalog # 562432)

Example 1 Expression of VSIG4 in Macrophages

VSIG4 is expressed in M2 macrophages. Figures 3A and 3B show correlation data for VSIG4 expression (as measured by mRNA) with expression of various genes associated with type 2 (M2) macrophages and tumor-associated macrophages. As shown in Figure 3A, VSIG4 expression is negatively correlated with [CXCL11, CXCL13, ZNMB, IFNAR1, IFNAR2] expression, but it is positively correlated with CCL19, IRF5 and IL1A expression. Figure 3B shows that the expression of VSIG 4 positively correlates with the expression of CD163, CSF1R, MSR1, TGFBR2, STAT6, IL1R1, IL10RA, MS4A4A, CCL2, CCL14, CCL17 and MS4A6A.

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

Example 2: Human anti-VSIG4 antibody induces secretion of cytokines and chemokines in M2 macrophages

M1 and M2 macrophages were treated with EU103.2 antibody and the secretion of pro-inflammatory cytokines and chemokines was measured. As shown in Figure 6, M2 treatment of EU103.2 macrophages resulted in the induction of cytokines and chemokines IL12, IFNγ, IL10 and IL23.

Example 3 Humanized Anti-VSIG4 Antibody Converts M2 Macrophages to M1 Macrophages

To further test the effects of EU103.2 on M2 macrophages, macrophages were treated with EU103.2 antibody and stained for the M2 macrophage marker CD163. As shown in Figure 7, EU103.2 treatment resulted in decreased expression of the M2 macrophage marker CD163 in M2 macrophages, indicating that blocking VSIG4 with EU103.2 resulted in the transformation of M2 macrophages into a different cell type.

The effect of EU103.2 antibody on macrophage-T cell interaction was then tested by co-incubation of M2 macrophages with CD8⁺ T cells with or without addition of EU103.2 antibody treatment. As shown in Figure 8 EU103.2 M2 treated macrophages co-incubated with CD8⁺ T cells caused CD8 proliferation⁺ T cells, this indicated that M2 macrophages are converted to M1 macrophages when treated with the EU103.2 antibody, since M1 macrophages induce CD8+ T cell proliferation while M2 macrophages suppress CD8+ T cell proliferation.

Then, to further investigate the effect of the EU103.2 antibody on human macrophages in the context of cancer biology, abdominal fluid samples were collected from patients with ovarian cancer and first analyzed for VSIG4 expression. As shown in Figure 9, macrophages obtained from the abdominal fluid of ovarian cancer patients contained M2 macrophages that coexpressed VSIG4 and CD14 and, as shown in Figures 10 and 11, induced CD8+ T cell proliferation.

Microscopic images then confirmed that treatment with EU103.2 antibody converted M2 macrophages into M1 macrophages as shown in Figure 12.

The role of VSIG4 signaling in macrophage-induced CD8+ T cell proliferation was further confirmed by co-culture of VSIG4-expressing HeLa cells with PBMCs, with or without the addition of an anti-VSIG4 antibody to block VSIG4, as shown in FIG. 13. This experiment showed that blocking the interaction between VSIG4 and CD8⁺ T cell leads to increased CD8 proliferation⁺ T cells.

To further explore the role of VSIG4 in relation to CD8 ⁺ T cells, THP-1 monocytic cells were co-incubated at various ratios with T cells, in the presence of either an anti-VSIG4 antibody or a control IgG antibody, and it was shown that the anti-VSIG4 antibody was able to increase the number of CD8 ⁺ T cells in 4 divisions, in contrast to the control IgG.

Example 4 Antitumor Effects of Blocking VSIG4 Signaling with an Anti-VSIG4 Antibody, or a VSIG4 Knockout Mouse Model

To determine the antitumor effects of an anti-VSIG4 antibody, three mouse tumor models were used: the MC38 mouse tumor model of colon adenocarcinoma, the B16F10 mouse tumor model of melanoma, and the 3LL mouse tumor model of lung carcinoma using VSIG4 knockout mice, and compared with wild-type mice. as shown in Figures 15A, 15B and 15C, respectively. Tumor growth was suppressed, especially in the MC38 and 3LL mouse tumor models, in VSIG4 knockout mice compared to wild-type mice (see Figures 15A and 15C, respectively), confirming that tumor growth is suppressed in the absence of VSIG4 signaling.

Similar inhibition of tumor growth was observed in the MC38 mouse tumor model in wild-type mice injected with anti-VSIG4 antibody compared to mice injected with control IgG, as shown in Figure 1. 16. The degree of tumor growth inhibition by the anti-VSIG4 antibody was at least as, if not more pronounced, than in VSIG4 knockout mice.

The activation status of lymphocytes from tumor-draining lymph nodes in VSIG4 knockout mice was then studied and compared with wild-type mice. As shown in Figures 17A and 17B, comparable levels of CD4⁺ and CD8⁺ lymphocytes were observed in VSIG4 knockout mice compared to wild type mice, and comparable CD62L expression was observed in these CD4 and CD8 expressing lymphocytes in VSIG4 knockout mice and wild type mice. In addition, VSIG4 knockout mice had elevated CD8β levels.⁺ T cells versus wild-type mice as shown in Figure 17C but comparable CD11β levels⁺/Gr-1-lymphocytes as shown in Figure 17D.

To further evaluate the role of VSIG4 signaling in tumor growth, a CD38 mouse tumor model of colon adenocarcinoma was used in both VSIG4 knockout and wild-type mice, with the chemotherapeutic agent Claforane (CTX) administered by intraperitoneal injection on days 18 and 23 after tumor injection. cells to mice as shown at the top of the schematic in Figure 18A. Claforane injection resulted in greater tumor volume reduction in VSIG4 knockout mice compared to wild-type mice, and this tumor volume reduction was maintained for 40 days after tumor cell injection. In contrast, in wild-type mice, injection of CTX resulted in a slight decrease in tumor volume, followed by a steady increase in tumor size, as shown in Figure 1. 18A. Images of mice and photomicrographs of sections of tumors in both VSIG4 knockout mice and wild-type mice at day 24 after tumor cell injection are presented in Figure 18B. Tumor sections were prepared for VSIG4 mice^+/+ and VSIG4^-/- C57BL/6 on day 24, and paraffin sections of tumor tissues were stained with H&E.

The effects of anti-VSIG4 on tumor suppression in a humanized mouse model were assessed by injection of 10 mg/kg anti-VSIG4 antibody on days 19, 22, 25, 28 and 31 after injection of HT29 cancer cells into humanized mice, as shown in Figure 19. Significant growth suppression tumors were observed in mice that received anti-VSIG4 antibody compared to those that received control IgG injection.

These experiments demonstrated, as shown schematically in Figure 20 that VSIG4 signaling modulates the suppression of M2 T cell proliferation by macrophages, and that blocking VSIG4 signaling results in (1) abolition of the suppression of T cell proliferation induced by M2 macrophages, leading to CD8+ T cell proliferation and tumor suppression; and (2) conversion of M2 macrophages to M1 macrophages.

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

Antibody clones A1, A2, A1.3 and A2.3 were generated by affinity maturation of EU103.2 antibodies. Protein profiles obtained by size exclusion HPLC are presented in Figures 22, 23, 24 and 25, respectively, and summary results are shown in Table 4.

TABLE 4: Size-exclusion HPLC data for antibodies A1, A2, A1.3 and A2.3

At Time Square Height Width % Area Log(MM) MM A1 7.551 8.57E+02 29.91175 0.4484 100,000 2.135 136.605 A2 7.567 2.62E+02 8.96929 0.4567 100,000 2.128 134.380 A1.3 7.525 2.02E+03 106.8666 0.2842 100,000 2.134 136.081 A2.3 7.542 6.61E+02 34.47963 0.2898 100,000 2.126 133.730

As shown in Figures 26A and 26B, A1 or A2 antibodies were used to treat differentiated M2 macrophages for 2 days and FACS analysis showed a decrease in CD163, a marker of M2 macrophages, and a significant increase in CD86, a marker of M1 macrophages. Treatment with LPS/IFNγ for two days was used as a positive control. Treatment with both A1 and A2 antibodies resulted in an increase in the M1/M2 ratio. In particular, the A2 antibody resulted in a ratio increase close to that which occurred in the positive control group.

Then, as shown in Figure 27, A1 or A2 antibodies were used to treat differentiated M2 macrophages for 2 days to change their polarization into M1 macrophages, and the change in cytokine and chemokine production in the culture medium was determined using LEGENDplex™. The aim was to confirm the change in polarization of M2 macrophages to M1 macrophages. Treatment with LPS/IFNγ for two days was used as a positive control. In both groups, A1 and A2, there was an increase in the production of M1-type cytokines/chemokines (TNFα, IL6, IFNγ, IP-10 and IL12 p40) compared to M2 macrophages, while the production of M2-type cytokines/chemokines (IL -10, arginase, TARC and IL-1RA) decreased. In particular, the increase in TNFα and IL6 production was comparable to the results in the positive control group.

In addition, as shown in Figure 28, A1 or A2 antibodies were used to treat differentiated M2 macrophages for 2 days, and a FACS analysis was performed to demonstrate a decrease in the expression of CD163, a marker of M2 macrophages, and a significant increase in the expression of CD86, a marker of M1 macrophages. Treatment with LPS/IFNγ for two days was used as a positive control.

To further confirm the change in polarization of M2 macrophages to M1 macrophages by A1 and A2 antibodies, A1 or A2 antibodies were used at different concentrations (5, 10 and 20 µg/mL) to treat differentiated M2 macrophages for 2 days, and the production of macrophages of cytokines and chemokines, as shown in Figure 29. The change in cytokine/chemokine production in the culture medium was determined using LEGENDplex™. Treatment with LPS/IFNγ for two days was used as a positive control. In both groups, A1 and A2, an increase in the production of TNFα, IL6 and IP-10, which are associated with M1 macrophages, was observed, and this trend was especially pronounced when M2 macrophages were treated with A2 antibody. The production of arginase, which is associated with M2 macrophages, decreased regardless of the concentration of antibodies.

The ability of macrophages to chemotaxis after conversion of M2 macrophages to M1 macrophages by antibody A2 was assessed in a chemotaxis assay. Using a 24-well Transwell chamber with a 5 µm pore size (Corning, cat. no. CLS3421-48EA), the chemoattractant rhCCL19 (Biolegend, cat. no. 582104), which is an M1-type chemokine, was added to the lower chamber at a concentration of 100 ng/mL ( volume 400 µl), and M1 repolarized macrophages were added to the upper chamber at 1.5~5×10 ⁵ cells/600 µl, where M2 or A2 was used for 2 days. (Treatment with LPS/IFNγ for two days was used as a positive control). After a 4 hour incubation period at 37° C. under 5% CO ₂ , 100 μl of cells from the lower chamber were transferred to a 96-well plate. Then, 10 μl of CCK-8 solution (Dojindo, cat. no. CK04) was added to each well, and absorbance (450 nm) was measured after incubation for 1 hour. As shown in Figure 30, M1 macrophages from group A2 showed the ability to chemotaxis, which confirmed the conversion of M2 to M1 macrophages as a result of exposure to antibody A2.

Gene array analysis was performed to determine changes in gene expression after conversion of M2 macrophages to M1 macrophages by A2 antibodies, as shown in Figure 42. M2 macrophages were treated with A2 antibodies and cells were harvested two days later. (Macrogen, Agilent Human GE 8×60K V3). The analysis showed an increase in the expression of the M1 phenotype marker and M1-type cytokines/chemokines, similar to cells treated with LPS/IFNγ as a positive control, and a decrease in the expression of the M2 phenotype marker and M2-type cytokines/chemokines.

Example 6 Antitumor Effects of Antibodies A1, A2, A1.3 and A2.3

The antitumor effect of A1 and A2 antibodies was evaluated using a humanized mouse model. Human CD34 cells were injected into NBSGW mice, after which blood samples were collected and human CD45 cells were quantified in PBMC for mouse humanization assay over a period of 12~14 weeks. HCT-15 colon cancer cells were injected at 1×10 ⁷ cells/mouse into humanized mice, and after 5 days the mice were distributed into three groups, each of which received injections of hIgG (Sigma-Aldrich, cat. no. I4506), antibodies A1 or A2 antibodies. The antibodies were injected every three days for a total of 5 injections, as shown schematically in Figure 31A. Tumor volumes were measured and, after mice were sacrificed, serum was used to measure IFNγ by ELISA (Invitrogen, cat. no. 88-7316-88) and tumor samples were used to analyze infiltrated leukocytes.

As shown in Figures 31B and 31C, there was no antitumor effect in the case of A1 antibodies, but a decrease in tumor size and an increase in IFNγ production were observed in the A2 antibody group, which confirmed the antitumor effect of A2 antibodies.

The effect of converting M2 macrophages to M1 macrophages by the A2 antibody in the context of tumor growth was then evaluated in vivo . As schematically depicted in Figure 32A, SW480 colon cancer cells were injected into mice (1×10 ⁷ cells/mouse), and after tumors grew to a certain size (~1000 mm ³ ), differentiated M2 macrophages (7×10 ⁵ cells/mouse) were injected. mouse) with hIgG or A2 antibody. Antibodies were injected every 2 days for a total of 5 injections, after the first injection, blood samples were collected on days 4, 7, and 11 after tumor cell injection, and blood samples were used for serum isolation or PBMC to analyze macrophage phenotype change by FACS.

On day 7 after the injection of tumor cells, a change in the phenotype of M1 macrophages in the A2 antibody group was observed, as shown in the Figure. 32b. While there were no changes in CD163, a marker of M2 macrophages, expression of CD86 and HLA-DR, markers of M1 macrophages, was markedly increased compared to the hIgG group, confirming the conversion of M2 to M1 macrophages by antibody A2, as shown in Figure 32C.

Then, an analysis was made of the effect of the transformation of M2 into M1 macrophages on tumor growth.

As shown schematically in Figure 33A, a mixture of HCT-15 colon cancer cells (8×10 ⁶ cells/mouse) and various amounts of M2 macrophages (2.5×10 ⁵ /5×10 ⁵ /1×10 ⁶ cells/mouse) were injected into mice. After 2 days, an injection of hIgG or antibody A2 was administered every 3 days for a total of 5 injections.

As shown in Figure 33B, antibody A2 reduced or retarded tumor growth in a dose-dependent manner on day 14 post-tumor cell injection compared to hIgG-treated control mice, and this effect was maintained at day 25 post-tumor cell injection.

The antitumor effects of the A2 antibody in a humanized mouse model were then evaluated using different mouse tumor models. As schematically shown in Figure 34A, human CD34 cells were injected into NBSGW mice, after which blood samples were collected and human CD45 cells were quantified in PBMC for mouse humanization assay over a period of 12~14 weeks. Colon cancer SW480 cells (1×10 ⁷ cells/mouse) were injected into mice, and after 5 days the mice were divided into two groups, in each of which the animals received an injection of hIgG or A2 antibody (20 mg/kg). Antibodies were injected every 3 days for a total of 5 injections in each group. Tumor volumes were measured, and after mice were sacrificed, serum was used to measure IFNγ by ELISA, and tumor samples were used to analyze infiltrated leukocytes.

There was no antitumor effect in the case of A1 antibodies, however, as shown in the Figures 34B and 34C, in the A2 antibody group, a decrease in tumor size and an increase in IFNγ production were observed, further confirming the antitumor effect of A2 antibody.

In general, in the group of animals that received the injection of antibody A2, we observed a smaller tumor size and an increased content of IFNγ in serum. In addition, as shown in Fig. 34D, an increase in CD8 content was observed⁺ T cells, in particular IFNγ secreting CD8γ T cells. As shown in Figure 34E, there was a decrease in the expression of CD93 and CD163 (macrophage M2 markers) and an increase in the expression of CD86 (macrophage M1 marker), with no change in HLA-DR. These data confirm that the A2 antibody mediates the cytotoxic activity of CD8⁺ T cells.

The antitumor effect of antibodies A2 and A2.3 was then compared in a humanized mouse model. As schematically shown in Figure 35A, human CD34 cells were injected into NBSGW mice, after which blood samples were collected and human CD45 cells were quantified in PBMC for mouse humanization assay over a period of 12~14 weeks. Colon cancer HCT-15 cells were injected at 1×10⁷ cells/mouse to humanized mice, and after tumors grow to a certain size (~1000 mm³) mice were divided into three groups, in each of which the animals received injections of antibodies hIgG, A2 or A2.3 (20 mg/kg). The antibodies were injected every three days for a total of 5 injections. The volume of tumors was measured, after the first injection, blood samples were collected at D5 and D13, and inflammatory cytokines were determined in the blood serum. As shown in Figure 35B, both antibodies, A2 and A2.3, resulted in a reduction in tumor size.

Then, four anti-VSIG4 antibodies, A1, A1.3, A2 and A2.3, were evaluated for their effect on CD8+ T cell proliferation. Antibodies A1, A1.3, A2, and A2.3 were added to donor-derived macrophages to convert M2 macrophages to M1 macrophages, and co-cultured with CD8⁺ T cells isolated from PBMCs from the same donor in a co-culture assay. CD8⁺ T cells were labeled with CFSE (Life technologies, cat. no. V12883) and co-cultured in an anti-CD3 coated 96-well plate (BD Biocoat, cat. no. 354725) at a 2:1 ratio with post-conversion macrophages (CD8 T : macrophage=2× ten⁵ cells/well : 1×10⁵ cells/well). After 5 days, harvested cells were stained with hCD8-V450 and analyzed by FACS. Proliferation of CD8⁺ T cells was confirmed by the fact that the levels of CFSE decreased. While M2 macrophages negatively regulated CD8+ T cell proliferation, co-cultivation with A1, A1.3, A2, and A2.3 antibodies resulted in the conversion of M2 macrophages to M1 macrophages and also resulted in increased CD8+ T cell proliferation, as shown on Figures 36A and 36B.

Similar experiments comparing the effects of A2 antibody and A2.3 antibody were performed using macrophages and T cells obtained from other donors than in the experiment described above (i.e., other donors than those for which the results are shown in Figure 36). Antibodies A2 and A2.3 were added to macrophages to convert M2 macrophages to M1 macrophages, and CD8 ⁺ T cells isolated from PBMCs from the same donor were used in the co-culture assay. CD8 T cells were labeled with CFSE (Life technologies, cat. no. V12883) and co-cultured in an anti-CD3 coated 96-well plate (BD Biocoat, cat. no. 354725) in a 1:1 or 2:1 ratio with macrophages after conversion ( CD8 ⁺ T : macrophage=2×10 ⁵ cells/well : 2×10 ⁵ cells/well or 2×10 ⁵ cells/well : 1×10 ⁵ cells/well). After 5 days, harvested cells were stained with hCD8-V450 and analyzed by FACS. The proliferation of CD8 ⁺ T cells was confirmed by the fact that the levels of CFSE decreased. While M2 macrophages negatively regulated CD8+ T cell proliferation, the addition of A2 and A2.3 antibodies to convert M2 macrophages to M1 macrophages resulted in increased CD8+ T cell proliferation.

hVSIG4-expressing HeLa cells (HeLa-hVSIG4 cells) were then used to confirm the effect of VSIG4 signaling on antibody A2 and antibody A2.3 mediated induction of CD8+ T cell proliferation.

CD8 ⁺ T cells were isolated from PBMC from a healthy donor, labeled with CFSE and added to an anti-CD3 coated plate (2×10 ⁵ cells/well). After 1 day, HeLa or HeLa-hVSIG4 cells were added to the wells after irradiation with 30 Gy (X-ray) (1×10 ⁵ or 0.5×10 ⁵ cells/well). After 5 days, CD8+ T cell proliferation was analyzed by measuring CFSE levels by FACS. CD8 ⁺ T cells were also treated with anti-CD3 (Miltenyi Biotech, catalog no. 130-093-387) at different concentrations, and after 1 day, HeLa or HeLa-hVSIG4 cells were added to wells after irradiation with 30 Gy (X-ray) - (1× ¹⁰⁵ cells/well). After 5 days, 100 μl of cultured cells were transferred to a separate 96-well plate and CCK-8 (10 μl/well) was added to each well. After 5 hours, the absorbance at 450 nm was measured, confirming the proliferation of CD8+ T cells. As shown in Figures 38A and 38B, HeLa-hVSIG4 negatively regulated CD8+ T cell proliferation, with A2 or A2.3 treatment inducing T cell proliferation.

The panel with human phosphokinase was used to study the signaling pathway through which the polarization of macrophages changes under the influence of antibody A2. As shown in Figure 39, conversion of M2 macrophages to M1 macrophages by treatment with A2 antibody resulted in a significant increase in JNK, MSK1/2 and p38a phosphorylation as measured using Proteome Profiler™ antibody panels (R&D Systems, catalog no. ARY003B).

ANTIBODY SEQUENCE AND BINDING AFFINITY INFORMATION

Antibody sequence and binding affinity information for the various anti-VSIG4 antibodies described herein is provided in Tables 5-12 below.

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

Name At Subsequence EU103.2_VH (amino acid) QVQLQESGPGLVKPSQTLSLTCSFSGISLTTSGMGVGWIRQPPGKGLEWLADIFWDDNKYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVRVYYKNDGYFDVWGQGTLVTVSS (SEQ ID NO: 2) EU103.2_VL (amino acid) EIVMTQSPATLSVSPGERATLSCRASKSVTTSGYSFMHWYQQKPGQAPRLLIYLASNLEPGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHSRELPYTFGQGTKLEIK (SEQ ID NO: 4)

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

Name At Subsequence EU103.3_VH
(hu6H8.3_VH)
(amino acid) QVTLKESGPTLVKPTQTLTLTCTFSGISLTTSGMGVGWIRQPPGKALEWLADIFWDDNKYYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCVRVYYKNDGYFDVWGKGTTVTVSS (SEQ ID NO: 6) EU103.3_VL
(hu6H8.3_VL)
(amino acid) DIVLTQSPLSLPVTLGQPASISCRASKSVTTSGYSFMHWYQQRPGQSPRLLIYLASNLEPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSRELPYTFGQGTKLEIK (SEQ ID NO: 8)

TABLE 7: VH and VL sequences of antibody A1

Name At Subsequence A1_VH (amino acid) QVTLKESGPTLVKPTQTLTLTCTFSGISLTTSGMGVGWIRQPPGKALEWLADIFWDDNKYYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCVRVYYKNDGYFDVWGKGTTVTVSS (SEQ ID NO: 6) A1_VL (amino acid) DIVLTQSPLSLPVTLGQPASISCRASKSVTTSGYSFMHWYQQRPGQSPRLLIYLASNLEPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSGELPYTFGQGTKLEIK (SEQ ID NO: 10)

TABLE 8: VH and VL sequences of antibody A2

Name At Subsequence A2_VH (amino acid) QVTLKESGPTLVKPTQTLTLTCTFSGISLTTSGMGVGWIRQPPGKALEWLADIFWDDNKYYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCVRVYYKNDGYFDVWGKGTTVTVSS (SEQ ID NO: 6) A2_VL (amino acid) DIVLTQSPLSLPVTLGQPASISCRASKSVTTSGYSFMHWYQQRPGQSPRLLIYLASNLEPGVPDRFSGSGSGTDFTLKIFRVEAEDVGVYYCQQSGELPYTFGQGTKLEIK (SEQ ID NO: 12)

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

Name At Subsequence A1.3_VH (amino acid) QVTLVESGPTLVKPGQTLTLTCTFSGISLTTSGMGVGWIRQPPGKALEWLADIFWDDNKYYNPSLKGRLTITKDTSKNQVYLTMTNMDPVDATYYCVRVYYKNDGYFDVWGKGTTVTVSS (SEQ ID NO: 14) A1.3_VL (amino acid) DIVLTQSPLSLPVTLGQPASISCRASKSVTTSGYSFMHWYQQRPGQSPRLLIYLASNLEPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSGELPYTFGQGTKLEIK (SEQ ID NO: 10)

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

Name At Subsequence A2.3_VH (amino acid) QVTLVESGPTLVKPGQTLTLTCTFSGISLTTSGMGVGWIRQPPGKALEWLADIFWDDNKYYNPSLKGRLTITKDTSKNQVYLTMTNMDPVTATYYCVRVYYKNDGYFDVWGKGTTVTVSS (SEQ ID NO: 16) A2.3_VL (amino acid) DIVLTQSPLSLPVTLGQPASISCRASKSVTTSGYSFMHWYQQRPGQSPRLLIYLASNLEPGVPDRFSGSGSGTDFTLKIFRVEAEDVGVYYCQQSGELPYTFGQGTKLEIK (SEQ ID NO: 12)

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

VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 EU103.2 GISLTT
(SEQ ID NO: 17) IFWDNK
(SEQ ID NO: 18) VRVYYKNDGYFDV
(SEQ ID NO: 19) KSVTTS
(SEQ ID NO: 20) LAS
(SEQ ID NO: 21) QHSRELPYT
(SEQ ID NO: 22) EU103.3 GISLTT
(SEQ ID NO: 17) IFWDNK
(SEQ ID NO: 18) VRVYYKNDGYFDV
(SEQ ID NO: 19) KSVTTS
(SEQ ID NO: 20) LAS
(SEQ ID NO: 21) QHSRELPYT
(SEQ ID NO: 22) A1 GISLTT
(SEQ ID NO: 17) IFWDNK
(SEQ ID NO: 18) VRVYYKNDGYFDV
(SEQ ID NO: 19) KSVTTS
(SEQ ID NO: 20) LAS
(SEQ ID NO: 21) QQSGELPYT
(SEQ ID NO: 23) A2 GISLTT
(SEQ ID NO: 17) IFWDNK
(SEQ ID NO: 18) VRVYYKNDGYFDV
(SEQ ID NO: 19) KSVTTS
(SEQ ID NO: 20) LAS
(SEQ ID NO: 21) QQSGELPYT
(SEQ ID NO 23) A1.3 GISLTT
(SEQ ID NO: 17) IFWDNK
(SEQ ID NO: 18) VRVYYKNDGYFDV
(SEQ ID NO: 19) KSVTTS
(SEQ ID NO: 20) LAS
(SEQ ID NO: 21) QQSGELPYT
(SEQ ID NO: 23) A2.3 GISLTT
(SEQ ID NO: 17) IFWDNK
(SEQ ID NO: 18) VRVYYKNDGYFDV
(SEQ ID NO: 19) KSVTTS
(SEQ ID NO: 20) LAS
(SEQ ID NO: 21) QQSGELPYT
(SEQ ID NO: 23)

TABLE 12: Binding affinity (K _D ) antibodies EU103.2, EU103.3, A1, A2, A1.3 and A2.3 for VSIG4

Antibody Ka (1/ms) Kd (1/s) KD (M) EU103.2 1.834E+5 0.01313 7.156E-8 A1 3.779E+5 0.003283 8.688E-9 A1.3 4.022E+5 0.003198 7.952E-9 A2 3.604E+5 0.002964 8.226E-9 A2.3 4.037E+5 0.003083 7.636E-9

OTHER IMPLEMENTATION OPTIONS

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

--->

SEQUENCE LIST

<110> UTAILEX CO., LTD.

<120> ANTIBODIES AGAINST HUMAN VSIG4 AND THEIR USE

<130> 47683-0034WO1

<140> PCT/US2019/053824

<141> 2019-09-30

<150> 62/776.523

<151> 2018-12-07

<150> 62/738.255

<151> 2018-09-28

<160> 31

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Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

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Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

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Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Pro Gly Ile Pro Ala

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Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser

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Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

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Trp Leu Ala Asp Ile Phe Trp Asp Asp Asn Lys Tyr Tyr Asn Pro Ser

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Leu Lys Gly Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val

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Tyr Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

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Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly

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Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala

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Ser Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr

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Trp Tyr Lys Gln Gln Thr Asn Asn Gln Glu Pro Ile Lys Val Ala Thr

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Tyr Phe Cys Thr Ala Lys Gly Gln Val Gly Ser Glu Gln His Ser Asp

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Ile Val Lys Phe Val Val Lys Asp Ser Ser Lys Leu Leu Lys Thr Lys

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Val Lys Gln Ser Trp Asp Trp Thr Thr Asp Met Asp Gly Tyr Leu Gly

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Leu Ile Ile Ser Leu Cys Cys Met Val Val Phe Thr Met Ala Tyr Ile

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Met Leu Cys Arg Lys Thr Ser Gln Gln Glu His Val Tyr Glu Ala Ala

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Arg Ala His Ala Arg Glu Ala Asn Asp Ser Gly Glu Thr Met Arg Val

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Ala Ile Phe Ala Ser Gly Cys Ser Ser Asp Glu Pro Thr Ser Gln Asn

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Leu Gly Asn Asn Tyr Ser Asp Glu Pro Cys Ile Gly Gln Glu Tyr Gln

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Ile Ile Ala Gln Ile Asn Gly Asn Tyr Ala Arg Leu Leu Asp Thr Val

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Pro Leu Asp Tyr Glu Phe Leu Ala Thr Glu Gly Lys Ser Val Cys

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Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu

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Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile

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Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp

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Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr

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Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly

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Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg

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Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr

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Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile

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Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val

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Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly Ile

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Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg Glu

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Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln Ser Leu Ser Glu Leu Val

35 40 45

Val Phe Trp Gln Asp Gln Glu Asn Leu Val Leu Asn Glu Val Tyr Leu

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Gly Lys Glu Lys Phe Asp Ser Val His Ser Lys Tyr Met Gly Arg Thr

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Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg Leu His Asn Leu Gln Ile

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Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile His His Lys Lys Pro Thr

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Gly Met Ile Arg Ile His Gln Met Asn Ser Glu Leu Ser Val Leu Ala

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Asn Phe Ser Gln Pro Glu Ile Val Pro Ile Ser Asn Ile Thr Glu Asn

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Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile His Gly Tyr Pro Glu Pro

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Lys Lys Met Ser Val Leu Leu Arg Thr Lys Asn Ser Thr Ile Glu Tyr

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Asp Gly Ile Met Gln Lys Ser Gln Asp Asn Val Thr Glu Leu Tyr Asp

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Val Ser Ile Ser Leu Ser Val Ser Phe Pro Asp Val Thr Ser Asn Met

195 200 205

Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys Thr Arg Leu Leu Ser Ser

210 215 220

Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln Pro Pro Pro Asp His Ile

225 230 235 240

Pro Trp Ile Thr Ala Val Leu Pro Thr Val Ile Ile Cys Val Met Val

245 250 255

Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys Lys Lys Arg Pro Arg Asn

260 265 270

Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu Arg Glu Glu Ser Glu Gln

275 280 285

Thr Lys Lys Arg Glu Lys Ile His Ile Pro Glu Arg Ser Asp Glu Ala

290 295 300

Gln Arg Val Phe Lys Ser Ser Lys Thr Ser Ser Cys Asp Lys Ser Asp

305 310 315 320

Thr Cys Phe

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Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile Ile Ser Ile Ile

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Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly Ile Ser

20 25 30

Ala Phe Ser Met Pro Glu Val Asn Val Asp Tyr Asn Ala Ser Ser Glu

35 40 45

Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln Pro Thr Val Val

50 55 60

Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser Glu Val Ser Asn

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Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met Lys Val Val Ser

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Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser Cys Met Ile Glu

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Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val Thr Glu Ser Glu

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Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser Lys Ala Ser Leu

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Cys Val Ser Ser Phe Phe Ala Ile Ser Trp Ala Leu Leu Pro Leu Ser

145 150 155 160

Pro Tyr Leu Met Leu Lys

165

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Met Ile Phe Leu Leu Leu Met Leu Ser Leu Glu Leu Gln Leu His Gln

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Ile Ala Ala Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile

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Glu His Gly Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser

35 40 45

His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn

50 55 60

Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu

65 70 75 80

Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp

85 90 95

Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr

100 105 110

Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr

115 120 125

His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln

130 135 140

Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val

145 150 155 160

Pro Ala Asn Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val

165 170 175

Thr Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys

180 185 190

Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp

195 200 205

Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu Leu His

210 215 220

Ile Phe Ile Pro Ser Cys Ile Ile Ala Phe Ile Phe Ile Ala Thr Val

225 230 235 240

Ile Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu Tyr Ser Ser Lys Asp

245 250 255

Thr Thr Lys Arg Pro Val Thr Thr Thr Lys Arg Glu Val Asn Ser Ala

260 265 270

ile

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Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

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Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Asn Glu Arg Thr His

225 230 235 240

Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr

245 250 255

Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys

260 265 270

Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu

275 280 285

Glu Thr

290

<210> 30

<211> 534

<212> PROTEIN

<213> Homo sapiens

<400> 30

Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala

1 5 10 15

Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln

20 25 30

Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu

35 40 45

Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn

50 55 60

Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala

65 70 75 80

Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe

85 90 95

Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val

100 105 110

Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp

115 120 125

Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys

130 135 140

Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr

145 150 155 160

Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val

165 170 175

Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr

180 185 190

Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu

195 200 205

Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn

210 215 220

Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro Gln

225 230 235 240

Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val

245 250 255

Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro

260 265 270

Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr

275 280 285

Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly

290 295 300

Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln

305 310 315 320

Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly

325 330 335

Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val

340 345 350

Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu

355 360 365

Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser

370 375 380

Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln

385 390 395 400

Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu

405 410 415

Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala

420 425 430

Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp

435 440 445

Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro

450 455 460

Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu

465 470 475 480

Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys

485 490 495

Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly

500 505 510

Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp

515 520 525

Asp Gly Gln Glu Ile Ala

530

<210> 31

<211> 302

<212> PROTEIN

<213> Homo sapiens

<400> 31

Met Arg Leu Gly Ser Pro Gly Leu Leu Phe Leu Leu Phe Ser Ser Leu

1 5 10 15

Arg Ala Asp Thr Gln Glu Lys Glu Val Arg Ala Met Val Gly Ser Asp

20 25 30

Val Glu Leu Ser Cys Ala Cys Pro Glu Gly Ser Arg Phe Asp Leu Asn

35 40 45

Asp Val Tyr Val Tyr Trp Gln Thr Ser Glu Ser Lys Thr Val Val Thr

50 55 60

Tyr His Ile Pro Gln Asn Ser Ser Leu Glu Asn Val Asp Ser Arg Tyr

65 70 75 80

Arg Asn Arg Ala Leu Met Ser Pro Ala Gly Met Leu Arg Gly Asp Phe

85 90 95

Ser Leu Arg Leu Phe Asn Val Thr Pro Gln Asp Glu Gln Lys Phe His

100 105 110

Cys Leu Val Leu Ser Gln Ser Leu Gly Phe Gln Glu Val Leu Ser Val

115 120 125

Glu Val Thr Leu His Val Ala Ala Asn Phe Ser Val Pro Val Val Ser

130 135 140

Ala Pro His Ser Pro Ser Gln Asp Glu Leu Thr Phe Thr Cys Thr Ser

145 150 155 160

Ile Asn Gly Tyr Pro Arg Pro Asn Val Tyr Trp Ile Asn Lys Thr Asp

165 170 175

Asn Ser Leu Leu Asp Gln Ala Leu Gln Asn Asp Thr Val Phe Leu Asn

180 185 190

Met Arg Gly Leu Tyr Asp Val Val Ser Val Leu Arg Ile Ala Arg Thr

195 200 205

Pro Ser Val Asn Ile Gly Cys Cys Ile Glu Asn Val Leu Leu Gln Gln

210 215 220

Asn Leu Thr Val Gly Ser Gln Thr Gly Asn Asp Ile Gly Glu Arg Asp

225 230 235 240

Lys Ile Thr Glu Asn Pro Val Ser Thr Gly Glu Lys Asn Ala Ala Thr

245 250 255

Trp Ser Ile Leu Ala Val Leu Cys Leu Leu Val Val Val Ala Val Ala

260 265 270

Ile Gly Trp Val Cys Arg Asp Arg Cys Leu Gln His Ser Tyr Ala Gly

275 280 285

Ala Trp Ala Val Ser Pro Glu Thr Glu Leu Thr Gly His Val

290 295 300

<---

Claims (62)

1. An isolated humanized anti-VSIG4 antibody, or its antigen-binding fragment, containing: a) a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 17, A heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 18, A heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 19; and b) a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 20, A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and A light chain CDR3 containing the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23. 2. An antibody, or antigen-binding fragment, according to claim 1, where the antibody, or antigen-binding fragment, contains any of the following: a) a heavy chain variable domain containing an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6 , SEQ ID NO: 14 or SEQ ID NO: 16; b) a light chain variable domain containing an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8 , SEQ ID NO: 10 or SEQ ID NO: 12; or c) a heavy chain variable domain containing an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6 , SEQ ID NO: 14 or SEQ ID NO: 16, and a light chain variable domain containing an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12. 3. An antibody, or antigen-binding fragment, according to any one of claims 1, 2, where the antibody, or antigen-binding fragment, contains any of the following: a) a heavy chain variable domain comprising the amino acid sequence of 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 of 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 of 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 of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. 4. An antibody or antigen-binding fragment according to claim 1, wherein the light chain CDR3 of the antibody contains the amino acid sequence of SEQ ID NO: 23. 5. The antibody, or antigen-binding fragment, according to claim 2, where the antibody, or antigen-binding fragment, contains a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 4. 6. The antibody, or antigen-binding fragment, according to claim 2, where the antibody, or antigen-binding fragment, contains a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 8. 7. The antibody, or antigen-binding fragment, according to claim 2, where the antibody, or antigen-binding fragment, contains a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 10. 8. An antibody or antigen-binding fragment according to claim 2, wherein the antibody or antigen-binding fragment contains a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 12. 9. An antibody or antigen-binding fragment according to claim 2, wherein the antibody or antigen-binding fragment contains a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 10. 10. The antibody, or antigen-binding fragment, according to claim 2, where the antibody, or antigen-binding fragment, contains a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 16, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 12. 11. An antibody or antigen-binding fragment according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment has a binding affinity (K _D ) for the V-set molecule and a human immunoglobulin domain containing protein 4 (VSIG4) of 1 ×10 ^-7 -1×10 ^-9 . 12. An antibody or antigen-binding fragment according to any one of claims 1-11, wherein the antibody or antigen-binding fragment has a binding affinity (K _D ) for the VSIG4 molecule of about 7.156 x 10 ^-8 to about 7.636 x 10 ^{- 9} . 13. An antibody or antigen binding fragment according to any one of claims 1 to 11, wherein the antibody or antigen binding fragment has a binding affinity (K _D ) for the VSIG4 molecule of about 7.156×10 ^-8 , about 7.636×10 ^-9 , about 7.952×10 ^-9 , about 8.226×10 ^-9 or about 8.688×10 ^-9 . 14. A nucleic acid molecule encoding an antibody or antigen-binding fragment according to any one of claims 1-13. 15. A recombinant vector for expressing an antibody according to any one of claims 1 to 13, containing a nucleic acid molecule according to claim 14. 16. The recombinant vector of claim 15, wherein the nucleic acid molecule of claim 14 is operably linked to a promoter. 17. A recombinant vector according to claim 15 or 16, which includes two separate vectors, each of which contains a nucleotide sequence corresponding to the heavy chain and light chain of the antibody, or antigen-binding fragment. 18. A host cell for producing an antibody according to any one of claims 1-13, containing a nucleic acid molecule according to claim 14 or a recombinant vector according to any one of claims 15-17. 19. A host cell according to claim 18, which is a mammalian cell, a yeast cell, or a bacterial cell. 20. A host cell according to claim 19, which is a cell selected from the group consisting of E. coli, P. pastoris , Sf9, COS, HEK293, Expi293, CHO-S, CHO-DG44, CHO-K1 and lymphocyte mammal. 21. The host cell of claim 20, which is an Expi293 cell. 22. Pharmaceutical composition for the treatment of a disease caused by VSIG4, containing an antibody or antigen-binding fragment according to any one of claims 1-13, a nucleic acid molecule according to claim 14, a recombinant vector according to any one of claims 15-17, or a host cell according to any one of claims 18-21, and a pharmaceutically acceptable carrier. 23. A method for treating a disease caused by VSIG4 in an individual, comprising the steps of: a) administering to an individual a composition that contains or delivers an antibody or antigen-binding fragment according to any one of claims 1-13, a nucleic acid molecule according to claim 14, a recombinant vector according to any one of claims 15-17, or a host cell according to any one of 18-21, whereby the disease or condition is treated. 24. The method of claim 23, wherein the individual has cancer or is at risk of developing it. 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 cancer and neck, hematologic, laryngeal, liver, lung, lymphoma, melanoma, mesothelioma, ovarian, primary peritoneal, salivary gland, sarcoma, gastric, thyroid, pancreatic, renal cell, glioblastoma, and prostate cancer. 26. The method according to any one of claims 23-25, wherein the individual has received, or will receive, one or more additional types of anti-cancer therapy selected from ionizing radiation, a chemotherapeutic agent, an antibody-based drug, and cell therapy, such that the individual receives both type of therapy. 27. The method of claim 26, wherein the one or more additional anti-cancer therapies include the use of an immune checkpoint inhibitor, IL-12, GM-CSF, an anti-CD4 agent, cisplatin, fluorouracil, doxorubicin, irinotecan, paclitaxel, an indolamine-2 inhibitor ,3-dioxygenase 1 (IDO1) or cyclophosphamide. 28. A method for increasing the secretion of cytokines or chemokines in M2 macrophages, comprising: a) creating contact M2 macrophages with the antibody, or antigen-binding fragment, according to any one of claims 1-13. 29. A method for inducing CD8+ T cell proliferation, comprising: a) bringing the M2 macrophage into contact with the antibody, or antigen-binding fragment, according to any one of claims 1-13; and b) co-incubation of M2 macrophage with CD8 ⁺ T cells. 30. A method for converting an M2 macrophage into an M1 macrophage, comprising contacting the M2 macrophage with an antibody, or antigen-binding fragment, according to any one of claims 1-13.

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