Hybridoma clones, monoclonal antibodies to VSIG-4, and methods of making and using

Abstract

Described herein are monoclonal antibodies that bind to VSIG‑4, including antibodies that block the binding of VSIG‑4 to SIGLEC‑7, compositions comprising the monoclonal antibodies, and methods of making and using those antibodies and compositions.

Description

[0001] Continue to apply for data

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 524,821, filed on June 26, 2017, which is incorporated herein by reference.

[0003] background

[0004] V-set and Ig domain-containing 4 (VSIG-4 or VSIG4) is a negative regulator of C3 complement receptor, B7 family-related protein and T cell activation. VSIG-4 expression has been observed to be restricted to tissue macrophages and has been shown to be downregulated in response to lipopolysaccharide (LPS) (Vogt et al., (2006) J. of Clin. Invest. 116:2817). In healthy tissues, VSIG-4 is expressed on tissue-resident macrophages and functions as a complement receptor to opsonize bacterial pathogens. VSIG-4 has been observed in patients diagnosed with non-small cell lung cancer. + The extensive infiltration of macrophages into the tumor microenvironment (Liao et al., (2014) Lab. Invest. 94:706), and high VSIG-4 expression is also associated with high-grade gliomas and poor patient prognosis (Xu et al., (2015) Am. J. Transl. Res. 7:1172). SUMMARY OF THE INVENTION

[0006] In some aspects, the disclosure describes antibodies that bind to VSIG-4. In some embodiments, the antibodies eliminate the binding of VSIG-4 to a VSIG-4 ligand. In some embodiments, the antibodies eliminate the binding of VSIG-4 to sialic acid-binding Ig-like lectin 7 (SIGLEC-7, SIGLEC7, Siglec-7, or Siglec7).

[0007] In some embodiments, the antibody can be produced by at least one of the following clones deposited on May 11, 2017 at the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 20110, USA): Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187); Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188); Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189); Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178); Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179); Ms x hVSIG4 Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182); Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183); Rt x hVSIG4489509.11 (ATCC Accession No. PTA-124184); Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186). The deposits were made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0008] In some embodiments, the antibody comprises at least one of a heavy chain variable region and a light chain variable region of a monoclonal antibody produced by at least one of the following clones: Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187); Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188); Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189); Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178); Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179); Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180); Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181); Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182); Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183); Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184); Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

[0009] In some embodiments, the antibody comprises at least one of the following heavy chain variable region and light chain variable region: the heavy chain variable region comprises the complementarity determining regions (CDRs) of the heavy chain variable region of a monoclonal antibody produced by at least one of the following clones, and the light chain variable region comprises the complementarity determining regions (CDRs) of the light chain variable region of a monoclonal antibody produced by at least one of the following clones: Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187); Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188); Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189); Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178); Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179); Ms x Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180); Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181); Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182); Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183); Rt x hVSIG4489509.11 (ATCC Accession No. PTA-124184); Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

[0010] In some embodiments, the antibody may be directly or indirectly coupled to a detectable label. In some embodiments, the antibody comprises an IgG antibody.

[0011] In some embodiments, the antibodies include antigen-binding fragments, including, for example, Fab fragments, Fab' fragments, F(ab)2 fragments, and Fv fragments.

[0012] In some embodiments, the antibody can eliminate the binding of VSIG-4 to a VSIG-4 ligand. In some embodiments, the VSIG-4 ligand may include SIGLEC-7. In some embodiments, at least one of VSIG-4 and the VSIG-4 ligand may be on the surface of a cell. In some embodiments, VSIG-4 may be expressed on the surface of a macrophage (including, for example, an M2c macrophage). In some embodiments, the antibody can eliminate cell-cell interactions.

[0013] In some embodiments, the antibody can bind to the extracellular domain of VSIG-4. In some embodiments, the antibody can bind to a VSIG-4 polypeptide. In some embodiments, the antibody can bind to glycosylated and unglycosylated VSIG-4.

[0014] The disclosure further describes compositions and kits comprising the antibodies described herein, and the antibodies described herein in methods of treating cancer in a mammal, detecting cancer in a mammal, or treating an autoimmune disease.

[0015] In another aspect, the disclosure describes a method of treating cancer in a mammal comprising exposing the mammal comprising mammalian cancer cells to an antibody described herein.

[0016] In some embodiments, VSIG-4 expression is amplified in a patient sample comprising mammalian cancer cells. In some embodiments, VSIG-4 expression is amplified in macrophages of a patient sample comprising mammalian cancer cells. In some embodiments, the mammalian cancer cells include lung cancer cells or gliomas.

[0017] In some embodiments, the mammalian cancer cells are also exposed to chemotherapy or radiation therapy.

[0018] In a further aspect, the present disclosure describes a method of detecting cancer in a mammal comprising exposing mammalian cancer cells to an antibody described herein.

[0019] In yet another aspect, the disclosure describes a method of treating an autoimmune disease in a mammal comprising exposing cells of the mammal to an antibody described herein.

[0020] In other aspects, the present disclosure relates to the following hybridoma cell lines and monoclonal antibodies produced by the following hybridoma cell lines:

[0021] Ms x hVSIG4 528902.11, deposited as ATCC Accession No. PTA-124187;

[0022] Ms x hVSIG4 528903.111, deposited as ATCC Accession No. PTA-124188;

[0023] Ms x hVSIG4 528905.11, deposited as ATCC Accession No. PTA-124189;

[0024] Ms x hVSIG4 528906.11, deposited as ATCC accession number PTA-124178;

[0025] Ms x hVSIG4 528908.11, deposited as ATCC Accession No. PTA-124179;

[0026] Ms x hVSIG4 528910.111, deposited as ATCC Accession No. PTA-124180;

[0027] Ms x hVSIG4 528912.11, deposited as ATCC accession number PTA-124181;

[0028] Ms x hVSIG4 528922.111, deposited as ATCC Accession No. PTA-124182;

[0029] Ms x hVSIG4 528927.111, deposited as ATCC accession number PTA-124183;

[0030] Rt x hVSIG4 489509.11, deposited as ATCC Accession No. PTA-124184;

[0031] Rt x hVSIG4 489517.111, deposited as ATCC Accession No. PTA-124185; and

[0032] Rt x hVSIG4 489518.11, deposited as ATCC Accession No. PTA-124186.

[0033] As used herein, the term "antibody" refers to a molecule comprising at least one antigen binding site that immunospecifically binds to a specific target antigen target. Therefore, the term "antibody" includes, but is not limited to, full-length antibodies and / or variants thereof, fragments thereof, peptibodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed by at least two complete antibodies, human antibodies, humanized antibodies, and antibody mimics or their specified fragments or portions, including single-chain antibodies and fragments thereof, that mimic the structure and / or function of antibodies. The binding of an antibody to a target can cause a variety of effects, such as, but not limited to, such binding regulates, reduces, increases, antagonizes, excites, mitigates, slows down, blocks, inhibits, eliminates, and / or interferes with at least one target activity or binding, or receptor activity or binding, in vitro, in situ, and / or in vivo. Thus, the antibodies of the present disclosure encompass antibody fragments capable of binding to a biomolecule (e.g., an antigen or receptor) or a portion thereof, including but not limited to Fab, Fab' and F(ab')2, pFc', Fd, single domain antibodies (sdAb), variable fragments (Fv), single chain variable fragments (scFv) or disulfide-linked Fv (sdFv); bifunctional antibodies or bivalent bifunctional antibodies; linear antibodies; single chain antibody molecules; multispecific antibodies formed from antibody fragments. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0034] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for a possible naturally occurring mutation that may be present in a small amount, the individual antibodies that make up the population are identical. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, compared with polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies can be synthesized by hybridoma cells that are not contaminated by other immunoglobulin-producing cells. Alternatively, monoclonal antibodies can be recombinantly produced, including, for example, by cells stably or transiently transfected with heavy and light chain genes encoding monoclonal antibodies.

[0035] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring engineering of the antibody by any particular method. In some embodiments, the term "monoclonal" as used herein refers to an antibody derived from a clonal population of cells, including any eukaryotic, prokaryotic or phage clone, rather than a method for engineering the antibody.

[0036] As used herein, the term "VSIG-4 ligand" refers to a protein binding partner of VSIG-4 and may include a ligand and / or a counter-receptor. The interaction between VSIG-4 and VSIG-4 may result in signal transduction in one or both directions.

[0037] As used herein, "isolated" refers to material that is removed from its original environment (eg, the natural environment if it is naturally occurring) and thus altered "by the hand of man" from its natural state.

[0038] As used herein, "room temperature" is 16° C. to 26° C., or more preferably 18° C. to 24° C. In some embodiments, "room temperature" is 20° C. to 22° C.

[0039] As used herein, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. When discussed herein, whether any particular polypeptide has at least 40% (%), at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to another polypeptide can be determined by using methods and computer programs / software known in the art, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman (1981) Advances in Applied Mathematics 2:482-489 to find the best homology fragment between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence has for example 95% identity with reference sequences according to the present invention, parameters are set so that the identity percentage is calculated on the total length of the reference polypeptide sequence total length, and in the homology, 5% intervals of the total amino acid in the reference sequence are allowed.

[0040] "Binding affinity" or "affinity binding" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic epitope). The affinity of a molecule X for its partner Y is given by the dissociation constant (K D), which can generally be determined using methods known in the art, for example, using a BIACORE biosensor available from BIACORE (GE Healthcare Worldwide, Chicago, IL). In some embodiments, antibodies of the present disclosure can be described according to their binding affinity for VSIG-4. In some embodiments, antibodies of the present disclosure include antibodies that interact with an antigen where the dissociation constant (K D ) is less than or equal to 5 X10 -6 M, less than or equal to 1 X 10 -6 M, less than or equal to 5 X 10 -7 M, less than or equal to 1 X 10 -7 M, less than or equal to 5 X 10 -8 M, less than or equal to 1 X 10 -8 M, less than or equal to 5 X 10 -9 M, less than or equal to 1 X 10 -9 M, less than or equal to 5 X 10 -10 M, less than or equal to 1 X 10 -10 M, less than or equal to 5 X 10 -11 M, less than or equal to 1 X 10 -11 M, less than or equal to 5 X 10 -12 M, less than or equal to 1 X 10 -12 M, less than or equal to 5 X 10 -13 M, less than or equal to 1X 10 -13 M, less than or equal to 5 X 10 -14 M, less than or equal to 1 X 10 -14 M, less than or equal to 5 X 10 -15 M, or less than or equal to 1 X 10 -15 M.

[0041] As used herein, the term "subject" includes, but is not limited to, humans and non-human vertebrates. In some embodiments, the subject is a mammal, particularly a human. The subject can be an individual. The subject can be an "individual," "patient," or "host." Non-human vertebrates include livestock animals, companion animals, and laboratory animals. Non-human subjects also include non-human primates and rodents, such as, but not limited to, rats or mice. Non-human subjects also include, but are not limited to, chickens, horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, minks, and rabbits.

[0042] As used herein, "in vitro" is in cell culture, while "in vivo" is within a subject. As used herein, "isolated" refers to material that has been removed from its natural environment (e.g., the natural environment where it is naturally present), produced using recombinant technology, or chemically or enzymatically synthesized, and thus altered "by the hand of man" from its natural state.

[0043] The words "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and other embodiments are not intended to be excluded from the scope of the invention.

[0044] Where the terms "comprises" and variations thereof appear in the description and claims, these terms do not have a limiting meaning.

[0045] Unless otherwise specified, "a", "an", "said", and "at least one" are used interchangeably to mean one or more than one.

[0046] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0047] For any method disclosed herein including discrete steps, the steps can be performed in any feasible order. In addition, any combination of two or more steps can be performed simultaneously when appropriate.

[0048] Unless otherwise indicated, all numerical values ​​used in the specification and claims, indicating components, molecular weights, etc., should be understood as being modified in all cases by the term "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate and can be varied depending on the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0049] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain ranges necessarily resulting from the standard deviations found in their respective testing measurements.

[0050] All headings are for the convenience of the reader and, unless so indicated, should not be used to limit the meaning of the text under the heading.

[0051] The above summary of the present invention is not intended to describe each disclosed embodiment or each implementation of the present invention. The following description more specifically illustrates exemplary embodiments. In several places throughout the application, guidance is provided by lists of examples, which can be used in various combinations. In each case, the narrated lists serve only as representative groups and should not be interpreted as exhaustive lists. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Cell-based assay showing Siglec-7 / VSIG-4 interaction. Human VSIG-4 (hVSIG4) / eGFP HEK transfectants were incubated with recombinant human Siglec-7 (rhSiglec-7) / Fc protein (2 μg / mL) plus anti-Fc APC, or negative control (anti-Fc-APC only). The percentages shown in the upper right quadrant indicate the level of interaction.

[0054] Figure 2 (AB) shows surface plasmon resonance measured using BIACORE (GE Healthcare Worldwide, Chicago, IL) to confirm VSIG-4-Siglec-7 protein-protein interaction. Figure 2A : Schematic diagram of determination; Figure 2B CM5 microarrays (GE Healthcare Worldwide, Chicago, IL) were covalently coated with protein A / G / L (Novus Biologicals, Littleton, CO). Fc-tagged Siglec7 proteins were captured on the protein A / G / L microarray. The microarray was then exposed to varying amounts of soluble histidine-tagged VSIG-4 recombinant protein to determine the K of the binding interaction between the two proteins. D。 The binding affinity was determined to be 29.7 nM.

[0055] Figure 3 Recombinant human Siglec-7 (rhSiglec-7) / Fc protein titration is shown on hVSIG-4 transfected cells. hVSIG4 / eGFP HEK transfected cells were incubated with varying amounts of rhSiglec-7 / Fc protein (between 500 ng / mL and 6 μg / mL) or a negative control (FAB110A). The percentages shown in the upper right quadrant represent the level of interaction.

[0056] Figure 4Recombinant human VSIG-4 (rhVSIG-4) / Fc protein titration on hSiglec-7 transfected cells is shown. hSiglec-7 / eGFP HEK transfected cells were incubated with varying amounts of rhVSIG-4 / Fc protein (between 500 ng / mL and 6 μg / mL) or a negative control (FAB110A). The percentages shown in the upper right quadrant represent the level of interaction.

[0057] Figure 5 Four anti-human VSIG-4 antibodies are shown to block the interaction of VSIG-4 with rhSiglec-7 / Fc. hVSIG4 / eGFP HEK transfectants were incubated with 2.5 μg / mL of four different anti-hVSIG-4 antibodies (produced by clones Ms x hVSIG4528903.111 ("528903"), Ms x hVSIG4 528906.11 ("528906"), Ms x hVSIG4 528908.11 ("528908"), and Ms x hVSIG4 528912.11 ("528912")) to test the ability of the antibodies to block the interaction with Siglec-7. 4 μg / mL of rhSiglec-7 / Fc protein was used. The percentages shown in the upper right quadrant represent the level of interaction.

[0058] Figure 6 (AB) shows that VSIG-4 was polarized with 30 ng / mL dexamethasone for 3 days ( Fig. 6A ) or polarized with 20 ng / mL IL-10 for 3 days ( Figure 6B ) are expressed on M2c monocytes. M2c cells were polarized as described in Example 3. Ms x hVSIG4 528903.111 ("528903"), Ms x hVSIG4 528906.11 ("528906"), Ms x hVSIG4 528908.11 ("528908"), Ms x hVSIG4 528912.11 ("528912") and Ms x hVSIG4 528922.111 ("528922") are clones expressing mouse anti-human VSIG-4 antibodies. The histograms shown are for CD14 + Zombie Violet - Gating was set on cells.

[0059] Figure 7Detection of Siglec-7 / VSIG-4 interaction in primary M2c polarized cells is shown. Polarized M2c cells were incubated with the indicated amounts of human Siglec-7 Fc protein (1 μg / mL to 10 μg / mL) or negative control (FAB110P) for 30 minutes at room temperature. Anti-Fc PE detection antibody was added for an additional 20 minutes. The cells were then washed and stained with anti-VSIG4 Alexa Fluor 647 and anti-CD14 FITC for 30 minutes. The cells were then washed with RDFII staining buffer and analyzed on an LSRII Fortessa flow cytometer (BD Biosciences, San Jose, CA).

[0060] Figure 8A-8C Diagram showing the architecture and structure of VSIG-4. Fig. 8A : VSIG-4 is a member of the immunoglobulin superfamily; this type I transmembrane receptor contains a 19 amino acid signal peptide followed by an extracellular domain (residues 20-283), a transmembrane domain (residues 284-304), and an intracellular domain (residues 305-399), wherein the residue numbering corresponds to Uniprot numbering: Q9Y279. The extracellular domain of VSIG-4 contains two tandem Ig domains followed by a juxtamembrane sequence of ~60 residues. Figure 8B : The structure of the N-terminal VSIG-4 Ig domain (PDB ID 2icc) shows an exemplary V-type Ig domain, the VSIG-4 domain responsible for binding to C3b / iC3b. Figure 8C : The structure prediction of the VSIG-4 extracellular domain shows a tandem Ig domain structure followed by a structureless juxtamembrane region of ~60 residues.

[0061] Figure 9A-9D Siglec7 was shown to bind glycosylated VSIG-4. Fig. 9A : Purified recombinant human VSIG-4 ectodomain (rhVSIG4) expressed in NS0 cells was deglycosylated with an enzyme cocktail, separated by SDS-PAGE, and visualized by silver staining. FIG. 9B to FIG. 9D Recombinant human Siglec7-Fc (rhSiglec7-Fc) was captured on a Biacore CM5 chip modified with Protein A / G / L at a capture density of ~700 RU and tested with fully glycosylated rhVSIG4 ( Fig. 9B ), deglycosylated VSIG-4 ( Fig. 9C ) and CD56 / NCAM ( Fig.9D ) combination.

[0062] Figures 10A-10D Rt x hVSIG4 489517.111 was shown to bind specifically to the VSIG-4 polypeptide regardless of glycosylation status as confirmed by Biacore. The blocking antibody (Rt x hVSIG4 489517.111) was captured on a protein A / G / L chip at ~350 RU and tested with mammalian NS0 cell-derived rhVSIG4 ( Fig. 10A ), mammalian-derived and fully deglycosylated rhVSIG4 ( Fig. 10B ), E. coli-derived non-glycosylated VSIG-4 ( Fig. 10C ) and the highly glycosylated protein CD56 / NCAM (Figure 10 D). All analyte proteins, rhVSIG4 and CD56 / NCAM were tested at a concentration range between 25 pM and 250 nM. Regardless of the glycosylation state of VSIG-4, Rt x hVSIG4 489517.111 binds to VSIG-4, but not to CD56 / NCAM, a highly glycosylated molecule with a similar glycoprofile to VSIG4, demonstrating the specificity of Rt x hVSIG4 489517.111 for the VSIG-4 polypeptide and showing that Rt xhVSIG4 489517.111 is not anti-glycosylation specific.

[0063] Figures 11A-11D Ms x hVSIG4 528906.11 was shown to bind specifically to the VSIG-4 polypeptide regardless of glycosylation status as confirmed by Biacore. Ms x hVSIG4 528906.11 was captured on a protein A / G / L chip at ~350 RU and tested with mammalian NS0 cell-derived rhVSIG4 ( Fig.11A ), mammalian-derived and fully deglycosylated rhVSIG4 ( Fig. 11B ), E. coli-derived and non-glycosylated VSIG-4 ( Fig. 11C ) and the highly glycosylated protein CD56 / NCAM ( Fig.11D ). All analyte proteins, rhVSIG4 and CD56 / NCAM were tested at a concentration range between 25 pM and 250 nM. Regardless of the glycosylation state of VSIG-4, Ms x hVSIG4 528906.11 bound to VSIG4, but not to CD56 / NCAM, a highly glycosylated molecule with a similar sugar profile to VSIG4, demonstrating the specificity of Ms x hVSIG4 528906.11 for the VSIG4 polypeptide and showing that Ms x hVSIG4 528906.11 is not anti-glycosylation specific.

[0064] Figures 12A-12D Ms x hVSIG4 528908.11 was shown to bind specifically to the VSIG4 polypeptide regardless of glycosylation status as confirmed by Biacore. Ms x hVSIG4 528908.11 was captured on a protein A / G / L chip at ~350 RU and tested against mammalian NS0 cell-derived rhVSIG4 ( Fig. 12A ), mammalian-derived and fully deglycosylated rhVSIG4 ( Fig. 12B ), E. coli-derived and non-glycosylated VSIG-4 ( Fig. 12C ) and the highly glycosylated protein CD56 / NCAM ( Fig.12D ). All analyte proteins, rhVSIG4 and CD56 / NCAM were tested at a concentration range between 25 pM and 250 nM. Regardless of its glycosylation state, the antibody binds to VSIG4, but not to CD56 / NCAM, a highly glycosylated molecule with a similar carbohydrate profile to VSIG4, demonstrating the specificity of Ms x hVSIG4 528908.11 for the VSIG4 polypeptide and showing that Ms x hVSIG4 528908.11 is not anti-glycosylation specific.

[0065] Figures 13A-13D Ms x hVSIG4 528912.11 was shown to bind specifically to the VSIG4 polypeptide regardless of glycosylation status, as confirmed by Biacore. Ms x hVSIG4 528912.11 was captured on a protein A / G / L chip at ~350 RU and tested with mammalian NS0 cell-derived rhVSIG4 ( Fig.13A ), mammalian-derived and fully deglycosylated rhVSIG4 ( Fig. 13B ), E. coli-derived and non-glycosylated VSIG-4 ( Fig. 13C ) and the highly glycosylated protein CD56 / NCAM ( Fig.13D ). All analyte proteins, rhVSIG4 and CD56 / NCAM were tested at a concentration range between 25 pM and 250 nM. Regardless of the glycosylation state of VSIG-4, Ms x hVSIG4 528912.11 bound to VSIG4, but not to CD56 / NCAM, a highly glycosylated molecule with a similar sugar profile to VSIG4, demonstrating the specificity of Ms x hVSIG4 528912.11 for the VSIG4 polypeptide and showing that Ms x hVSIG4 528912.11 is not anti-glycosylation specific.

[0066] Fig.14Recombinant human Siglec-7 was shown to specifically bind to HEK293 cells transfected with recombinant human VSIG-4 fused to eGFP in a dose-dependent manner, but not to the same cell type transfected with recombinant human VSIG3-eGFP.

[0067] Fig.15 Representative anti-human VSIG-4 antibodies are shown blocking the interaction of VSIG-4 with recombinant human Siglec-7 / Fc on adherent M2c macrophages polarized with dexamethasone (adherent cells). M2c macrophages were incubated with 10 μg / mL, 40 μg / mL, or 200 μg / mL of Ms x hVSIG4 528908.11 to test the ability of the antibodies to block the interaction with 100 μg / mL, 25 μg / mL, or 5 μg / mL of rhSiglec-7. Histograms for CD14 + CD206 + Cells were gated.

[0068] Fig.16 Shown are the titrations of Ms x hVSIG4 528908.11 and recombinant human Siglec-7 Fig.15 Representative dot plots of the histograms shown in Figure 2. Data shown are for CD14 + and CD206 + Cells were gated.

[0069] Fig.17 Representative anti-human VSIG-4 antibodies are shown blocking the interaction of VSIG-4 with recombinant human Siglec-7 / Fc on suspended M2c macrophages polarized with dexamethasone (suspension cells). M2c macrophages were incubated with 10 μg / mL, 40 μg / mL, or 200 μg / mL of Ms x hVSIG4 528908.11 to test the ability of the antibodies to block the interaction with 100 μg / mL, 25 μg / mL, or 5 μg / mL of rhSiglec-7. Histograms for CD14 + CD206 + Cells were gated.

[0070] Fig.18 Shown are the titrations of Ms x hVSIG4 528908.11 and recombinant human Siglec-7 Fig.17 Representative dot plots of the histograms shown in Figure 2. Data shown are for CD14 + and CD206 + Cells were gated.

[0071] Figure 19A-Figure 19C Representative data of ELISA results obtained from direct ELISA testing are shown. Fig.19AIt was shown that Msx hVSIG4 528902.11 recognized monomeric VSIG-4 expressed in E. coli but not dimeric VSIG-4 expressed in NSO. Fig.19B It was shown that Ms x hVSIG4 528906.11 recognized glycosylated and unglycosylated VSIG-4 proteins equally. Fig.19C It was shown that Ms x hVSIG4 528905.11 recognized the glycosylated VSIG-4 protein but not the E. coli expressed VSIG-4.

[0072] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0073] The present disclosure describes antibodies capable of specifically binding to V-binding and Ig domain-containing 4 (VSIG-4 or VSIG4, also known as complement receptor Ig (CRIg) and Z391G); capable of blocking interactions between VSIG-4 and its ligands (including, for example, the recently observed interaction between VSIG-4 and SIGLEC-7); compositions comprising the antibodies described herein; and methods of making and using these antibodies and compositions.

[0074] VSIG-4 acts as a negative regulator of T cell activation and is likely to play a role in maintaining T cell tolerance. Thus, overexpression of VSIG-4 in the inflammatory tumor microenvironment may promote tumor tolerance. Therefore, antibodies that recognize VSIG-4 have potential diagnostic value to, for example, allow identification of the aggressive grade of tumors, provide an indication of potential long-term prognosis in animals with tumors, or serve as an aid in guiding treatment. In addition, neutralizing antibodies that abolish VSIG-4-induced inhibition of T cell activation may allow for increased immune responses against tumor cells.

[0075] Using a protein-protein interaction screen, sialic acid-binding Ig-like lectin 7 (Siglec7) was identified as a potential ligand for VSIG-4.

[0076] Siglec7 is a key regulator of natural killer (NK) cells and CD8 + A family of immunoglobulin monomers expressed on the surface of T cells (Nicoll et al., (1999) J. Biol. Chem. 274:34). Siglec7 is expressed on a highly functional subset of NK cells, and ligation of Siglec7 inhibits NK cell-mediated functions, suggesting that Siglec7 may act as an inhibitory receptor (Shao et al., (2016) Scand. J. Immunol. 84:182). In addition, the expression of Siglec7 ligands in the tumor microenvironment affects NK cell tumor immune surveillance (Jandus (2014) J. Clin. Invest. 124:1810).

[0077] Sialic acid-binding Ig-like lectins (SIGLECS or Siglecs) are typically activated by binding to sialic acid-modified proteins (or lipids), and the specificity of the interaction is determined by both the Siglec target molecule (including, for example, the precise glycan modification of a specific target protein that is usually not limited to a single site) and the Siglec molecule itself (for example, different Siglecs can bind to different sets of glycan modifications), and it is therefore difficult to determine the specificity or promiscuity of Siglec-target interactions.

[0078] As further described herein, therapeutic blockade of the VSIG-4 / Siglec7 interaction with neutralizing antibodies can be used to release VSIG4-induced inhibition and increase immune responses against tumor cells.

[0079] Antibody

[0080] In some embodiments, the disclosure describes antibodies that bind to VSIG-4 (ie, anti-VSIG-4 antibodies). In some embodiments, the antibodies bind to human VSIG-4 (hVSIG-4).

[0081] In some embodiments, the antibody that binds to VSIG-4 is a monoclonal antibody. In some embodiments, the antibody that binds to VSIG-4 includes a monoclonal antibody produced by a hybridoma cell line (also referred to herein as a clone) listed in Table 1 and / or by recombinant methods.

[0082] In some embodiments, the antibody is an isolated antibody.In some embodiments, the antibody can be isolated or purified by conventional immunoglobulin purification procedures, such as protein A- or G-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

[0083] In some embodiments, the antibody that binds to VSIG-4 recognizes a VSIG-4 polypeptide. In some embodiments, the VSIG-4 polypeptide is human VSIG-4 (Uniprot number: Q9Y279; Gene ID 11326) or a fragment thereof. In some embodiments, the VSIG-4 polypeptide is mouse VSIG-4 (Uniprot number: F6TUL9) or a fragment thereof. In some embodiments, the antibody that binds to VSIG-4 recognizes a non-reduced VSIG-4 polypeptide.

[0084] In some embodiments, the antibodies that bind to VSIG-4 bind to both glycosylated and unglycosylated VSIG-4. In some embodiments, the antibodies that bind to VSIG-4 recognize VSIG-4 polypeptides without recognizing glycan modifications of VSIG-4. In some embodiments, the antibodies that bind to VSIG-4 recognize glycan modifications of VSIG-4. In some embodiments, the antibodies that bind to VSIG-4 do not bind to α(2,8) linked polysialic acid. In some embodiments, the antibodies that bind to VSIG-4 bind to α(2,8) linked polysialic acid.

[0085] In some embodiments, an antibody that binds to VSIG-4 preferably binds to the extracellular domain of VSIG-4. In some embodiments, the extracellular domain of VSIG-4 includes amino acids 20-283 of Uniprot numbering Q9Y279. The extracellular domain of VSIG-4 may include an Ig domain, as shown in Figure 8 in some embodiments. In some embodiments, an antibody that binds to VSIG-4 may bind to Ig domain 1 of VSIG-4 (amino acids 21-131 of Q9Y279). In some embodiments, an antibody that binds to VSIG-4 may bind to Ig domain 2 of VSIG-4 (amino acids 143-226 of Q9Y279).

[0086] In some embodiments, antibodies that bind to VSIG-4 may include antibody derivatives that have been modified or conjugated by covalent attachment of any type of molecule to the antibody. Such antibody derivatives include, for example, antibodies that have been modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, toxins, or attachment to cellular ligands or other proteins. Any of a number of chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage of tunicamycin, acetylation, formylation, and metabolic synthesis. In addition, the derivative may contain one or more non-classical amino acids.

[0087] The antibody that binds to VSIG-4 can be directly or indirectly coupled to a detectable label by techniques well known in the art. A detectable label is a reagent that can be detected by, for example, spectroscopy, photochemistry, biochemistry, immunochemistry or chemical means. Useful detectable labels include, but are not limited to, fluorescent dyes, chemiluminescent compounds, radioisotopes, electron-dense reagents, enzymes, coenzymes, colored particles, biotin or digoxin. Detectable labels typically produce measurable signals, such as radioactivity, fluorescence, color or enzyme activity. Antibodies conjugated to detectable agents can be used for diagnostic or therapeutic purposes. Examples of detectable agents include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. Detectable substances can be directly coupled or conjugated to antibodies using techniques known in the art, or indirectly coupled or conjugated to antibodies through intermediates, for example, such as joints known in the art. See, for example, U.S. Pat. No. 4,741,900, which describes the conjugation of metal ions to antibodies for diagnostic purposes. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinamine fluorescein, dansyl chloride, and phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferin and aequorin; and examples of suitable radioactive materials include iodine ( 121 I, 123 I, 125 I, 131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 111 In, 112 In, 113 mIn, 115 mIn), technetium ( 99 Tc, 99 mTc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), Mo( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh and 97 Ru. The techniques for conjugating such therapeutic moieties to antibodies are well known.

[0088] In some embodiments, an antibody that binds to VSIG-4 comprises a monoclonal antibody produced by at least one of the following hybridoma cell lines (also referred to herein as clones): Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187); Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188); Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189); Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178); Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179); Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180); Ms x hVSIG4 Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182); Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183); Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184); Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); or Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

[0089] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187).

[0090] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188).

[0091] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189).

[0092] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178).

[0093] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179).

[0094] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180).

[0095] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181).

[0096] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182).

[0097] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183).

[0098] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184).

[0099] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185).

[0100] In some embodiments, an antibody that binds VSIG-4 comprises a monoclonal antibody produced by Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

[0101] Also included in the present disclosure are monoclonal antibodies produced by progeny or derivatives of these hybridoma cell lines, monoclonal antibodies produced by equivalent or similar hybridoma cell lines, and / or recombinant derivatives thereof. In some embodiments, antibodies that bind to VSIG-4 include recombinantly derived monoclonal antibodies, including, for example, rabbit B cell-derived monoclonal antibodies.

[0102] A complete antibody molecule has two heavy (H) chain variable regions (abbreviated herein as V H ) and two light (L) chain variable regions (abbreviated herein as V L ). H and V L The regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDRs"), interspersed with regions that are more conserved, termed "framework regions" ("FRs"). The extent of FRs and CDRs has been precisely defined (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242 and Chothia et al., J. Mol. Biol . 1987;196: 901-917). Each V H and V L It is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0103] In some embodiments, the monoclonal antibody comprises a monoclonal antibody having the same heavy chain as a monoclonal antibody produced by at least one of the following hybridoma cell lines: Ms x hVSIG4 528902.11; Ms x hVSIG4528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4 528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms xhVSIG4 528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4489518.11. In some embodiments, the monoclonal antibody comprises a monoclonal antibody having the same light chain as a monoclonal antibody produced by at least one of the following hybridoma cell lines: Ms x hVSIG4 528902.11; Ms x hVSIG4528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4 528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms xhVSIG4 528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4489518.11. In some embodiments, the monoclonal antibody includes a monoclonal antibody having the same heavy chain and the same light chain as a monoclonal antibody produced by at least one of the following hybridoma cell lines: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4528908.11; Ms x hVSIG4 528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4528922.111; Ms x hVSIG4 528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4 489518.11.In some embodiments, the monoclonal antibody may comprise 1, 2, 3, 4, 5, 6 or more amino acid substitutions in the heavy chain and / or light chain identified above, wherein the amino acid substitutions do not substantially affect the binding of the antibody to VSIG-4.

[0104] In some embodiments, the monoclonal antibody comprises a monoclonal antibody having the same V as a monoclonal antibody produced by at least one of the following hybridoma cell lines: H monoclonal antibodies against the HER2 domain: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4 528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms xhVSIG4 528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4 489518.11. In some embodiments, the monoclonal antibody comprises a monoclonal antibody having the same V as a monoclonal antibody produced by at least one of the following hybridoma cell lines: L monoclonal antibodies against the HER2 domain: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4 528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms xhVSIG4 528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4 489518.11. In some embodiments, the monoclonal antibody comprises a monoclonal antibody having the same V as a monoclonal antibody produced by at least one of the following hybridoma cell lines: H domain and the same V LIn some embodiments, the monoclonal antibody may be a monoclonal antibody against a V domain identified above: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4 528910.111; Ms x hVSIG4 528912.11; Ms xhVSIG4 528922.111; Ms x hVSIG4 528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4 489518.11. H Domain and / or V L The domain contains 1, 2, 3, 4, 5, 6 or more amino acid substitutions that do not substantially affect the binding of the antibody to VSIG-4.

[0105] In some embodiments, the monoclonal antibody comprises a V having a monoclonal antibody produced by at least one of the following hybridoma cell lines: H or Rt x hVSIG4 489518.11. In some embodiments, the monoclonal antibody comprises a V having a monoclonal antibody produced by at least one of the following hybridoma cell lines: Hor Rt x hVSIG4 489518.11. In some embodiments, the monoclonal antibody comprises a V having a monoclonal antibody produced by at least one of the following hybridoma cell lines: H or Rt x hVSIG4. The invention further comprises a monoclonal antibody comprising at least three CDRs of a domain of the antibody: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms x hVSIG4528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4489518.11.

[0106] Additionally or alternatively, in some embodiments, the monoclonal antibody comprises a V having a monoclonal antibody produced by at least one of the following hybridoma cell lines: Lor Rt x hVSIG4 489518.11. In some embodiments, the monoclonal antibody comprises a V having a monoclonal antibody produced by at least one of the following hybridoma cell lines: L or Rt x hVSIG4 489518.11. In some embodiments, the monoclonal antibody comprises a V having a monoclonal antibody produced by at least one of the following hybridoma cell lines: L or Rt x hVSIG4. The invention further comprises a monoclonal antibody comprising at least three CDRs of a domain of the antibody: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms x hVSIG4528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4489518.11.

[0107] In some embodiments, the monoclonal antibody may comprise 1, 2, 3, 4, 5, 6 or more amino acid substitutions in one or more of the CDRs identified above that do not substantially affect the binding of the antibody to VSIG-4.

[0108] In some embodiments, the monoclonal antibody may comprise 1, 2, 3, 4, 5, 6 or more amino acid substitutions in one or more framework regions (FR). In some embodiments, the substitution or substitutions in the framework region (FR) do not substantially affect the binding of the antibody to VSIG-4.

[0109] In some embodiments, the monoclonal antibodies include monoclonal antibodies having an amino acid sequence that is identical to the V sequence of a monoclonal antibody produced by at least one of the following hybridoma cell lines: H The amino acid sequence of at least one CDR of the domain has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms x hVSIG4528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4 489518.11.

[0110] In some embodiments, the monoclonal antibodies include monoclonal antibodies having an amino acid sequence that is identical to the V sequence of a monoclonal antibody produced by at least one of the following hybridoma cell lines: HThe amino acid sequences of at least two CDRs of the domain are at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms x hVSIG4528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4 489518.11.

[0111] In some embodiments, the monoclonal antibodies include monoclonal antibodies having an amino acid sequence that is identical to the V sequence of a monoclonal antibody produced by at least one of the following hybridoma cell lines: H The amino acid sequences of at least three CDRs of the domain are at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms x hVSIG4528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4 489518.11.

[0112] In some embodiments, the monoclonal antibodies include antibodies having an amino acid sequence that is identical to the V sequence of an antibody expressed by at least one of the following hybridoma cell lines: Lat least one CDR of the domain has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4 528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms x hVSIG4 528927.111; Rt x hVSIG4489509.11; Rt x hVSIG4489501. 489517.111; or Rt x hVSIG4 489518.11.

[0113] In some embodiments, the monoclonal antibodies include antibodies having an amino acid sequence that is identical to the V sequence of an antibody expressed by at least one of the following hybridoma cell lines: L At least two CDRs of the domain have at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4 528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms x hVSIG4 528927.111; Rt x hVSIG4489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4 489518.11.

[0114] In some embodiments, the monoclonal antibodies include antibodies having an amino acid sequence that is identical to the V sequence of an antibody expressed by at least one of the following hybridoma cell lines: LAt least three CDRs of the domain have at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4 528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms x hVSIG4 528927.111; Rt x hVSIG4489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4 489518.11.

[0115] The antibody can be an antibody from any suitable species. In some embodiments, the antibody can be a mouse antibody. In some embodiments, the antibody can be a rat antibody. In some embodiments, the antibody can be a rabbit antibody.

[0116] In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody can be an antibody or IgG subclass, including, for example, IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody can be a mouse IgG of one of the following subclasses: IgG1, IgG2A, IgG2B, IgG2C, and IgG3. In some embodiments, the antibody can be a rat IgG of one of the following subclasses: IgG1, IgG2A, IgG2B, or IgG2C.

[0117] In some embodiments, the antibody may comprise a kappa light chain. In some embodiments, the antibody may comprise a lambda light chain.

[0118] In some embodiments, the monoclonal antibody includes an antigen-binding fragment, including a Fab fragment, a Fab' fragment, a F(ab)2 fragment and / or a Fv fragment.

[0119] Monoclonal antibodies can be obtained by any suitable technique. In some embodiments, antibodies that bind to VSIG-4 can be prepared by recombinant DNA methods, produced by phage display and / or produced by combinatorial methods. DNA encoding antibodies that bind to VSIG-4 can be easily separated and sequenced using conventional procedures. In some embodiments, the hybridoma cells described herein can be used as a source of such DNA. Once isolated, the DNA can be transfected into host cells (including, for example, simian COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK), or myeloma cells that do not otherwise produce immunoglobulins), or introduced into host cells by genome editing (e.g., using a CRISPR-Cas system) to obtain the synthesis of monoclonal antibodies in recombinant host cells. The DNA encoding antibodies that bind to VSIG-4 can be modified, for example, to humanize the antibodies.

[0120] In some embodiments, the antibody may be a humanized antibody. Antibodies that bind to VSIG-4 may be humanized by any suitable method. Techniques for producing humanized monoclonal antibodies are described, for example, in Jones et al., (1986). Nature 321:522 and Singer et al., (1993) J. Immunol . 150:2844. For example, humanization of an antibody may include changes to the antibody to reduce the immunogenicity of the antibody when used in humans. In some embodiments, a humanized antibody that binds to VSIG-4 may include at least a portion of an immunoglobulin constant region (Fc) of a human immunoglobulin. In some embodiments, a humanized antibody that binds to VSIG-4 may include a human immunoglobulin (acceptor antibody) in which residues from one or more complementary determining regions (CDRs) of the acceptor antibody are replaced with residues from one or more CDRs of a non-human species antibody (donor antibody, such as a mouse, rat, or rabbit antibody) that binds to VSIG-4. In some embodiments, the Fv framework residues of a human immunoglobulin may be replaced by corresponding non-human residues from an antibody that binds to VSIG-4.

[0121] In some embodiments, monoclonal antibodies include chimeric antibodies, i.e., antibodies in which different portions are derived from different animal species. Chimeric antibodies can be obtained, for example, by splicing together genes from a mouse antibody molecule with the appropriate antigenic specificity with genes from a human antibody molecule with the appropriate biological specificity. See, e.g., Takeda et al., (1985) Nature 314:544.

[0122] In some embodiments, the antibody comprises a bispecific or bifunctional antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or connection of F(ab') fragments. See, e.g., Songsivilai and Lachmann (1990) Clin. Exp. Immunol . 79:315; Kostelny et al. (1992) J. Immunol .148:1547. In addition, bispecific antibodies can be used as "bifunctional antibodies" (Holliger et al. (1993) PNAS USA 90:6444), or "Janusins" (Traunecker et al., (1991) EMBO J. 10:3655; Traunecker et al. (1992) Int. J. Cancer Suppl . 7:51) formed.

[0123] In some embodiments, the antibody is produced by an animal (including but not limited to humans, mice, rats, rabbits, hamsters, goats, horses, chickens or turkeys), produced by cells from animals, chemically synthesized, or recombinantly expressed. Antibodies can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, and fractionated column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. In addition, the antibody can be fused to a heterologous polypeptide sequence, as described herein or otherwise known in the art, including, for example, to facilitate purification.

[0124] Can be by the method described herein and any suitable method known in the art, measure the immunospecific binding of other antibody.Usable immunoassay includes but is not limited to the competitive and / or non-competitive assay system using following technology, described technology such as BIACORE analysis, fluorescence activated cell sorting (FACS) analysis, immunofluorescence, immunocytochemistry, Western blot, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), "sandwich" immunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay and protein A immunoassay.Such determination is conventional and well-known in the art (referring to, people such as Ausubel edit, Current Protocols in Molecular Biology, first volume, John Wiley & Sons, Inc., NY (1994)).

[0125] In some embodiments, antibodies that bind to VSIG-4 include antibodies that eliminate the binding of VSIG-4 to a VSIG-4 ligand. In some embodiments, antibodies include antibodies that eliminate the binding of VSIG-4 to Siglec-7. In some embodiments, Siglec-7 is human Siglec-7 (Uniprot No.: Q9Y286). In some embodiments, the antibody can reduce the binding of VSIG-4 to a VSIG-4 ligand (including, for example, Siglec-7) by at least 10 percent (%), at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98%.

[0126] In some embodiments, the VSIG-4 ligand may be anchored to the cell via a transmembrane domain. In some embodiments, the VSIG-4 ligand may be in solution.

[0127] In some embodiments, an antibody that abolishes binding of VSIG-4 to Siglec-7 comprises a monoclonal antibody produced by at least one of the following hybridoma cell lines: Ms x hVSIG4 528903.111; Ms x hVSIG4 528908.11; Ms x hVSIG4 528912.11; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt xhVSIG4 489518.11.

[0128] In some embodiments, at least one of VSIG-4 and the VSIG-4 ligand may be on the cell surface. For example, VSIG-4 may be expressed on the surface of a macrophage (including, for example, an M2 macrophage). M2 macrophages may include one or more M2 subtypes, including M2a, M2b, M2c, and M2d subtypes. In some embodiments, the macrophage may preferably include an M2c macrophage. When at least one of VSIG-4 and the VSIG-4 ligand is on the cell surface, an antibody that binds to VSIG-4 can eliminate the binding of VSIG-4 to a cell expressing the VSIG-4 ligand, the binding of a cell expressing VSIG-4 to a VSIG-4 ligand, and / or the binding of a cell expressing VSIG-4 to a cell expressing the VSIG-4 ligand. For example, when VSIG-4 and the VSIG-4 ligand Siglec-7 are expressed on the cell surface, an antibody that binds to VSIG-4 can eliminate cell-cell binding.

[0129] In some embodiments, antibodies that bind to VSIG4 can be prepared by immunizing an animal with the extracellular domain of VSIG4. In some embodiments, antibodies that bind to VSIG4 can be prepared by immunizing an animal with amino acids 1-283 of human VSIG-4 (Uniprot number: Q9Y279; gene ID 11326). In some embodiments, antibodies that bind to VSIG4 can be prepared by immunizing an animal with a portion of human VSIG-4. In some embodiments, the animal can be a mammal. For example, the animal can be a rabbit, a mouse, a goat, a sheep, a llama, or a rat. In some embodiments, the animal can be a chicken.

[0130] On the other hand, the disclosure describes isolated polynucleotide molecules. In some embodiments, the isolated polynucleotide molecules include nucleotide sequences encoding antibodies. In some embodiments, the isolated polynucleotide molecules include nucleotide sequences with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the nucleotide sequences encoding antibodies described herein. In some embodiments, the isolated polynucleotide molecules include polynucleotides or their complements that hybridize to the nucleotide sequences encoding antibodies under high stringency. As used herein, "stringent conditions" refers to the ability of a first polynucleotide molecule to hybridize and remain bound to a second polynucleotide molecule bound to a filter at 65°C in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), and 1 mM EDTA, followed by washing in 0.2 X SSC / 0.1% SDS at 42°C (see Ausubel et al., (eds.), Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY (1989), at p. 2.10.3). In some embodiments, the isolated polynucleotide molecule includes a polynucleotide encoding one or more CDRs or heavy and / or light chains of a monoclonal antibody of the invention. General techniques for cloning and sequencing immunoglobulin variable domains and constant regions are well known. See, e.g., Orlandi et al., (1989) Proc. Nat'l Acad. Sci. USA 86:3833.

[0131] On the other hand, the present disclosure describes a recombinant vector comprising an isolated polynucleotide of the present invention. The vector can be in the form of, for example, a plasmid, a viral particle, or a bacteriophage. The appropriate DNA sequence can be inserted into the vector by a variety of procedures. Typically, the DNA sequence is inserted into a suitable restriction endonuclease site in the vector by procedures known in the art. Such procedures are considered to be within the capabilities of those skilled in the art. A large number of suitable vectors and promoters are known to those skilled in the art and are commercially available. The following vectors are provided by way of example. Bacterial vectors include, for example, pQE70, pQE60, pQE-9, pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5. Eukaryotic vectors include, for example, pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, and pSVL. However, any other plasmid or vector may be used.

[0132] In a further aspect, the present disclosure also includes a host cell containing at least one of the above-mentioned vectors. The host cell can be a higher eukaryotic cell, such as a mammalian or insect cell, or a lower eukaryotic cell, such as a yeast cell. Alternatively, the host cell can be a prokaryotic cell, such as a bacterial cell or a plant cell. The vector construct can be introduced into the host cell by any suitable technique, for example, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, or electroporation (Davis et al., Basic Methods in Molecular Biology (1986)).

[0133] Antibodies of the present invention can be expressed in mammalian cells, yeast, bacteria or other cells under the control of an appropriate promoter. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY (1989).

[0134] The present invention also includes phage display libraries expressing one or more hypervariable regions from antibodies of the present invention, and clones obtained from such phage display libraries. Phage display libraries are used to produce antibody-derived molecules. Gene fragments encoding the antigen-binding variable domains of antibodies are fused to genes encoding phage coat proteins. Phages containing such gene fusions are used to infect bacteria, and the resulting phage particles have a coat expressing the antibody fusion protein, wherein the antigen-binding domain is displayed on the outside of the phage. Phage display libraries can be prepared, for example, using a Ph.D.-7 phage display peptide library kit (Catalog No. E8100S) or a Ph.D.-12 phage display peptide library kit (Catalog No. E8110S) available from New England Biolabs Inc. Ipswich, MA. See, for example, Smith and Petrenko (1997) Chem Rev. 97:391-410.

[0135] Hybridoma cell lines

[0136] The present disclosure further describes hybridoma cell lines (also referred to herein as "clones") that express monoclonal antibodies, including, for example, the following hybridoma cell lines: Ms x hVSIG4 528902.11; Ms x hVSIG4 528903.111; Ms x hVSIG4 528905.11; Ms x hVSIG4 528906.11; Ms x hVSIG4 528908.11; Ms x hVSIG4528910.111; Ms x hVSIG4 528912.11; Ms x hVSIG4 528922.111; Ms x hVSIG4528927.111; Rt x hVSIG4 489509.11; Rt x hVSIG4 489517.111; or Rt x hVSIG4 5289529. hVSIG4489518.11. In some embodiments, the monoclonal antibody produced by the hybridoma cell line binds to VSIG-4. In some embodiments, the monoclonal antibody produced by the hybridoma cell line abolishes the binding of VSIG-4 to Siglec-7.

[0137] Hybridoma cell lines can be obtained by various techniques familiar to those skilled in the art. For example, cells from an animal immunized with a desired antigen are immortalized, usually by fusion with myeloma cells (see, e.g., Kohler and Milstein (1976)). Eur. J. Immunol. 6:511; J. Goding in "Monoclonal Antibodies: Principles and Practice," Academic Press, pp 59-103 (1986); and Harlow et al., Antibodies: A Laboratory Manual, 726 pages (Cold Spring Harbor Pub. 1988). In some embodiments, the immunized animal is preferably a mammal. In some embodiments, the immunized animal is a rat, including, for example, a Wistar rat, or a mouse, including, for example, a BALB / C mouse. In some embodiments, the cell from the animal is a spleen cell. In some embodiments, the cell from the animal is preferably a lymphocyte. In some embodiments, the myeloma cell includes a P3X63Ag8.653 cell.

[0138] Other known methods for generating transformed B cell lines that produce monoclonal antibodies may also be used.

[0139] Recombinant Antibodies

[0140] The present disclosure further describes recombinantly derived monoclonal antibodies. Recombinantly derived monoclonal antibodies may include, for example, rabbit B cell-derived monoclonal antibodies. The cloned antibodies of the present disclosure may be produced by any suitable recombinant technology, including, for example, by phage display, or by a combinatorial approach. See, for example, U.S. Patent No. 5,223,409; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; or WO 90 / 02809. Such methods may be used to produce human monoclonal antibodies.

[0141] Uses of anti-VSIG-4 antibodies

[0142] As described herein, antibodies that bind to VSIG-4 can be used in any suitable application. For example, monoclonal antibodies can be used in in vitro and in vivo diagnostic and therapeutic methods.

[0143] In some embodiments, the antibodies can be used to determine the expression level of VSIG-4 protein in vitro or in vivo. In some embodiments, determining the level of VSIG-4 protein expression can be used to detect cancer. In some embodiments, the antibodies can be used to label cells in vivo or in vitro. In some embodiments, the antibodies can be used to determine the expression level of VSIG-4 protein in a patient sample. In some embodiments, the patient sample can include mammalian cancer cells. In some embodiments, the mammalian cancer cells can include lung cancer cells or glioma cells. In some embodiments, the patient sample (including, for example, a patient sample including mammalian cancer cells) can include macrophages.

[0144] In some embodiments, the antibody can be labeled. For example, the antibody can be used to label cells, and the labeled cells can be imaged directly or indirectly by secondary methods. In certain embodiments, the cells are mammalian cells.

[0145] In some embodiments, the antibody can be used to identify the presence or absence of VSIG-4 protein in a sample from a subject. In some embodiments, identifying the presence of VSIG-4 can include identifying the amount of VSIG-4 in a sample from a subject. In some embodiments, identifying the presence of VSIG-4 can be used to detect cancer.

[0146] VSIG-4 has been previously reported to be expressed on tissue-resident macrophages. VSIG-4 has been observed in patients diagnosed with non-small cell lung cancer. + The massive infiltration of macrophages into the tumor microenvironment (Liao et al., (2014) Lab. Invest. 94:706), and high VSIG-4 expression is also associated with high-grade gliomas and poor patient prognosis (Xu et al., (2015) Am. J. Transl. Res. 7:1172). As shown in Example 3, VSIG-4 expression was detected on CD14-positive human PBMC monocytes polarized in vitro with dexamethasone or recombinant human IL-10 (rhIL-10) to differentiate into the M2c macrophage lineage (Figure 6).

[0147] In some embodiments, anti-VSIG-4 antibodies can be used to determine the expression level of VSIG-4 on cancer cells, tumor cells, or patient samples including cancer cells or tumor cells. In some embodiments, the patient sample including cancer cells or tumor cells may further include macrophages. In some embodiments, anti-VSIG-4 antibodies can be used to determine the expression level of VSIG-4 in or on cells of a patient sample including, for example, macrophages. In some embodiments, the level of VSIG-4 expression will be amplified. In some embodiments, anti-VSIG-4 antibodies can be used to determine the expression level of VSIG-4 in and / or on macrophages. In some embodiments, identifying the expression or expression level of VSIG-4 can be used to identify cancer or tumors; assist in the grading of cancer or tumors; and / or provide an indication of long-term prognosis. In some embodiments, the cancer may include lung cancer or glioma.

[0148] In some embodiments, anti-VSIG-4 antibodies can be used to treat mammalian cancer. In some embodiments, treating mammalian cancer can include exposing a mammal comprising mammalian cancer cells to an anti-VSIG-4 antibody. In some embodiments, VSIG-4 expression can be amplified in a patient sample comprising mammalian cancer cells. For example, VSIG-4 expression can be amplified in macrophages of a patient sample comprising mammalian cancer cells. In some embodiments, the mammalian cancer can include lung cancer or glioma.

[0149] In some embodiments, anti-VSIG-4 antibodies can be used to treat autoimmune diseases. Autoimmune diseases can include, for example, autoimmune type 1 diabetes, rheumatoid arthritis, psoriasis, or lupus. Without wishing to be bound by theory, macrophages are known to play a role in the pathogenesis of certain autoimmune diseases, see, for example, Campos Navegantes et al., (2017) J. Transl. Med. 15:36, and, as shown herein (see Example 5), anti-VSIG-4 antibodies can block the interaction between VSIG-4 naturally expressed on M2c macrophages and Siglec-7, and anti-VSIG-4 antibodies can be used, for example, to treat autoimmune diseases that involve interaction with VSIG-4 expressed on M2c macrophages.

[0150] Soluble recombinant human VSIG-4 was plated as a capture reagent in a standard ELISA assay as described in Example 1. Recombinant, biotinylated human SIGLEC-7 was found to bind to VSIG-4 ( Figure 1& Table 1). Putative protein-protein interactions were then examined in a cell-based assay. HEK cells transfected to overexpress human VSIG-4 and eGFP were cultured with rhSIGLEC-7 / Fc. Binding of rhSIGLEC-7 / Fc to cells was detected by flow cytometry using an APC-conjugated anti-Fc antibody (Figure 2). Binding of SIGLEC-7 to VSIG-4 was further analyzed via surface plasmon resonance (SPR) / BIACORE ( Figure 3 ).

[0151] A cell-based assay was used to titrate the amount of rhSIGLEC-7 on VSIG-4-transfected cells and the interaction was shown to be dose-dependent ( Figure 4 To further confirm this interaction, the assay was flipped and it was shown that rhVSIG-4 bound to cells expressing SIGLEC-7 in a dose-dependent manner ( Figure 5 ).

[0152] As shown in Examples 2 and 3, specific binding of rhSIGLEC-7 to VSIG-4 was confirmed using anti-VSIG-4 antibodies to block the interaction ( Figure 5 ). Four clones were shown to specifically block the SIGLEC-7 / VSIG-4 interaction. The binding affinities of these and other monoclonal anti-VSIG-4 antibodies were determined via surface plasmon resonance (SPR) / BIACORE (Table 2).

[0153] As shown in Example 4, VSIG-4 can be glycosylated ( FIG. 9 ). As shown in Example 4 and Example 6, in some embodiments, anti-VSIG-4 antibodies can bind to VSIG-4 polypeptides without binding to glycan modifications of VSIG-4 ( FIG. 10 - FIG. 13 , FIG. 19 , Table 3), resulting in anti-VSIG-4 antibodies that can bind to glycosylated and unglycosylated VSIG-4. In some embodiments, anti-VSIG4 antibodies can bind to glycosylated VSIG-4, but not to unglycosylated polypeptides.

[0154] In some embodiments, anti-VSIG-4 antibodies or compositions comprising anti-VSIG-4 antibodies can be used to eliminate the binding of VSIG-4 to Siglec-7. For example, as shown in Example 5, anti-VSIG-4 antibodies or compositions comprising anti-VSIG-4 antibodies can be used to eliminate cell-cell interactions ( Figure 15-18 Without wishing to be bound by theory, it is believed that antibodies that abolish the binding of VSIG-4 to Siglec-7 may be activated by Siglec-7 in CD8 +The connection on T cells and NK cells is used to eliminate or inhibit T cell / NK cell immune surveillance. Therefore, therapeutic blockade of the VSIG-4-Siglec7 interaction using neutralizing antibodies can be used to release VSIG-4-induced inhibition and enhance immune function against tumor cells or inhibit immune responses in autoimmune diseases. Additionally or alternatively, antibodies that eliminate the binding of VSIG-4 to Siglec-7 can eliminate or inhibit macrophage signaling by inhibiting the binding of Siglec-7 to VSIG-4 on macrophages.

[0155] The present disclosure further describes kits comprising antibodies. For example, the kit can include a composition comprising an anti-VSIG-4 monoclonal antibody. The antibodies in the kit can be labeled with one or more detectable markers, as described herein.

[0156] The test kit may include one or more containers filled with one or more monoclonal antibodies of the present invention. In addition, the test kit may include other reagents, such as buffers, and also include solutions required for implementing the present invention. Optionally associated with such containers may be instructions for notification or printing. As used herein, the phrase "packaging material" refers to one or more physical structures for containing the contents of the test kit. The packaging material is constructed by known methods, preferably providing an aseptic, non-contaminated environment. As used herein, the term "packaging" refers to a solid matrix or material, such as glass, plastic, paper, foil, etc., that can keep the polypeptide within fixed limits.

[0157] Compositions comprising antibodies

[0158] In some embodiments, the present disclosure describes compositions comprising at least one antibody described herein.

[0159] In some embodiments, the composition may also include, for example, a buffer to help maintain the pH within an acceptable range, or a preservative to prevent the growth of microorganisms. The composition may also include, for example, a carrier, excipient, stabilizer, chelating agent, salt, or antimicrobial agent. Acceptable carriers, excipients, stabilizers, chelating agents, salts, preservatives, buffers, or antimicrobial agents include, but are not limited to, buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives, such as sodium azide, octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and meta-cresol; polypeptides; proteins, such as serum albumin; Protein, gelatin or nonspecific immunoglobulin; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc (Zn)-protein complexes); and / or nonionic surfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG).

[0160] In some embodiments, the composition is a pharmaceutical composition and includes a monoclonal antibody and a pharmaceutically acceptable carrier, diluent or excipient. As will be apparent to those skilled in the art, a variety of vehicles and excipients may be used in the preparation of pharmaceutical compositions comprising the antibodies described in the teachings herein.

[0161] The pharmaceutical composition will generally comprise a pharmaceutically acceptable carrier and a pharmacologically effective amount of the antibody or antibody mixture.

[0162] The pharmaceutical composition can be formulated as a powder, granules, solution, suspension, aerosol, solid, pill, tablet, capsule, gel, topical cream, suppository, transdermal patch, and / or another formulation known in the art.

[0163] For the purposes described herein, pharmaceutically acceptable salts of antibodies are intended to include any pharmaceutically acceptable salts recognized in the art, including organic and inorganic acids and / or bases. Examples of salts include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and primary, secondary and tertiary amines, esters of lower hydrocarbons, such as methyl, ethyl and propyl. Other salts include, but are not limited to, organic acids, such as acetic acid, propionic acid, pyruvic acid, maleic acid, succinic acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, salicylic acid, and the like.

[0164] As used herein, "pharmaceutically acceptable carrier" includes any standard pharmaceutically acceptable carrier known to those of ordinary skill in the art of formulating pharmaceutical compositions. For example, the antibody can be prepared as a formulation in a pharmaceutically acceptable diluent, the diluent including, for example, saline, phosphate buffered saline (PBS), aqueous ethanol, or glucose, mannitol, dextran, propylene glycol, oil (e.g., vegetable oil, animal oil, synthetic oil, etc.), microcrystalline cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, magnesium stearate, calcium phosphate, gelatin, polysorbate 80 solution, or prepared as a solid preparation in an appropriate excipient.

[0165] The pharmaceutical composition will generally further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids, such as glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, etc.), bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), solutes that make the preparation isotonic, hypotonic or weakly hypertonic with the blood of the recipient, suspending agents, thickening agents and / or preservatives. Alternatively, the composition of the present invention can be formulated as a lyophilized agent.

[0166] Any suitable carrier known to those of ordinary skill in the art can be used in a composition comprising at least one antibody described herein.Antibody compositions can be formulated for any suitable mode of administration, including, for example, oral, nasal, mucosal, intravenous, intraperitoneal, intradermal, subcutaneous, and intramuscular administration.

[0167] Administration and treatment

[0168] The compositions of the present disclosure can be formulated into various forms of pharmaceutical preparations suitable for the selected route of administration. It will be appreciated by the technician that the composition will vary depending on the mode of administration and dosage unit. For example, for parenteral administration, isotonic saline can be used. For topical administration, a cream can be used, which contains a carrier, such as dimethyl sulfoxide (DMSO), or other agents that are commonly found in topical creams and do not block or inhibit peptide activity. Other suitable carriers include, but are not limited to, alcohol, phosphate buffered saline and other balanced salt solutions. The compounds of the present invention can be administered in a variety of ways, including but not limited to intravenous, topical, oral, subcutaneous, intraperitoneal and intramuscular delivery. In some aspects, the compounds of the present invention can be formulated for controlled release or sustained release. In some aspects, the preparations for controlled release or sustained release are suitable for subcutaneous implantation. In some aspects, the preparations for controlled release or sustained release include patches. The compound can be formulated for enteral administration, such as capsules or tablets.

[0169] Can be administered as a single dose or multiple doses. In some embodiments, the dosage is an effective amount determined by standard methods, including but not limited to those described herein. Technicians in the field of clinical trials will be able to optimize the dosage of a particular compound through standard studies. In addition, the appropriate dosage of the composition can be determined using a standard dose-response protocol without excessive experimentation. Administration includes but is not limited to any dosage and dosing regimen, dosing interval and / or mode of administration described in the examples included herein.

[0170] Compositions comprising antibodies according to the present disclosure can be administered by any suitable means, including, but not limited to, e.g., orally, rectally, nasally, topically (including transdermal, aerosol, buccal and / or sublingual), vaginally, parenterally (including subcutaneous, intramuscular and / or intravenous), intradermally, intravesically, intraarticularly, intraarterioleally, intraventricularly, intracranially, intraperitoneally, intranasally, by inhalation, or intralesionally (e.g., by injection into or around a tumor).

[0171] For parenteral administration in aqueous solution, for example, the solution should be appropriately buffered (if necessary), and the liquid diluent should first be made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous medium that can be used is known to those skilled in the art. Depending on the patient's condition to be treated, some changes in dosage will inevitably occur. In any case, the person responsible for administration will determine the appropriate dose for the individual subject. In addition, for human administration, the preparation should meet the sterility, pyrogenicity and general safety and purity standards required by the FDA. Such preparations can be pyrogen-free.

[0172] Many suitable formulations are known, including polymer or protein microparticles encapsulating the drug to be released, ointments, gels or solutions that can be used externally or topically to administer the drug, and even patches that provide controlled release over extended periods of time. These can also take the form of implants. Such implants can be implanted into the tumor.

[0173] The compounds of the present invention can also be provided in lyophilized form. Such compositions can include a buffer, such as bicarbonate, for reconstitution prior to administration, or a buffer can be included in the lyophilized composition to be reconstituted using, for example, water. The lyophilized composition can further include a suitable vasoconstrictor, such as epinephrine. The lyophilized composition can be provided in a syringe, optionally packaged in combination with a buffer for reconstitution, so that the reconstituted composition can be administered immediately to the patient.

[0174] As used herein, "treating" or "treatment" may include therapeutic and / or preventive treatment. As used herein, "treating a disease" is not intended to be an absolute term. Treatment may result in an improved prognosis, or a reduction in the frequency or severity of symptoms. As used herein, a "therapeutically effective" concentration or amount is an amount that provides some improvement or benefit to the subject. Desired therapeutic effects include preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. Likewise, as used herein, the term "prevention" is not intended to be an absolute term. Instead, prevention refers to delaying the onset, reducing the frequency of symptoms, or reducing the severity of symptoms associated with the disease. Therefore, prevention refers to a wide range of preventive measures that will be understood by those skilled in the art. In some cases, the frequency and severity of symptoms are reduced to non-pathological levels. In some cases, symptoms in an individual receiving a composition of the invention are only 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% of the frequency or severity of symptoms experienced by an untreated individual with the disease.

[0175] Therapeutically effective concentrations and amounts for each application herein can be determined empirically by testing the compounds in known in vitro and in vivo systems (such as those described herein), and dosages for humans or other animals can then be extrapolated from there.

[0176] It should be understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known test protocols or by inference from in vivo or in vitro test data. It should be noted that concentrations and dosage values ​​may also vary with the severity of the condition to be alleviated. It should be further understood that for any particular subject, a specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the concentration ranges set forth herein are merely exemplary and are not intended to limit the scope or implementation of the claimed compositions and methods.

[0177] Toxicity and therapeutic efficacy of the compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as LD 50 and ED 50 The ratio between ED and 4 is preferably between ED and 4. Compositions that exhibit high therapeutic indices may be preferred. Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compositions may preferably be within the range that includes ED50 The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage may be selected by individual physicians taking into account the patient's condition.

[0178] Compositions as described herein can be administered once, or can be divided into multiple smaller doses, administered at intervals. For example, the composition can be repeatedly administered, such as at least 2, 3, 4, 5, 6, 7, 8 or more times, or can be administered by continuous infusion. It should be understood that the precise dosage and duration of treatment are a function of the disease being treated, and can be determined empirically using known test protocols or by inferring from in vivo or in vitro test data. It should be noted that concentrations and dosage values ​​can also vary with the severity of the condition to be alleviated. It should be further understood that for any specific subject, a specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the concentration ranges set forth herein are merely exemplary and are not intended to limit the scope or implementation of the claimed composition and method.

[0179] In some therapeutic embodiments, an "effective amount" of an agent is an amount that results in a reduction in at least one pathological parameter. Thus, for example, in some aspects of the present disclosure, an effective amount is an amount that is effective to achieve at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% reduction compared to the expected reduction in the parameter in an individual not treated with the agent.

[0180] In some aspects of the methods of the present disclosure, the methods further include the administration of one or more additional therapeutic agents. One or more additional therapeutic agents may be administered before, after, and / or simultaneously with the administration of the monoclonal antibody as described herein. Additional therapeutic agents may include, for example, chemotherapy, radiotherapy, etc. Additional therapeutic agents may be administered alone or as part of a mixture or cocktail. In some aspects of the present disclosure, the administration of antibodies may allow for the effectiveness of other treatments at lower doses, providing relief from toxicity observed in other treatments at higher doses, compared to the administration of other treatments alone.

[0181] In some aspects of the methods of the present disclosure, administration of the compositions described herein and at least one additional therapeutic agent demonstrates therapeutic synergy. In some aspects of the methods of the present disclosure, a measure of response to treatment observed after administration of both an antibody as described herein and an additional therapeutic agent is improved compared to the same measure of response to treatment observed after administration of either the antibody or the additional therapeutic agent alone.

[0182] Exemplary embodiments

[0183] Embodiment 1: Monoclonal antibodies that bind to VSIG-4.

[0184] Embodiment 2: A monoclonal antibody produced by at least one of the following clones:

[0185] Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

[0186] Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

[0187] Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

[0188] Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

[0189] Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

[0190] Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

[0191] Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

[0192] Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

[0193] Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

[0194] Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

[0195] Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

[0196] Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

[0197] Embodiment 3: Monoclonal antibody, wherein the monoclonal antibody comprises at least one of the following:

[0198] The heavy chain variable region of a monoclonal antibody produced by at least one of the following clones:

[0199] Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

[0200] Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

[0201] Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

[0202] Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

[0203] Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

[0204] Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

[0205] Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

[0206] Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

[0207] Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

[0208] Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

[0209] Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

[0210] Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186);

[0211] and

[0212] The light chain variable region of a monoclonal antibody produced by at least one of the following clones:

[0213] Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

[0214] Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

[0215] Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

[0216] Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

[0217] Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

[0218] Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

[0219] Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

[0220] Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

[0221] Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

[0222] Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

[0223] Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

[0224] Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

[0225] Embodiment 4: Monoclonal antibody, wherein the monoclonal antibody comprises at least one of the following:

[0226] A heavy chain variable region comprising the complementarity determining regions (CDRs) of the heavy chain of a monoclonal antibody produced by at least one of the following clones:

[0227] Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

[0228] Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

[0229] Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

[0230] Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

[0231] Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

[0232] Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

[0233] Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

[0234] Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

[0235] Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

[0236] Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

[0237] Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

[0238] Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186);

[0239] and

[0240] A light chain variable region comprising the complementarity determining regions (CDRs) of a light chain of a monoclonal antibody produced by at least one of the following clones:

[0241] Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

[0242] Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

[0243] Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

[0244] Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

[0245] Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

[0246] Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

[0247] Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

[0248] Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

[0249] Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

[0250] Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

[0251] Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

[0252] Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

[0253] Embodiment 5: The monoclonal antibody of embodiment 4, wherein the monoclonal antibody comprises each CDR of the heavy chain of a monoclonal antibody produced by one of the following clones:

[0254] Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

[0255] Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

[0256] Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

[0257] Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

[0258] Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

[0259] Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

[0260] Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

[0261] Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

[0262] Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

[0263] Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

[0264] Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

[0265] Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

[0266] Embodiment 6: The monoclonal antibody of any one of Embodiments 4 or 6, wherein the monoclonal antibody comprises each CDR of the light chain of a monoclonal antibody produced by one of the following clones:

[0267] Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

[0268] Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

[0269] Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

[0270] Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

[0271] Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

[0272] Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

[0273] Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

[0274] Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

[0275] Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

[0276] Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

[0277] Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

[0278] Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

[0279] Embodiment 7: A monoclonal antibody, wherein the monoclonal antibody comprises an amino acid sequence:

[0280] Having at least 80% identity to the amino acid sequence of at least one complementarity determining region (CDR) of the heavy chain variable region of a monoclonal antibody produced by at least one of the following clones:

[0281] Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

[0282] Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

[0283] Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

[0284] Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

[0285] Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

[0286] Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

[0287] Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

[0288] Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

[0289] Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

[0290] Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

[0291] Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

[0292] Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186);

[0293] and / or

[0294] Having at least 80% identity to the amino acid sequence of at least one complementarity determining region (CDR) of the light chain variable region of a monoclonal antibody produced by at least one of the following clones:

[0295] Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

[0296] Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

[0297] Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

[0298] Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

[0299] Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

[0300] Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

[0301] Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

[0302] Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

[0303] Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

[0304] Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

[0305] Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

[0306] Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

[0307] Embodiment 8: The monoclonal antibody of Embodiment 7, wherein the monoclonal antibody comprises an amino acid sequence, wherein:

[0308] Having at least 80% identity with the amino acid sequence of each complementarity determining region (CDR) of the heavy chain variable region of a monoclonal antibody produced by at least one of the following clones:

[0309] Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

[0310] Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

[0311] Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

[0312] Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

[0313] Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

[0314] Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

[0315] Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

[0316] Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

[0317] Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

[0318] Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

[0319] Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

[0320] Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186);

[0321] and

[0322] The antibody is at least 80% identical to the amino acid sequence of each complementarity determining region (CDR) of the light chain variable region of a monoclonal antibody produced by at least one of the following clones:

[0323] Ms x hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

[0324] Ms x hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

[0325] Ms x hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

[0326] Ms x hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

[0327] Ms x hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

[0328] Ms x hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

[0329] Ms x hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

[0330] Ms x hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

[0331] Ms x hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

[0332] Rt x hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

[0333] Rt x hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

[0334] Rt x hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

[0335] Embodiment 9: The monoclonal antibody of any one of Embodiments 2 to 8, wherein said antibody binds to VSIG-4.

[0336] Embodiment 10: The monoclonal antibody of any one of embodiments 1 to 9, wherein said monoclonal antibody is directly or indirectly conjugated to a detectable label.

[0337] Embodiment 11: The monoclonal antibody of any one of Embodiments 1 to 10, wherein the monoclonal antibody comprises an IgG antibody.

[0338] Embodiment 12: The monoclonal antibody of any one of Embodiments 1 to 11, wherein the monoclonal antibody abolishes the binding of VSIG-4 to a VSIG-4 ligand.

[0339] Embodiment 13: The monoclonal antibody of any one of Embodiments 1 to 12, wherein the VSIG-4 ligand comprises SIGLEC-7.

[0340] Embodiment 14: The monoclonal antibody of Embodiment 12 or 13, wherein at least one of VSIG-4 and the VSIG-4 ligand is on the surface of a cell.

[0341] Embodiment 15: The monoclonal antibody of any one of Embodiments 12 to 14, wherein VSIG-4 is on the surface of M2c macrophages.

[0342] Embodiment 16: The monoclonal antibody of any one of Embodiments 12 to 15, wherein said monoclonal antibody abrogates cell-cell interactions.

[0343] Embodiment 17: The monoclonal antibody of any one of Embodiments 1 to 16, wherein said monoclonal antibody binds to the extracellular domain of VSIG-4.

[0344] Embodiment 18: The monoclonal antibody of any one of Embodiments 1 to 17, wherein said monoclonal antibody binds to a VSIG-4 polypeptide.

[0345] Embodiment 19: The monoclonal antibody of any one of Embodiments 1 to 18, wherein said monoclonal antibody binds to both glycosylated and unglycosylated VSIG-4.

[0346] Embodiment 20: The monoclonal antibody of any one of Embodiments 1 to 19, wherein the monoclonal antibody comprises an antigen binding fragment, the antigen binding fragment comprising at least one of a Fab fragment, a Fab' fragment, a F(ab)2 fragment and a Fv fragment.

[0347] Embodiment 21: A composition comprising the monoclonal antibody of any one of embodiments 1 to 20.

[0348] Embodiment 22: A kit comprising the monoclonal antibody of any one of embodiments 1 to 20.

[0349] Embodiment 23: The monoclonal antibody of any one of Embodiments 1 to 20, for use in a method of treating cancer in a mammal.

[0350] Embodiment 24: The monoclonal antibody of any one of embodiments 1 to 20, for use in a method of treating an autoimmune disease.

[0351] Embodiment 25: A method of treating cancer in a mammal comprising exposing the mammal comprising mammalian cancer cells to the monoclonal antibody of any one of embodiments 1 to 20.

[0352] Embodiment 26: The method of Embodiment 25, wherein VSIG-4 expression is amplified in a patient sample comprising mammalian cancer cells.

[0353] Embodiment 27: The method of Embodiment 25, wherein VSIG-4 expression is amplified in macrophages of a patient sample comprising mammalian cancer cells.

[0354] Embodiment 28: The method of any one of Embodiments 25 to 27, wherein the mammalian cancer cells comprise lung cancer cells or gliomas.

[0355] Embodiment 29: The method of any one of Embodiments 25 to 28, wherein the mammalian cancer cells are also exposed to chemotherapy or radiation therapy.

[0356] Embodiment 30: A method of detecting cancer in a mammal, comprising exposing mammalian cancer cells to the monoclonal antibody of any one of embodiments 1 to 20.

[0357] Embodiment 31: A method of treating an autoimmune disease in a mammal, comprising exposing cells of the mammal to the monoclonal antibody of any one of embodiments 1 to 20.

[0358] Embodiment 32: The method of Embodiment 31, wherein VSIG-4 expression is amplified in a patient sample comprising mammalian cells.

[0359] Embodiment 33: The method of Embodiment 32, wherein VSIG-4 expression is amplified in macrophages of a patient sample comprising mammalian cells.

[0360] Embodiment 34: The method of any one of Embodiments 31 to 33, wherein the autoimmune disease comprises autoimmune type 1 diabetes, rheumatoid arthritis, psoriasis, or lupus.

[0361] Embodiment 35: The monoclonal antibody of any one of Embodiments 1 to 20, for use in a method of treating an autoimmune disease.

[0362] Exemplary Hybridoma Cell Line Embodiments

[0363] Embodiment 1: The hybridoma cell line Ms x hVSIG4 528902.11 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124187.

[0364] Embodiment 2: The hybridoma cell line Ms x hVSIG4 528903.111 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124188.

[0365] Embodiment 3: The hybridoma cell line Ms x hVSIG4 528905.11 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124189.

[0366] Embodiment 4: The hybridoma cell line Ms x hVSIG4 528906.11 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124178.

[0367] Embodiment 5: The hybridoma cell line Ms x hVSIG4 528908.11 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124179.

[0368] Embodiment 6: The hybridoma cell line Ms x hVSIG4 528910.111 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124180.

[0369] Embodiment 7: The hybridoma cell line Ms x hVSIG4 528912.11 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124181.

[0370] Embodiment 8: The hybridoma cell line Ms x hVSIG4 528922.111 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124182.

[0371] Embodiment 9: The hybridoma cell line Ms x hVSIG4 528927.111 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124183.

[0372] Embodiment 10: The hybridoma cell line Rt x hVSIG4 489509.11 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124184.

[0373] Embodiment 11: The hybridoma cell line Rt x hVSIG4 489517.111 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124185.

[0374] Embodiment 12: The hybridoma cell line Rt x hVSIG4 489518.11 is deposited in the American Type Culture Collection (ATCC) with ATCC accession number PTA-124186.

[0375] Exemplary Monoclonal Antibodies Produced by Hybridoma Cell Line Embodiments

[0376] Embodiment 1: Monoclonal antibodies produced by the hybridoma cell line Ms x hVSIG4528902.11 deposited under ATCC Accession No. PTA-124187.

[0377] Embodiment 2: Monoclonal antibodies produced by the hybridoma cell line Ms x hVSIG4528903.111 deposited under ATCC Accession No. PTA-124188.

[0378] Embodiment 3: Monoclonal antibodies produced by the hybridoma cell line Ms x hVSIG4528905.11 deposited under ATCC Accession No. PTA-124189.

[0379] Embodiment 4: Monoclonal antibodies produced by the hybridoma cell line Ms x hVSIG4528906.11 deposited under ATCC Accession No. PTA-124178.

[0380] Embodiment 5: Monoclonal antibodies produced by the hybridoma cell line Ms x hVSIG4528908.11 deposited under ATCC Accession No. PTA-124179.

[0381] Embodiment 6: Monoclonal antibodies produced by the hybridoma cell line Ms x hVSIG4528910.111 deposited under ATCC Accession No. PTA-124180.

[0382] Embodiment 7: Monoclonal antibodies produced by the hybridoma cell line Ms x hVSIG4528912.11 deposited under ATCC Accession No. PTA-124181.

[0383] Embodiment 8: Monoclonal antibodies produced by the hybridoma cell line Ms x hVSIG4528922.111 deposited under ATCC Accession No. PTA-124182.

[0384] Embodiment 9: Monoclonal antibodies produced by the hybridoma cell line Ms x hVSIG4528927.111 deposited under ATCC Accession No. PTA-124183.

[0385] Embodiment 10: Monoclonal antibodies produced by the hybridoma cell line Rt x hVSIG4489509.11 deposited under ATCC Accession No. PTA-124184.

[0386] Embodiment 11: Monoclonal antibodies produced by the hybridoma cell line Rt x hVSIG4489517.111 deposited under ATCC Accession No. PTA-124185.

[0387] Embodiment 12: Monoclonal antibodies produced by the hybridoma cell line Rt x hVSIG4489518.11 deposited under ATCC Accession No. PTA-124186.

[0388] Embodiment 13: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Ms x hVSIG4 528902.11 deposited under ATCC Accession No. PTA-124187.

[0389] Embodiment 14: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Ms x hVSIG4 528903.111 deposited under ATCC Accession No. PTA-124188.

[0390] Embodiment 15: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Ms x hVSIG4 528905.11 deposited under ATCC Accession No. PTA-124189.

[0391] Embodiment 16: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Ms x hVSIG4 528906.11 deposited under ATCC Accession No. PTA-124178.

[0392] Embodiment 17: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Ms x hVSIG4 528908.11 deposited under ATCC Accession No. PTA-124179.

[0393] Embodiment 18: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Ms x hVSIG4 528910.111 deposited under ATCC Accession No. PTA-124180.

[0394] Embodiment 19: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Ms x hVSIG4 528912.11 deposited under ATCC Accession No. PTA-124181.

[0395] Embodiment 20: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Ms x hVSIG4 528922.111 deposited under ATCC Accession No. PTA-124182.

[0396] Embodiment 21: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Ms x hVSIG4 528927.111 deposited under ATCC Accession No. PTA-124183.

[0397] Embodiment 22: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Rt x hVSIG4 489509.11 deposited under ATCC Accession No. PTA-124184.

[0398] Embodiment 23: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Rt x hVSIG4 489517.111 deposited under ATCC Accession No. PTA-124185.

[0399] Embodiment 24: A monoclonal antibody comprising the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line Rt x hVSIG4 489518.11 deposited under ATCC Accession No. PTA-124186.

[0400] The present invention is illustrated by the following examples. It is to be understood that the specific examples, materials, amounts and methods are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0401] Example

[0402] Unless otherwise noted, all incubations in the following examples were performed at room temperature, and all reagents, starting materials, and solvents used were purchased from commercial suppliers (e.g., Sigma-Aldrich, St. Louis, MO) and used without further purification unless otherwise noted.

[0403] Example 1: Characterization of VSIG4-Siglec7 interaction

[0404] Human Siglec7 (hSiglec7) (Uniprot number: Q9Y286) and human VSIG4 (hVSIG4) (Uniprot number: Q9Y279; gene ID 11326) were recombinantly expressed in HEK293, NSO or CHO cells to provide recombinant human Siglec7 (rhSiglec7) and recombinant human VSIG4 (rhVSIG4). As indicated, the proteins were tagged with various N- or C-terminal tags (such as His, Fc or GFP) to enable purification and analysis in various assays.

[0405] Interaction of rhVSIG4 with biotinylated rhSiglec7. Soluble rhVSIG4 was coated on a standard ELISA plate and incubated with soluble rhSIGLEC 7 after removing excess protein and blocking other binding sites on the plate with BSA. Biotinylated rhSIGLEC-7 was detected using streptavidin-horseradish peroxidase (HRP). After background correction, an absorbance of 2.3 was detected at 452 nm in a standard microplate reader.

[0406] HEK / eGFP transfectants and Fc protein flow screening. HEK293 cells transfected with eGFP expressing human VSIG-4 or human Siglec-7 were harvested, washed with RDFII staining buffer (0.5% (w / v) BSA, 10 mM HEPES, 100 mM sodium chloride, 0.01% sodium azide, pH 7.2), counted, aliquoted at 100,000 cells / sample, and incubated with different amounts of recombinant human Siglec-7 or VSIG-4 proteins containing human IgG Fc regions (rhSiglec-7 / Fc or rhVSIG-4 / Fc). The protein concentrations tested ranged from 500 ng / mL to 6 μg / mL. The transfectants and Fc proteins were incubated at room temperature for 30 minutes, and then anti-human IgG Fc-APC detection antibody (FAB110A, R&D Systems, Minneapolis, MN) was added for another 20 minutes. The cells were then washed with RDFII staining buffer and analyzed on a Novocyte flow cytometer (ACEA Biosciences, Inc., San Diego, CA) or a LSRII FORTESSA flow cytometer (BD Biosciences, San Jose, CA).

[0407] Example 2: Identification of anti-VSIG4 antibodies and VSIG4-Siglec7 blocking antibodies

[0408] Using a panel of monoclonal antibodies raised against human or mouse VSIG4 protein (Uniprot number: Q9Y279 or F6TUL9, respectively), antibodies binding to VSIG-4 were identified by direct ELISA and flow cytometry. Table 1 lists clones producing VSIG-4 binding antibodies.

[0409] The ability of these anti-VSIG-4 antibodies to abrogate the binding of VSIG-4 to Siglec-7 protein was also determined, as further described below.

[0410] VSIG4-Siglec7 blocking assay. Human VSIG-4 HEK / eGFP transfectants were harvested, washed with RDFII staining buffer, counted, aliquoted at 100,000 cells / sample, and incubated with 2.5 μg / mL anti-VSIG-4 antibody for 30 minutes at room temperature. Cells were washed with RDFII staining buffer and 4 μg / mL of hSiglec-7 / Fc protein was added to each tube for 30 minutes. After this incubation, anti-Fc APC detection antibody (FAB110A) was added for an additional 20 minutes. Cells were then washed with RDFII staining buffer and analyzed on an Aceabio Novocyte flow cytometer or LSRII Fortessa flow cytometer (BD Biosciences, San Jose, CA). Exemplary results are shown in Figure 5 Other results are shown in Table 1.

[0411] Table 1: Anti-VSIG-4 monoclonal antibodies and their characteristics

[0412] Host species Antibody Type application Kd Blocking clone Ms x hVSIG4 528902.11 Mouse IgG3 WB (negative), FC ND N clone Ms x hVSIG4 528903.111 Mouse IgG2A WB, FC 7.7 nM Y clone Ms x hVSIG4 528905.11 Mouse IgG2B WB, FC ND N clone Ms x hVSIG4 528906.11 Mouse IgG2A FC 4.6 nM N clone Ms x hVSIG4 528908.11 Mouse IgG2A WB, FC 6.3 nM Y clone Ms x hVSIG4 528910.111 Mouse IgG3 WB, FC ND N clone Ms x hVSIG4 528912.11 Mouse IgG2A FC 60.6 nM Y clone Ms x hVSIG4 528922.111 Mouse IgG2A WB (negative), FC ND N clone Ms x hVSIG4 528927.111 Mouse IgG2B WB, FC ND N clone Rt x hVSIG4 489509.11 Rat IgG2A FC 1 nM Y clone Rt x hVSIG4 489517.111 Rat IgG2A FC 2.1 nM Y clone Rt x hVSIG4 489518.11 Rat IgG2A FC 0.3 nM Y

[0413] ND (not determined, no binding observed in SPR assay); FC (flow cytometry), WB (Western blot).

[0414] Example 3: Characterization of VSIG4-Siglec7 blocking antibodies

[0415] Generation of polarized M2c cells. PBMCs were isolated using a SEPMATE / Ficoll gradient separation protocol according to standard protocols. CD14 was then purified using a positive selection kit for CD14 (Cat. No. MAGH105, R&D Systems, Minneapolis, MN). +One million CD14-positive cells / mL (total volume 4 mL) were plated for 4 days in complete medium (RPMI medium supplemented with 10% fetal bovine serum (ThermoFisher Scientific, Waltham, MA), 1.0 mM sodium pyruvate (Invitrogen, Carlsbad, CA), 50 mM 2-mercaptoethanol (Invitrogen), 1x Glutamax (Life Technologies, Grand Island, NY), 1x penicillin / streptomycin (Life Technologies, Grand Island, NY), and 1x nonessential amino acids (Life Technologies, Grand Island, NY)) supplemented with 50 ng / mL hM-CSF (Cat. No. 216-MC R&D Systems, Minneapolis, MN). On day 5, cells were transferred to fresh medium containing either 30 ng / mL dexamethasone (Catalog #1126, Tocris Biosciences, Minneapolis, MN) or 20 ng / mL rhIL-10 (Catalog #217-IL, R&D Systems, Minneapolis, MN) for an additional 3 days to allow polarization of cells to M2c macrophages.

[0416] Flow cytometry detection of VSIG-4 expression in polarized M2c cells. On day 7, dexamethasone-polarized M2c cells were harvested, washed twice with 1x PBS, and incubated with 1 μL Zombie Violet (BioLegend, San Diego, CA) per 100 μL cells at room temperature for 30 minutes to allow the exclusion of dead cells. The cells were then washed twice with RDFII staining buffer and blocked with human IgG and goat anti-human IgG (Catalog No. 1597, R&D Systems, Minneapolis, MN) at 4°C for 10 minutes. The cells were then surface stained for 30 minutes at 4°C with mouse anti-human VSIG4 Alexa Fluor 647, anti-CD14 FITC, and anti-Mer PE (Catalog Nos. FAB982F, FAB8912P, R&D Systems, Minneapolis, MN). The cells were washed one final time with RDFII staining buffer and analyzed on a LSRII Fortessa flow cytometer (BD Biosciences, San Jose, CA). The results are shown in FIG6 .

[0417] Detection of Siglec-7 / VSIG-4 interaction in primary M2c cells polarized with 20 ng / mL IL-10. Polarized M2c cells were incubated with varying amounts of hSiglec-7 Fc protein (1 μg / mL to 10 μg / mL) for 30 min at room temperature. After this incubation, anti-Fc PE detection antibody (FAB110P, R&D Systems, Minneapolis, MN) was added for an additional 20 min. After incubation, cells were washed with RDFII staining buffer and co-stained with anti-VSIG4 Alexa Fluor 647 and anti-CD14 FITC antibodies for 30 min. After incubation with these co-stains, cells were washed again with RDFII staining buffer and analyzed on an LSRII Fortessa flow cytometer (BD Biosciences, San Jose, CA). Results are shown in Figure 7 middle.

[0418] Antibody pairing and co-inhibition studies for antigen recognition. Antibody pairs were identified by ELISA using a biotinylated antibody detection method and streptavidin-HRP as detection. The results are shown in Table 2. Based on the ability to identify capture / detection antibody pairs against VSIG4, these clones recognize different epitopes in the protein.

[0419] Table 2: Antibody pairs targeting VSIG-4

[0420] <![CDATA[ Capture antibody clone ]]> <![CDATA[ Detection antibody clone ]]> Rt x hVSIG4 489509.11 Ms x hVSIG4 528903.111 Ms x hVSIG4 528906.11 Ms x hVSIG4 528908.11 Ms x hVSIG4 528912.11 Rt x hVSIG4 489517.111 Rt x hVSIG4 489518.11 Rt x hVSIG4 489517.111 Rt x hVSIG4 489509.11 Rt x hVSIG4 489518.11 Rt x hVSIG4 489509.11 Ms x hVSIG4 528903.111 Rt x hVSIG4 489509.11 Ms x hVSIG4 528906.11 Rt x hVSIG4 489509.11 Ms x hVSIG4 528908.11 Rt x hVSIG4 489509.11

[0421] Example 4: Glycosylation of VSIG4 and characterization of anti-VSIG4 antibodies binding to glycosylated VSIG4

[0422] To determine whether Siglec7 binds to glycosylated VSIG4 via post-translationally modified glycan moieties, rhVSIG4 was expressed in E. coli lacking the glycosyltransferase machinery and producing recombinant protein without any glycan modifications. Despite retaining its known function of binding to complement component iC3b, VSIG4 produced in E. coli lost the ability to bind to Siglec7, suggesting that Siglec7 binds to the glycosylated form of VSIG4.

[0423] However, enzymatic deglycosylation of VSIG4 using a standard deglycosylase kit containing a typical sialidase that cleaves α(2,3)- and α(2,6)-linked N-acetylneuraminic acid (NANA or sialic acid) showed that the binding between VSIG4 and Siglec7 was preserved. By incorporating a specific sialidase that cleaves the relatively rare α(2,8)-linked NANA in addition to α(2,3)- and α(2,6)-linked NANA, a ~10 kD shift in the molecular weight of rhVSIG4 was observed, from ~42 kD to the predicted molecular weight of ~32 kD. In addition, treatment with an extended deglycosylase kit abolished the binding interaction between VSIG4 and Siglec7.

[0424] These results suggest that VSIG4 is likely heavily modified by polysialic acid chains via α(2,8)-linked N-acetylneuraminic acid, a relatively rare polysaccharide bond. The most well-known protein containing α(2,8)-linked polysialic acid is CD56 / NCAM (Schnaar et al., Physiological Reviews 2014, 94(2): 461-518). The interaction between Siglec7 and CD56 / NCAM was confirmed using Biacore, and the interaction affinity between Siglec7-VSIG4 was roughly equivalent to the interaction affinity between Siglec7-CD56 NCAM.

[0425] To confirm that antibodies against VSIG4 derived using mammalian derived and fully glycosylated rhVSIG4 bind to the VSIG4 polypeptide and not to glycan modifications on VSIG4, antibody binding to fully glycosylated rhVSIG4, deglycosylated VSIG4, E. coli derived VSIG4 and CD56 / NCAM was tested. Strong binding of the blocking antibody to all VSIG4 species was observed, regardless of glycosylation status, but not to CD56 / NCAM.

[0426] Taken together, these results indicate that VSIG4 is heavily modified with a rare glycan bond (α(2,8)-linked polysialic acid) and that this modification mediates the interaction between VSIG4 and Siglec7. Furthermore, these results indicate that the blocking antibody specifically binds to the VSIG4 polypeptide but not to the glycan outer layer of VSIG4.

[0427] Purified recombinant human VSIG4 ectodomain (rhVSIG4) expressed in NS0 cells was deglycosylated using an enzyme mixture containing recombinant Cp neuraminidase (R&D Systems catalog number 5080-NM), recombinant Fm PNGase F (R&D Systems catalog number 9109-GH), recombinant Ef O-glycosidase (R&D Systems catalog number 8886-GH), and recombinant Sp B4 galactosidase (R&D Systems catalog number 5549-GH), supplemented with recombinant Mv neuraminidase (R&D Systems catalog number 5084-NM). Fully glycosylated and deglycosylated purified rhVSIG4 were separated by SDS-PAGE and visualized by silver staining. The results are shown in Fig. 9A In the absence of post-translational modifications, the predicted molecular weight of the rhVSIG4 extracellular domain is 32 kD, and the observed molecular weight of the fully glycosylated rhVSIG4 ECD is ~41 kD. Therefore, it is believed that rhVSIG4 derived from the NS0 mammalian cell line is highly glycosylated and the deglycosylation enzyme mixture removes almost all sugar molecules (where Mv neuraminidase cleaves 2-8 linked sialic acids).

[0428] Recombinant human Siglec7-Fc (rhSiglec7-Fc) was captured on a protein A / G / L modified Biacore CM5 chip at a capture density of ~700 RU and tested for binding to fully glycosylated rhVSIG4, deglycosylated VSIG4, and CD56 / NCAM at concentrations ranging from 0.5 nM to 500 nM. FIG. 9B to FIG. 9D As shown in , Siglec7 interacts with rhVSIG4, but not with deglycosylated VSIG4. In addition, Siglec7 does not interact with VSIG4 expressed in E. coli. CD56 / NCAM is heavily glycosylated, has a similar sugar profile to VSIG4, and the interaction between Siglec7 and CD56 / NCAM further suggests that the interaction between Siglec7 and VSIG4 is mediated by a unique glycosylation profile.

[0429] Anti-VSIG-4 antibodies that bind to VSIG4 polypeptides regardless of glycosylation status were tested using Biacore. Blocking antibodies (Rt x hVSIG4 489517.111, Ms x hVSIG4 528906.11,) were captured on a protein A / G / L chip at ~350 RU and tested for binding to rhVSIG4, mammalian NS0 cell-derived rhVSIG4, mammalian-derived and completely deglycosylated VSIG4, E. coli-derived and not glycosylated, and highly glycosylated protein CD56 / NCAM. All analyte proteins, rhVSIG4 and CD56 / NCAM were tested at concentrations ranging from 25 pM to 250 nM. Binding to VSIG4 regardless of the glycosylation state of VSIG4 and not binding to CD56 / NCAM, a heavily glycosylated molecule with a similar carbohydrate profile to VSIG4, demonstrated the specificity of the antibody to the amino acid sequence of VSIG4 and indicated that the antibody is not anti-glycosylation specific. The results are shown in Figures 10 to 13.

[0430] Example 5: Further characterization of VSIG4-Siglec7 interaction and VSIG4-Siglec7 blocking antibodies

[0431] HEK293 cells were transfected with recombinant human VSIG4-eGFP or recombinant human VSIG3-eGFP. Then, the cells were incubated with the indicated doses of no protein (negative control) or recombinant human Siglec-7-Fc. Then, the cells were incubated with anti-Fc allophycocyanin (APC). Fig.14 As shown in , recombinant human Siglec-7 specifically binds to HEK293 cells transfected with recombinant human VSIG4 fused to eGFP in a dose-dependent manner, but recombinant human Siglec-7 does not bind to HEK293 cell types transfected with recombinant human VSIG3-eGFP.

[0432] Adherent M2c macrophages were polarized with dexamethasone as described in Example 3. M2c macrophages were incubated with 10 µg / mL, 40 µg / mL, or 200 µg / mL of anti-human VSIG-4 antibodies to test the ability of the antibodies to block interaction with rhSiglec-7. rhSiglec-7 was tested at 100 µg / mL, 25 µg / mL, or 5 µg / mL. Ms x hVSIG4528906.11 blocked the interaction of VSIG-4 naturally expressed on M2c macrophages with recombinant human Siglec-7 / Fc in a dose-dependent manner, as shown in Figures 15 to 18 as shown in .

[0433] Example 6: Direct ELISA test for anti-VSIG-4 antibodies

[0434] Plates were coated with a 4-fold serial dilution, ranging from 400 ng / ml to 1.56 ng / ml of a serial titration of recombinant human VSIG4 protein. 1 mg / ml of anti-VSIG-4 antibody was added and incubated for 1 hour, followed by the addition of goat anti-mouse or goat anti-rat HRP-conjugated secondary antibodies (to match the primary clone) at a dilution of 1:10,000. HRP activity was detected by the addition of TMB and stop solution after 10 minutes. Results were evaluated by absorbance at 540 nm. Representative data are shown in Figure 19 and summarized in Table 3.

[0435] Table 3: Representative ELISA results

[0436] <![CDATA[ Clone number ]]> <![CDATA[ VSIG4 from NSO ]]> <![CDATA[ VSIG from Escherichia coli ]]> Ms x hVSIG4 528902.11 Negative weak Ms x hVSIG4 528903.111 Positive Positive Ms x hVSIG4 528905.11 Positive Negative Ms x hVSIG4 528906.11 Positive Positive Ms x hVSIG4 528908.11 Positive Positive Ms x hVSIG4 528910.111 Positive Negative Ms x hVSIG4 528912.11 Positive Positive Ms x hVSIG4 528922.111 Negative Positive Ms x hVSIG4 528927.111 Positive Positive Rt x hVSIG4 489509.11 Positive Positive Rt x hVSIG4 489517.111 Positive Positive Rt x hVSIG4 489518.11 Positive Positive

[0437] As shown in Table 3, most of the antibodies tested detected both glycosylated human VSIG4 expressed in NSO as well as unglycosylated recombinant human VSIG4 protein expressed in E. coli, indicating that the antibodies detect the amino acid components of the protein and not secondary modifications.

[0438] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence submissions in GenBank and RefSeq, and amino acid sequence submissions in, for example, SwissProt, PIR, PRF, PDB, and translations of annotated coding regions from GenBank and RefSeq) are incorporated by reference. In the event of any inconsistency between the disclosure of the present application and the disclosure of any document incorporated herein, the disclosure of the present application shall prevail. The foregoing detailed description and examples are given only for clarity of understanding. No unnecessary limitations are to be understood therefrom. The present invention is not limited to the exact details shown and described, as variations obvious to those skilled in the art will be included in the invention defined by the claims.

Claims

1. A monoclonal antibody that binds to VSIG-4 and eliminates the binding of VSIG-4 to SIGLEC-7, wherein the monoclonal antibody is produced by at least one of the following clones: ATCC accession number PTA-124188; ATCC accession number PTA-124178; ATCC accession number PTA-124179; ATCC accession number PTA-124181; ATCC Accession No. PTA-124185; and ATCC Accession No. PTA-124186.

2. A monoclonal antibody, wherein the monoclonal antibody binds to VSIG-4 and eliminates the binding of VSIG-4 to SIGLEC-7, and wherein the monoclonal antibody comprises: A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs of the heavy chain of the monoclonal antibody produced by ATCC Accession No. PTA-124188, and the light chain variable region comprises the three CDRs of the light chain of the monoclonal antibody produced by ATCC Accession No. PTA-124188; A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs of the heavy chain of the monoclonal antibody produced by ATCC Accession No. PTA-124178, and the light chain variable region comprises the three CDRs of the light chain of the monoclonal antibody produced by ATCC Accession No. PTA-124178; A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs of the heavy chain of the monoclonal antibody produced by ATCC Accession No. PTA-124179, and the light chain variable region comprises the three CDRs of the light chain of the monoclonal antibody produced by ATCC Accession No. PTA-124179; A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs of the heavy chain of the monoclonal antibody produced by ATCC Accession No. PTA-124181, and the light chain variable region comprises the three CDRs of the light chain of the monoclonal antibody produced by ATCC Accession No. PTA-124181; A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs of the heavy chain of the monoclonal antibody produced by ATCC Accession No. PTA-124185, and the light chain variable region comprises the three CDRs of the light chain of the monoclonal antibody produced by ATCC Accession No. PTA-124185; or A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs of the heavy chain of the monoclonal antibody produced by ATCC Accession No. PTA-124186, and the light chain variable region comprises the three CDRs of the light chain of the monoclonal antibody produced by ATCC Accession No. PTA-124186.

3. The monoclonal antibody of claim 1 or 2, wherein at least one of VSIG-4 and SIGLEC-7 is on the surface of a cell.

4. The monoclonal antibody of claim 3, wherein VSIG-4 is on the surface of M2c macrophages.

5. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody abrogates cell-cell interactions.

6. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody binds to the extracellular domain of VSIG-4.

7. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody binds to a VSIG-4 polypeptide.

8. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody binds to glycosylated and unglycosylated VSIG-4.

9. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody is directly or indirectly coupled to a detectable label.

10. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody comprises an IgG antibody.

11. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody comprises an antigen-binding fragment, and the antigen-binding fragment comprises at least one of a Fab fragment, a Fab' fragment, a F(ab)2 fragment and a Fv fragment.

12. A composition comprising the monoclonal antibody of any one of claims 1-11.

13. A kit comprising the monoclonal antibody according to any one of claims 1 to 11.

14. The monoclonal antibody of any one of claims 1-11 for use in a method of treating cancer in a mammal.

15. The monoclonal antibody of any one of claims 1-11 for use in a method of detecting cancer in a mammal.

16. The monoclonal antibody of any one of claims 1 to 11 for use in a method of treating an autoimmune disease.

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