Immunomodulatory antibodies and methods of use thereof

Abstract

SOLUTION: Provided herein are antibodies and methods of use thereof. The antibodies disclosed herein bind to CD163 + on cells such as macrophages. These antibodies can be used in methods of treatment, such as methods of treating cancer.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 876,580, filed July 19, 2019, U.S. Provisional Application No. 62 / 876,579, filed July 19, 2019, and U.S. Provisional Application No. 62 / 878,265, filed July 24, 2019, the entire contents of each of which are incorporated herein by reference. Summary of the Invention

[0002] Provided herein are antibodies useful for treating cancer and other disorders, including antigen-binding fragments and other antigen-binding polypeptides. In some embodiments, the antibodies specifically bind to M2 and M2-like immunosuppressive macrophages but not to M1 and M1-like antitumor macrophages. The disclosed antibodies bind to M2 and M2-like tumor-associated macrophages and modulate the physical and functional characteristics of tumor-associated M2 and M2-like macrophages, alleviating immunosuppression in the tumor microenvironment and increasing the activation and proliferation of cytotoxic T cells, thereby promoting tumor cell killing. The antibody molecules of the present disclosure specifically bind to human CD163, which is expressed on the surface of M2 and M2-like macrophages.

[0003] In certain embodiments, disclosed herein is an antibody or recombinant antibody comprising a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO:8.

[0004] In certain embodiments, disclosed herein is an antibody or recombinant antibody comprising a heavy chain variable region (VH) having at least 90% identity to the amino acid sequence of SEQ ID NO:8.

[0005] In certain embodiments, disclosed herein is an antibody or recombinant antibody comprising a heavy chain variable region (VH) having at least 95% identity to the amino acid sequence of SEQ ID NO:8.

[0006] In certain embodiments, disclosed herein is an antibody or recombinant antibody comprising a heavy chain variable region (VH) having at least 99% identity to the amino acid sequence of SEQ ID NO:8.

[0007] Disclosed herein in certain embodiments is an antibody or recombinant antibody comprising a heavy chain variable region (VH) having at least 100% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the antibody or recombinant antibody further comprises a light chain variable region (VL) having at least 80% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody or recombinant antibody further comprises a light chain variable region (VL) having at least 90% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody or recombinant antibody further comprises a light chain variable region (VL) having at least 95% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody or recombinant antibody further comprises a light chain variable region (VL) having at least 99% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody or recombinant antibody further comprises a light chain variable region (VL) having at least 100% identity to the amino acid sequence of SEQ ID NO:7.

[0008] In certain embodiments, disclosed herein is a light chain variable region (VL) having at least 80% identity to the amino acid sequence of SEQ ID NO:7.

[0009] In certain embodiments, disclosed herein is an antibody or recombinant antibody comprising a light chain variable region (VL) having at least 90% identity to the amino acid sequence of SEQ ID NO:7.

[0010] In certain embodiments, disclosed herein is an antibody or recombinant antibody comprising a light chain variable region (VL) having at least 95% identity to the amino acid sequence of SEQ ID NO:7.

[0011] In certain embodiments, disclosed herein is an antibody or recombinant antibody comprising a light chain variable region (VL) having at least 99% identity to the amino acid sequence of SEQ ID NO:7.

[0012] Disclosed herein in certain embodiments is an antibody or recombinant antibody comprising a light chain variable region (VL) having at least 100% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody or recombinant antibody further comprises a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the antibody or recombinant antibody further comprises a heavy chain variable region (VH) having at least 90% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the antibody or recombinant antibody further comprises a heavy chain variable region (VH) having at least 95% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the antibody or recombinant antibody further comprises a heavy chain variable region (VH) having at least 99% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the antibody or recombinant antibody further comprises a heavy chain variable region (VH) having at least 100% identity to the amino acid sequence of SEQ ID NO:8.

[0013] In certain embodiments herein, a heavy chain variable region (V) having at least 80% identity to the amino acid sequence of SEQ ID NO:8 is provided. H ) and a light chain variable region (V) having at least 80% identity to the amino acid sequence of SEQ ID NO: 7. L ) and a light chain variable region (V) having at least 85% identity to the amino acid sequence of SEQ ID NO:7. L In some embodiments, the antibody or recombinant antibody comprises a light chain variable region (V) having at least 90% identity to the amino acid sequence of SEQ ID NO:7. L In some embodiments, the antibody or recombinant antibody comprises a light chain variable region (V) having at least 95% identity to the amino acid sequence of SEQ ID NO:7. LIn some embodiments, the antibody or recombinant antibody comprises a light chain variable region (V) having at least 99% identity to the amino acid sequence of SEQ ID NO:7. L In some embodiments, the antibody or recombinant antibody comprises a light chain variable region (V) having at least 99% identity to the amino acid sequence of SEQ ID NO:7. L In some embodiments, the antibody or recombinant antibody comprises a light chain variable region (V) having at least 85% identity to the amino acid sequence of SEQ ID NO:8. H In some embodiments, the antibody or recombinant antibody comprises a heavy chain variable region (V) having at least 90% identity to the amino acid sequence of SEQ ID NO:8. H In some embodiments, the antibody or recombinant antibody comprises a light chain variable region (V) having at least 95% identity to the amino acid sequence of SEQ ID NO:8. H In some embodiments, the antibody or recombinant antibody comprises a heavy chain variable region (V) having at least 99% identity to the amino acid sequence of SEQ ID NO:8. H In some embodiments, the antibody or recombinant antibody comprises a light chain variable region (V) having at least 100% identity to the amino acid sequence of SEQ ID NO:8. H ) is included.

[0014] In certain embodiments, disclosed herein is an antibody or recombinant antibody comprising a heavy chain sequence comprising a complementarity determining region (CDR) H1 having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 80% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 80% identity to the amino acid sequence of SEQ ID NO: 6, and a light chain sequence comprising a CDR L1 having at least 80% identity to the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 80% identity to the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 80% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody or recombinant antibody comprises a light chain sequence comprising a CDR L1 having at least 85% identity to the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 85% identity to the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 85% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody or recombinant antibody comprises a light chain sequence comprising a CDR L1 having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 90% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody or recombinant antibody comprises a light chain sequence comprising a CDR L1 having at least 95% identity to the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 95% identity to the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 95% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody or recombinant antibody comprises a light chain sequence comprising a CDR L1 having at least 99% identity to the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 99% identity to the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 99% identity to the amino acid sequence of SEQ ID NO: 3.In some embodiments, the antibody or recombinant antibody comprises a light chain sequence comprising CDR L1 having at least 100% identity to the amino acid sequence of SEQ ID NO: 1, CDR L2 having at least 100% identity to the amino acid sequence of SEQ ID NO: 2, and CDR L3 having at least 100% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody or recombinant antibody comprises a heavy chain sequence comprising CDR H1 having at least 85% identity to the amino acid sequence of SEQ ID NO: 4, CDR H2 having at least 85% identity to the amino acid sequence of SEQ ID NO: 5, and CDR H3 having at least 85% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or recombinant antibody comprises a heavy chain sequence comprising CDR H1 having at least 90% identity to the amino acid sequence of SEQ ID NO: 4, CDR H2 having at least 90% identity to the amino acid sequence of SEQ ID NO: 5, and CDR H3 having at least 90% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or recombinant antibody comprises a heavy chain sequence comprising a CDR H1 having at least 95% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 95% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 95% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or recombinant antibody comprises a heavy chain sequence comprising a CDR H1 having at least 99% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 99% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 99% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or recombinant antibody comprises a heavy chain sequence comprising a CDR H1 having at least 100% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 100% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 100% identity to the amino acid sequence of SEQ ID NO: 6.

[0015] In certain embodiments, disclosed herein is an antibody or recombinant antibody comprising a heavy chain sequence comprising at least one complementarity determining region (CDR) H1 having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 80% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 80% identity to the amino acid sequence of SEQ ID NO: 6.

[0016] In certain embodiments herein, an antibody or recombinant antibody is disclosed comprising a heavy chain sequence comprising at least one complementarity determining region (CDR) H1 having at least 90% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 90% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 90% identity to the amino acid sequence of SEQ ID NO: 6.

[0017] In certain embodiments herein, an antibody or recombinant antibody is disclosed comprising a heavy chain sequence comprising at least one complementarity determining region (CDR) H1 having at least 95% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 95% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 95% identity to the amino acid sequence of SEQ ID NO: 6.

[0018] In certain embodiments herein, an antibody or recombinant antibody is disclosed comprising a heavy chain sequence comprising at least one complementarity determining region (CDR) H1 having at least 99% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 99% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 99% identity to the amino acid sequence of SEQ ID NO: 6.

[0019] In certain embodiments herein, an antibody or recombinant antibody is disclosed comprising a heavy chain sequence comprising at least one complementarity determining region (CDR) H1 having at least 100% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 100% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 100% identity to the amino acid sequence of SEQ ID NO: 6.

[0020] In some embodiments, the antibody or recombinant antibody further comprises a light chain sequence comprising at least one complementarity determining region (CDR) L1 having at least 80% identity to the amino acid sequence of SEQ ID NO: 1, CDR L2 having at least 80% identity to the amino acid sequence of SEQ ID NO: 2, and CDR L3 having at least 80% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, CDR L1 has at least 90% identity to the amino acid sequence of SEQ ID NO: 1, CDR L2 has at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and CDR L3 has at least 90% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, CDR L1 has at least 95% identity to the amino acid sequence of SEQ ID NO: 1, CDR L2 has at least 95% identity to the amino acid sequence of SEQ ID NO: 2, and CDR L3 has at least 95% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, CDR L1 has at least 99% identity to the amino acid sequence of SEQ ID NO: 1, CDR L2 has at least 99% identity to the amino acid sequence of SEQ ID NO: 2, and CDR L3 has at least 99% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, CDR L1 has at least 100% identity to the amino acid sequence of SEQ ID NO: 1, CDR L2 has at least 100% identity to the amino acid sequence of SEQ ID NO: 2, and CDR L3 has at least 100% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the light chain variable region (VL) has at least 80% identity to the amino acid sequence of SEQ ID NO: 7.

[0021] In some embodiments, the antibody or recombinant antibody further comprises a light chain variable region (VL) having at least 80% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or recombinant antibody comprises a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 8.

[0022] In certain embodiments herein, an antibody or recombinant antibody is disclosed comprising a light chain sequence comprising a complementarity determining region (CDR) L1 having at least 80% identity to the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 80% identity to the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 80% identity to the amino acid sequence of SEQ ID NO: 3.

[0023] In certain embodiments herein, an antibody or recombinant antibody is disclosed comprising a light chain sequence comprising a complementarity determining region (CDR) L1 having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 90% identity to the amino acid sequence of SEQ ID NO: 3.

[0024] In certain embodiments herein, an antibody or recombinant antibody is disclosed comprising a light chain sequence comprising a complementarity determining region (CDR) L1 having at least 95% identity to the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 95% identity to the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 95% identity to the amino acid sequence of SEQ ID NO: 3.

[0025] In certain embodiments herein, an antibody or recombinant antibody is disclosed comprising a light chain sequence comprising a complementarity determining region (CDR) L1 having at least 99% identity to the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 99% identity to the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 99% identity to the amino acid sequence of SEQ ID NO: 3.

[0026] In certain embodiments herein, an antibody or recombinant antibody is disclosed comprising a light chain sequence comprising a complementarity determining region (CDR) L1 having at least 100% identity to the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 100% identity to the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 100% identity to the amino acid sequence of SEQ ID NO: 3.

[0027] In some embodiments, the antibody or recombinant antibody further comprises a heavy chain sequence comprising at least one complementarity determining region (CDR) H1 having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, CDR H2 having at least 80% identity to the amino acid sequence of SEQ ID NO: 5, and CDR H3 having at least 80% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, CDR H1 has at least 90% identity to the amino acid sequence of SEQ ID NO: 4, CDR H2 has at least 90% identity to the amino acid sequence of SEQ ID NO: 5, and CDR H3 has at least 90% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, CDR H1 has at least 95% identity to the amino acid sequence of SEQ ID NO: 4, CDR H2 has at least 95% identity to the amino acid sequence of SEQ ID NO: 5, and CDR H3 has at least 95% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, CDR H1 has at least 99% identity to the amino acid sequence of SEQ ID NO: 4, CDR H2 has at least 99% identity to the amino acid sequence of SEQ ID NO: 5, and CDR H3 has at least 99% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, CDR H1 has at least 100% identity to the amino acid sequence of SEQ ID NO: 4, CDR H2 has at least 100% identity to the amino acid sequence of SEQ ID NO: 5, and CDR H3 has at least 100% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy chain variable region (VH) has at least 80% identity to the amino acid sequence of SEQ ID NO: 8.

[0028] In some embodiments, the antibody or recombinant antibody further comprises a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the light chain variable region (VL) has at least 80% identity to the amino acid sequence of SEQ ID NO: 7.

[0029] In some embodiments, the antibody or recombinant antibody further comprises a human heavy chain constant region or a human light chain constant region. In some embodiments, the human heavy chain constant region is IgG1 or IgG4, or a fragment thereof. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the light chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody or recombinant antibody comprises a human variable framework region and a mouse constant region. In some embodiments, the antibody or recombinant antibody further comprises a mouse heavy chain constant region or a mouse light chain constant region. In some embodiments, the antibody or mouse heavy chain constant region is IgG2A. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the light chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody or recombinant antibody is an antibody fragment comprising a single heavy chain, a single light chain, a Fab, Fab', F(ab)', F(ab')2, Fd, scFv, a variable heavy domain, a variable light domain, a variable NAR domain, a bispecific scFv, a bispecific Fab2, a trispecific Fab3, a single-chain binding polypeptide, a dAb fragment, or a diabody.

[0030] In some embodiments, the antibody or recombinant antibody specifically binds to CD163 protein expressed on immunosuppressive human myeloid cells, and binding of the antibody or recombinant antibody to the myeloid cells is determined by (i) CD4 + T cells, CD8 + (ii) activation of T cells, NK cells, or any combination thereof; and (iii) activation of CD4 + T cells, CD8 + Promotes immune cell function as measured by one or both of the proliferation of T cells, NK cells, or any combination thereof. In some embodiments, CD4 + T cells, CD8 +Activation of T cells, NK cells, or any combination thereof is measured as an increase in the levels of IFN-γ, TNF-α, or perforin, or any combination thereof. In some embodiments, the immunosuppressive human myeloid cells are macrophages or myeloid-derived suppressor cells. In some embodiments, the immune cell function is in a tumor microenvironment. In some embodiments, the immune cell function is in vivo. In some embodiments, the antibody or recombinant antibody specifically binds to CD163 protein expressed on human macrophages, and binding of the antibody to the macrophages increases immunostimulatory activity in the tumor microenvironment. In some embodiments, the tumor microenvironment is in vivo. In some embodiments, binding of the antibody or recombinant antibody to macrophages reduces immunosuppressive activity of the macrophages. In some embodiments, binding of the antibody or recombinant antibody to macrophages reduces tumor promoting activity of macrophages. In some embodiments, the macrophages are tumor-associated macrophages. In some embodiments, the antibody or recombinant antibody modifies expression of at least one marker on macrophages. In some embodiments, the CD163 protein is a glycoform of CD163. In some embodiments, the CD163 protein is a 150 kDa glycoform of CD163. In some embodiments, the antibody does not specifically bind to the 130 kDa glycoform of CD163 expressed by human macrophages.

[0031] In some embodiments, the antibody or recombinant antibody has a constant domain that enables binding to an Fc receptor. In some embodiments, the Fc receptor is expressed on macrophages. In some embodiments, the antibody or recombinant antibody has an antibody fragment comprising a single heavy chain, a single light chain, a Fab, a Fab', a F(ab)', a F(ab')2, a Fd, an scFv, a variable heavy domain, a variable light domain, a variable NAR domain, a bispecific scFv, a bispecific Fab2, a trispecific Fab3, a single-chain binding polypeptide, a dAb fragment, or a diabody. In some embodiments, the at least one marker on human macrophages is CD16, CD64, TLR2, or Siglec-15. In some embodiments, the human macrophages are M2 macrophages or M2-like macrophages. In some embodiments, the human macrophages are M2a, M2b, M2c, or M2d macrophages. In some embodiments, the CD163 protein is a component of a cell surface complex comprising at least one other protein expressed by macrophages. In some embodiments, the at least one other protein is a galectin-1 protein, a LILRB2 protein, a casein kinase II protein, or any combination thereof. In some embodiments, upon binding to the CD163 protein, the antibody or antibodies are internalized by human macrophages. In some embodiments, binding to the CD163 protein is not cytotoxic to macrophages. In some embodiments, binding to the CD163 protein is not cytotoxic to CD4 + T cell activation, CD4 + T-cell proliferation, or CD4 + In some embodiments, binding to the CD163 protein promotes both activation and proliferation of T cells. + In some embodiments, binding to the CD163 protein promotes the expression of CD8, CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by T cells. + T cell activation, CD8 + T-cell proliferation, or CD8 +In some embodiments, binding to the CD163 protein promotes both activation and proliferation of CD8 T cells. + In some embodiments, binding to the CD163 protein promotes expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by T cells. In some embodiments, binding to the CD163 protein reduces immunosuppression in the tumor microenvironment. In some embodiments, binding to the CD163 protein promotes tumor cell killing in the tumor microenvironment. In some embodiments, binding to the CD163 protein promotes cytotoxic lymphocyte-mediated cancer cell killing. In some embodiments, binding to the CD163 protein promotes NK cell-mediated tumor cell killing. In some embodiments, binding to the CD163 protein promotes expression of IL-2 by T cells. In some embodiments, binding to the CD163 protein promotes CD4 + T cells, CD196 - T cells, CXCR3 + T cells, CCR4 - T cells, or any combination thereof.

[0032] In some embodiments, binding to CD163 reduces immunosuppression in the tumor microenvironment caused by macrophages. In some embodiments, the antibody or recombinant antibody specifically binds to the CD163 protein expressed on human macrophages, and the binding results in the following effects: (a) reduction in expression of at least one marker by macrophages that is CD16, CD64, TLR2, or Siglec-15; (b) internalization of the antibody by macrophages; (c) reduction in CD4 + T cells, CD8 + (d) activation of T cells, NK cells, or any combination thereof; + T cells, CD8 +and (e) promoting tumor cell death in the tumor microenvironment. In some embodiments, the binding results in two or more of (a)-(e), three or more of (a)-(e), four or more of (a)-(e), or all of (a)-(e). In some embodiments, the antibody or recombinant antibody inhibits CD163 in the tumor microenvironment. + The antibody or recombinant antibody specifically binds to immunosuppressive myeloid cells, reducing the inhibition of cytotoxic T cell-mediated killing of tumor cells in the tumor microenvironment. In some embodiments, the antibody or recombinant antibody specifically binds to the CD163 protein expressed on human tumor-associated macrophages and reduces the expression of CD16, CD64, TLR2, Siglec-15, or any combination thereof, by the macrophages for use in cancer treatment methods. In some embodiments, the binding of the antibody or recombinant antibody to macrophages regulates the immune function of cells in the tumor microenvironment. In some embodiments, the binding of the antibody or recombinant antibody to macrophages promotes anti-tumor immune function.

[0033] In some embodiments, the antibody or recombinant antibody specifically binds to a CD163 epitope comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody or recombinant antibody specifically binds to a CD163 epitope comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody or recombinant antibody specifically binds to a CD163 epitope comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or recombinant antibody specifically binds to a CD163 epitope comprising SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively. In some embodiments, the antibody or recombinant antibody has a K D In some embodiments, the antibody or recombinant antibody specifically binds to CD163 with a K D In some embodiments, the antibody or recombinant antibody specifically binds to CD163 with a K DIn some embodiments, the antibody or CD163 specifically binds to CD163 with a K of 1 nM to 100 nM. In some embodiments, the antibody or recombinant antibody specifically binds to CD163 with a K of 1 nM to 100 nM. D In some embodiments, the antibody or recombinant antibody specifically binds to M2c macrophages with a K D In some embodiments, the antibody or recombinant antibody specifically binds to M2c macrophages with a K D In some embodiments, the M2c macrophage specifically binds to an M2c macrophage. In some embodiments, the M2c macrophage is a human M2c macrophage.

[0034] In certain embodiments, disclosed herein are compositions comprising an antibody or recombinant antibody and an excipient.

[0035] In certain embodiments, disclosed herein is a pharmaceutical composition comprising an antibody or recombinant antibody according to any one of the preceding embodiments and a pharmaceutically acceptable carrier.

[0036] In certain embodiments disclosed herein is the use of an antibody or recombinant antibody according to any one of the preceding embodiments in the manufacture of a medicament for treating cancer in a human subject.

[0037] In certain embodiments herein, there is disclosed the use of an antibody or recombinant antibody according to any one of the preceding embodiments in the manufacture of a medicament for reducing immunosuppression by tumor-associated macrophages in a human subject with cancer.

[0038] In certain embodiments herein, there is disclosed the use of an antibody or recombinant antibody according to any one of the preceding embodiments in the manufacture of a medicament for promoting T cell-mediated tumor cell killing in a human subject with cancer.

[0039] In certain embodiments, a method for promoting immune cell function is disclosed herein, comprising administering to an individual in need thereof an antibody or recombinant antibody according to any one of the preceding embodiments, and measuring the following parameters: (i) CD4 + T cells, CD8 + (ii) activation of T cells, NK cells, or any combination thereof; and (iii) activation of CD4 + T cells, CD8 + and promoting immune cell function as measured by one or both of T cells, NK cells, or any combination thereof. In some embodiments, CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof is measured as an increase in the level of IFN-γ, TNF-α, or perforin, or any combination thereof. In some embodiments, the immunosuppressive human myeloid cells are macrophages. In some embodiments, the immunosuppressive human myeloid cells are myeloid-derived suppressor cells. In some embodiments, the antibody or recombinant antibody is the antibody or recombinant antibody of any one of the preceding embodiments.

[0040] In certain embodiments herein, a method of treating cancer in an individual in need thereof is disclosed, the method comprising administering to the individual a therapeutically effective amount of an antibody or recombinant antibody described in any one of the preceding embodiments, thereby treating the cancer in the individual.

[0041] In certain embodiments, disclosed herein is a method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody or recombinant antibody described in any one of the preceding embodiments, whereby immunosuppression by tumor-associated macrophages in the individual is reduced.

[0042] In certain embodiments herein, a method of treating cancer in an individual in need thereof is disclosed, comprising administering to the individual a therapeutically effective amount of an antibody or recombinant antibody described in any one of the preceding embodiments, thereby increasing T cell-mediated tumor cell killing in the individual.

[0043] In certain embodiments, disclosed herein are methods for reducing tumor-promoting activity of tumor-associated macrophages in an individual in need thereof, the methods comprising: + T cell activation, CD4 + T cell proliferation, CD8 + T cell activation, CD8 + The method comprises administering to an individual an amount of a pharmaceutical composition according to any one of the preceding embodiments effective to modulate T cell proliferation, T cell proliferation, or any combination thereof.

[0044] In certain embodiments herein, a method is disclosed for promoting lymphocyte-mediated tumor cell killing in an individual in need thereof, the method comprising administering to the individual an effective amount of an antibody or recombinant process as described in any one of the preceding embodiments, or a pharmaceutical composition as described in any one of the preceding embodiments.

[0045] In some embodiments, the method further comprises promoting tumor cell death in the tumor microenvironment. In some embodiments, the cancer is lung cancer or sarcoma. In some embodiments, the lung cancer is lung carcinoma or lung adenocarcinoma. In some embodiments, the method further comprises administering to the individual an additional anti-cancer therapeutic or anti-cancer therapy. In some embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, and cytokine therapy, and combinations thereof. In some embodiments, the additional anti-cancer therapy is immunotherapy. In some embodiments, the immunotherapy is a composition comprising a checkpoint inhibitor.

[0046] In certain embodiments herein, a method for modulating the activity of tumor-associated macrophages in a tumor microenvironment is disclosed, the method comprising contacting tumor-associated macrophages with an antibody or recombinant antibody, wherein the antibody or recombinant antibody binds to CD163-expressing human macrophages and has the following effects: (a) binding of the antibody reduces the expression on the macrophages of at least one marker, which is CD16, CD64, TLR2, or Siglec-15; (b) upon binding of the antibody to the CD163 protein, the antibody is internalized by the human macrophages; (c) binding of the antibody is not cytotoxic to macrophages; and (d) binding of the antibody or recombinant antibody reduces the expression on the macrophages of at least one marker, which is CD16, CD64, TLR2, or Siglec-15. + T cells, CD8 + (e) the binding of an antibody or recombinant antibody to CD4 + T cells, CD8 + (f) binding of the antibody or recombinant antibody promotes activation of CD4 + T cells, CD8 + and (g) binding of the antibody or recombinant antibody promotes cell killing in the tumor microenvironment. In some embodiments, the binding results in two or more of (a)-(g), three or more of (a)-(g), four or more of (a)-(g), five or more of (a)-(g), six or more of (a)-(g), or all of (a)-(f).

[0047] In some embodiments, the method is performed in a tumor microenvironment. In some embodiments, the method is performed in vivo. In some embodiments, binding of the antibody or recombinant antibody to macrophages modulates cellular immune function in the tumor microenvironment. In some embodiments, binding of the antibody or recombinant antibody to macrophages promotes anti-tumor immune function. In some embodiments, the antibody or recombinant antibody comprises a constant domain, and the constant domain binds to an Fc receptor. In some embodiments, the Fc receptor is expressed on macrophages. In some embodiments, the method further comprises internalizing the antibody or recombinant antibody by human macrophages upon binding to CD163 protein. In some embodiments, binding to CD163 protein is not cytotoxic to human macrophages. In some embodiments, the method further comprises binding to CD4 + In some embodiments, the method further comprises promoting expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by the T cells. + In some embodiments, the method further comprises promoting expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by T cells. In some embodiments, the method comprises reducing immunosuppression in the tumor microenvironment. In some embodiments, the method comprises promoting cytotoxic lymphocyte-mediated cancer cell killing. In some embodiments, the method comprises promoting NK cell-mediated tumor cell killing. In some embodiments, the method comprises promoting IL-2 expression by T cells. In some embodiments, the method comprises promoting CD4 + T cells, CD196 - T cells, CXCR3 + T cells, CCR4 - T cells, or any combination thereof.

[0048] In certain embodiments, disclosed herein are antibodies that specifically bind to the CD163 protein expressed on immunosuppressive human myeloid cells, and the binding of the antibody to the myeloid cells is determined by: (i) CD4 + T cells, CD8+ (ii) activation of T cells, NK cells, or any combination thereof; and (iii) activation of CD4 + T cells, CD8 + In some embodiments, the immunosuppressive human myeloid cells are macrophages or myeloid-derived suppressor cells. In some embodiments, the immunosuppressive human myeloid cells are CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof is measured as an increase in the production of IFN-γ, TNF-α, perforin, or any combination thereof.

[0049] In certain embodiments, disclosed herein are antibodies that specifically bind to the CD163 protein expressed on human macrophages, and the binding of the antibody to macrophages is determined by: (i) CD4 + T cells, CD8 + (ii) activation of T cells, NK cells, or any combination thereof; and (iii) activation of CD4 + T cells, CD8 + In some embodiments, the immune cell function is enhanced as measured by one or both of proliferation of T cells, NK cells, or any combination thereof. In some embodiments, the immune cell function is in a tumor microenvironment. In some embodiments, the immune cell function is in vivo. In some embodiments, the immune cell function is enhanced by CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof is measured as an increase in the production of IFN-γ, TNF-α, perforin, or any combination thereof.

[0050] In certain embodiments, disclosed herein are antibodies that specifically bind to CD163 protein expressed on human macrophages, wherein binding of the antibody to the macrophages increases immunostimulatory activity in the tumor microenvironment. In some embodiments, the tumor microenvironment is in vivo. In some embodiments, binding of the antibody to the macrophages reduces immunosuppressive activity of the macrophages. In some embodiments, binding of the antibody to the macrophages reduces tumor promoting activity of the macrophages. In some embodiments, the macrophages are tumor-associated macrophages. In some embodiments, the antibody modifies the expression of at least one marker on the macrophages. In some embodiments, the CD163 protein is a glycoform of CD163. In some embodiments, the CD163 protein is a 150 kDa glycoform of CD163. In some embodiments, the antibody does not specifically bind to the 130 kDa glycoform of CD163 expressed by human macrophages. In some embodiments, the antibody has a constant domain that enables binding to an Fc receptor. In some embodiments, the Fc receptor is expressed on a macrophage. In some embodiments, the antibody fragment comprises a single heavy chain, a single light chain, a Fab, a Fab', a F(ab)', a F(ab')2, a Fd, an scFv, a variable heavy domain, a variable light domain, a variable NAR domain, a bispecific scFv, a bispecific Fab2, a trispecific Fab3, a single-chain binding polypeptide, a dAb fragment, or a diabody. In some embodiments, the at least one marker on a human macrophage is CD16, CD64, TLR2, or Siglec-15. In some embodiments, the human macrophage is an M2 macrophage or an M2-like macrophage. In some embodiments, the human macrophage is an M2a, M2b, M2c, or M2d macrophage. In some embodiments, the CD163 protein is a component of a cell surface complex comprising at least one other protein expressed by a macrophage.In some embodiments, the at least one other protein is a galectin-1 protein, a LILRB2 protein, a casein kinase II protein, or any combination thereof. In some embodiments, upon binding to the CD163 protein, the antibody is internalized by human macrophages. In some embodiments, the binding to the CD163 protein is not cytotoxic to macrophages. In some embodiments, the binding to the CD163 protein is not cytotoxic to CD4. + T cell activation, CD4 + T-cell proliferation, or CD4 + In some embodiments, binding to the CD163 protein promotes both activation and proliferation of T cells. + In some embodiments, binding to the CD163 protein promotes the expression of CD8, CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by T cells. + T cell activation, CD8 + T-cell proliferation, or CD8 + In some embodiments, binding to the CD163 protein promotes both activation and proliferation of CD8 T cells. + In some embodiments, binding to the CD163 protein promotes expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by T cells. In some embodiments, binding to the CD163 protein reduces immunosuppression in the tumor microenvironment. In some embodiments, binding to the CD163 protein promotes tumor cell killing in the tumor microenvironment. In some embodiments, binding to the CD163 protein promotes cytotoxic lymphocyte-mediated cancer cell killing. In some embodiments, binding to the CD163 protein promotes NK cell-mediated tumor cell killing. In some embodiments, binding to the CD163 protein promotes expression of IL-2 by T cells. In some embodiments, binding to the CD163 protein promotes CD4 + T cells, CD196-T cells, CXCR3 + T cells, CCR4 -In some embodiments, binding to CD163 reduces immune suppression in the tumor microenvironment caused by macrophages.

[0051] In certain embodiments, disclosed herein are antibodies that specifically bind to the CD163 protein expressed on human macrophages, and that result in the following effects as a result of the binding: (a) a decrease in the expression by the macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) internalization of the antibody by the macrophages; (c) a decrease in the expression of CD4 + T cells, CD8 + (d) activation of T cells, NK cells, or any combination thereof; + T cells, CD8 + and (e) promoting tumor cell death in the tumor microenvironment. In some embodiments, the binding results in two or more of (a)-(e), three or more of (a)-(e), four or more of (a)-(e), or all of (a)-(e). In some embodiments, the binding results in CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof is measured as an increase in the production of IFN-γ, TNF-α, perforin, or any combination thereof.

[0052] In certain embodiments herein, CD163 in the tumor microenvironment + Antibodies are disclosed that specifically bind to immunosuppressive myeloid cells, which binding reduces the inhibition of cytotoxic T cell-mediated killing of tumor cells in the tumor microenvironment.

[0053] In certain embodiments, disclosed herein are antibodies that specifically bind to the CD163 protein expressed on human tumor-associated macrophages and reduce the expression of CD16, CD64, TLR2, Siglec-15, or any combination thereof, by the macrophages, for use in methods for treating cancer. In some embodiments, binding of the antibody to macrophages regulates the immune function of cells in the tumor microenvironment. In some embodiments, binding of the antibody to macrophages promotes anti-tumor immune function.

[0054] In some embodiments, the composition comprises an antibody according to an embodiment as disclosed herein and an excipient.

[0055] In some embodiments, the pharmaceutical composition comprises an antibody according to an embodiment as disclosed herein and a pharmaceutically acceptable carrier.

[0056] In some embodiments, the use of the antibodies disclosed herein is for the manufacture of a medicament for treating cancer in a human subject.

[0057] In some embodiments, the use of the antibodies disclosed herein is for the manufacture of a medicament for reducing immunosuppression by tumor-associated macrophages in a human subject with cancer.

[0058] In some embodiments, the use of the antibodies disclosed herein is for the manufacture of a medicament to promote T cell-mediated tumor cell killing in a human subject with cancer.

[0059] In certain embodiments, a method for promoting immune cell function is disclosed herein, the method comprising the steps of specifically binding an antibody to the CD163 protein expressed on immunosuppressive human myeloid cells and measuring the following parameters: (i) CD4 + T cells, CD8 + (ii) activation of T cells, NK cells, or any combination thereof; and (iii) activation of CD4 + T cells, CD8 +and promoting immune cell function as measured by one or both of T cells, NK cells, or any combination thereof. In some embodiments, the immunosuppressive human myeloid cells are macrophages. In some embodiments, the immunosuppressive human myeloid cells are myeloid-derived suppressor cells. In some embodiments, the antibody is any embodiment disclosed herein.

[0060] In certain embodiments disclosed herein, there is provided a method of treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of an antibody according to any one of the embodiments disclosed herein, thereby treating the cancer in the individual.

[0061] In certain embodiments disclosed herein is a method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody according to any one of the embodiments disclosed herein, whereby immunosuppression by tumor-associated macrophages in the individual is reduced.

[0062] In certain embodiments herein, a method for treating cancer in an individual in need thereof is disclosed, the method comprising administering to the individual a therapeutically effective amount of an antibody according to any one of the embodiments disclosed herein, thereby increasing T cell-mediated tumor cell death in the individual.In some embodiments, the cancer is lung cancer or sarcoma.In some embodiments, the lung cancer is lung carcinoma or lung adenocarcinoma.

[0063] In certain embodiments, disclosed herein are methods for reducing tumor-promoting activity of tumor-associated macrophages in an individual in need thereof, the methods comprising: + T cell activation, CD4 + T cell proliferation, CD8 + T cell activation, CD8 +44. The method of claim 43, further comprising administering to the individual an amount of the pharmaceutical composition of claim 43 effective to modulate T cell proliferation, T cell proliferation, or any combination thereof. In some embodiments, the method further comprises promoting tumor cell death in the tumor microenvironment.

[0064] In certain embodiments herein, a method is disclosed for promoting lymphocyte-mediated tumor cell killing in an individual in need thereof, the method comprising administering to the individual an effective amount of a pharmaceutical composition according to any one of the embodiments disclosed herein.

[0065] In certain embodiments herein, a method of modulating the activity of tumor-associated macrophages in a tumor microenvironment is disclosed, the method comprising contacting tumor-associated macrophages with an antibody, wherein the antibody binds to CD163-expressing human macrophages and has the following effects: (a) binding of the antibody reduces the expression on the macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) upon binding of the antibody to the CD163 protein, the antibody is internalized by the human macrophages; (c) binding of the antibody is not cytotoxic to macrophages; and (d) binding of the antibody reduces the expression of CD4 + T cells, CD8 + (e) binding of the antibody promotes activation of CD4 + T cells, CD8 + and (f) binding of the antibody promotes cell killing in the tumor microenvironment. In some embodiments, binding results in two or more of (a)-(f), three or more of (a)-(f), four or more of (a)-(f), five or more of (a)-(f), or all of (a)-(f). In some embodiments, binding results in CD4 + T cells, CD8 +Activation of T cells, NK cells, or any combination thereof is measured as increased production of IFN-γ, TNF-α, perforin, or any combination thereof. In some embodiments, the method is performed in a tumor microenvironment. In some embodiments, the method is performed in vivo. In some embodiments, binding of the antibody to macrophages modulates cellular immune function in the tumor microenvironment. In some embodiments, binding of the antibody to macrophages promotes anti-tumor immune function. In some embodiments, the antibody comprises a constant domain, and the constant domain binds to an Fc receptor. In some embodiments, the Fc receptor is expressed on macrophages. In some embodiments, the method further comprises internalizing the antibody by human macrophages upon binding to CD163 protein. In some embodiments, binding to CD163 protein is not cytotoxic to human macrophages. In some embodiments, the method comprises binding to CD4 + In some embodiments, the method further comprises promoting expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by the T cells. + In some embodiments, the method further comprises promoting expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by T cells. In some embodiments, the method comprises reducing immunosuppression in the tumor microenvironment. In some embodiments, the method comprises promoting cytotoxic lymphocyte-mediated cancer cell killing. In some embodiments, the method comprises promoting NK cell-mediated tumor cell killing. In some embodiments, the method comprises promoting IL-2 expression by T cells. In some embodiments, the method comprises promoting CD4 + T cells, CD196 - T cells, CXCR3 + T cells, CCR4 - T cells, or any combination thereof. [Brief explanation of the drawings]

[0066] The novel features of the present disclosure are set forth with particularity in the appended claims. The features and advantages of the present disclosure will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings, in which: [Figure 1] FIG. 1 shows that the AB101 antibody binds to the human MDSC population. [Figure 2] FIG. 1 shows the binding of AB101 to CD163Hi cells. [Figure 3] FIG. 10 shows AB101 binding to human M2C, M1, and M0 compared to isotype control. [Figure 4] Figure showing AB101 binding to human peripheral blood T cells, B cells, NKT cells, neutrophils, monocytes, and dendritic cells. Isotype control is shown in gray, and AB101 binding is shown in black. [Figure 5] FIG. 1 shows the lack of binding of AB101 to a panel of human primary cells including small airway epithelial cells (SAECs), renal proximal tubule epithelial cells (RPTECs), pulmonary microvascular endothelial cells (HMVECs), umbilical vein endothelial cells (HUVECs), aortic smooth muscle cells (AOSMCs), and keratinocytes. [Figure 6A] FIG. 1 shows the top 20 targets for AB102 based on mass analysis of post-immunoprecipitation samples. [Figure 6B] FIG. 1 shows the top cell surface targets for AB102 based on mass analysis of post-immunoprecipitation samples. [Figure 6C] FIG. 1 shows the top cell surface targets of AB102 compared to ISO (isotype negative control) based on mass analysis of AB102 and ISO samples after immunoprecipitation. [Figure 7] FIG. 1 shows that AB102 co-immunoprecipitates distinct high molecular weight glycoforms of CD163. [Figure 8] FIG. 1 shows that AB101, AB102, and a control CD163 antibody bound to huCD163, while the isotype control showed no appreciable binding. [Figure 9]FIG. 1 shows that neither AB101 nor the control anti-huCD163 bound to recombinant mouse CD163, compared to a commercially available anti-muCD163 antibody that did not bind to mouse CD163. [Figure 10] FIG. 1 shows that pretreatment of M2c macrophages with polyclonal anti-CD163 antibody blocked binding of the AB101 antibody compared to treatment with a goat control polyclonal antibody that did not block binding of AB101. [Figure 11] FIG. 1 shows that pretreatment of M2c macrophages with polyclonal anti-CD163 antibody blocked binding of the control monoclonal anti-huCD163 antibody compared to treatment with a goat control polyclonal antibody, which did not block binding of the control monoclonal anti-huCD163 antibody. [Figure 12] FIG. 3 shows that treatment of polarized M2C macrophages with siRNA against CD163 substantially reduced binding of the AB102 antibody compared to M2C macrophages treated with scrambled siRNA (siScramb) or no siRNA. Three replicates are represented. [Figure 13] This figure shows that siRNA knockdown of CD163 reduced binding of the AB102 antibody, with a slight reduction in AB102 antibody binding observed after knockdown with siRNA against FCGR2A+FCGR3A (1 of 3 donors), FCGR2C, or FCGR3A, and no reduction in AB102 binding observed after knockdown with siCD206, siCD163L1, siPPIA, siLGALS1, siLGALS3, siLILRB2, and siUPAR. [Figure 14] FIG. 1 shows that treatment of cultured M2 macrophages with LPS resulted in loss of binding by both the AB101 antibody and a control anti-CD163 antibody. [Figure 15] FIG. 1 shows an increase in IL-2 production after treatment of bone marrow cells with the AB101 antibody. [Figure 16] FIG. 1 shows that AB101 antibody treatment during polarization promoted CD4+ T cell proliferation. [Figure 17]FIG. 1 shows that AB101 antibody treatment during polarization promoted CD8+ T cell proliferation. [Figure 18] FIG. 1 shows that treatment with AB101 antibody during polarization (labeled "pre" on the graph), after polarization (labeled "post" on the graph), or a combination of during and after polarization (labeled "pre and post" on the graph) resulted in enhanced IL-2 production compared to isotype antibody treatment. [Figure 19] FIG. 1 shows that treatment of M2 macrophages with AB101 antibody reduced the expression of CD16, CD64, calreticulin, and Siglec-15. [Figure 20] FIG. 1 shows that treatment of M2c cells with AB101 increased the Th1 / Th2 ratio compared to the isotype control. [Figure 21] FIG. 1 shows that treatment of M2c cells with ABlOl increased the expression of CD69 on CD4 T cells compared to the isotype control. [Figure 22] FIG. 1 shows that treatment of M2c cells with ABlOl increased the expression of ICOS on CD4 T cells compared to the isotype control. [Figure 23] FIG. 1 shows that treatment of M2c cells with ABlOl increased the expression of OX40 on CD4 T cells compared to the isotype control. [Figure 24] FIG. 1 shows that expansion of CTLs in the presence of Bite resulted in increased Raji tumor cell killing compared to isotype control. [Figure 25] FIG. 1 shows that the AB102 antibody was internalized in a similar manner to a commercially available anti-CD163 antibody (R&D Systems MAB1607-100), and that the AB101 antibody was internalized approximately twice as much as the commercially available CD163 antibody. [Figure 26]Figure 1 shows tumor volumes plotted over 30 days for A549 tumors. Arrows indicate injections with antibody treatment. Each point represents the average measurement from 7 mice. Error bars indicate the standard error of the mean (SEM). Statistical significance was calculated using the Mann-Whitney test. [Figure 27] Figure 1 shows tumor volumes plotted over 30 days for H1975 tumors. Arrows indicate injections with antibody treatment. Each point represents the average measurement from 7 mice. Error bars indicate the standard error of the mean (SEM). Statistical significance was calculated using the Mann-Whitney test. [Figure 28] FIG. 1 shows the experimental design of the M2c / T cell co-culture assay to evaluate the effect of AB101 treatment on T cell proliferation and IL-2 production. [Figure 29] FIG. 1 shows that treatment with ABlOl during M2c macrophage polarization restored T cell proliferation in an M2c / T cell co-culture assay. [Figure 30] FIG. 1 shows that treatment with ABlOl during M2c macrophage polarization enhanced IL-2 secretion by OKT3-activated T cells in an M2c / T cell co-culture assay. [Figure 31] FIG. 1 shows that pre-, pre- / post-regimen, and post-regimen treatment with AB101 increased CD8+ T cell proliferation in an M2c / T cell co-culture assay. [Figure 32] FIG. 1 shows that pre-, pre- / post-regimen, and post-regimen treatment with AB101 increased CD8+ T cell proliferation in an M2c / T cell co-culture assay for individual subjects. [Figure 33] FIG. 1 shows that treatment with AB101 enhanced CD8+ T cell proliferation in M2c / co-cultures in multiple subjects. [Figure 34] FIG. 1 shows that treatment with AB101 enhanced CD4+ T cell proliferation in M2c / co-cultures in multiple subjects. [Figure 35]FIG. 1 shows that treatment with ABlOl enhanced IL-2 production by multiple human T cells in M2c / co-culture. [Figure 36] FIG. 1 shows that AB101 is more potent than AB104 and AB102 isotypes in enhancing T cell proliferation in an M2c / T cell co-culture assay. [Figure 37] FIG. 1 shows that AB101, but not AB104, rescues CD8+ T cell proliferation from M2c-mediated immunosuppression pre- / post-regimen and post-regimen. [Figure 38] FIG. 1 shows that AB101 restored CD8+ T cell cytokine responses in an M2c / T cell co-culture assay. [Figure 39] FIG. 1 shows that ABlOl rescued CD4+ T cell, IFN-γ, TNF-α, and perforin responses from M2c macrophage-mediated immunosuppression. [Figure 40] FIG. 1 shows that treatment with AB101 enhanced the cytotoxic activity of CD8+ T cells. [Figure 41] FIG. 1 shows that treatment with AB101 enhanced BiTE®-assisted cytotoxic activity of CD8+ T cells. [Figure 42] FIG. 1 shows that treatment with AB101 enhanced the cytotoxic activity of non-HLA-restricted CD8+ T cells. [Figure 43] FIG. 1 shows that treatment with ABlOl alleviates M2c cell-mediated immunosuppression and induces a more specific expression pattern in activated CD4+ T cells. [Figure 44] FIG. 1 shows that treatment with AB101 alleviates M2c macrophage immunosuppression and enhances CD4+ and CD8+ T cell activation. [Figure 45] FIG. 1 shows CXCR3 expression by activated CD4+ T cells. [Figure 46] FIG. 1 shows that treatment with ABlOl during M2c macrophage polarization reduced the expression of CD16, CD64, Siglex-15, and TLR2 by M2c macrophages. [Figure 47] FIG. 1 shows a summary of changes upon pepsin digestion of AB101 bound to huCD163. [Figure 48] FIG. 1 shows a summary of changes upon nepenthesin II digestion of AB101 bound to huCD163. [Figure 49] FIG. 1 shows a summary of AB101 binding to human CD163 ECD based on HDX-MS studies. [Figure 50] FIG. 1 shows that AB101 binds to a truncated CD163 ECD composed of SRCR domains 1 to 5. [Figure 51] Figure 1 shows the alignment of human CD163 to cynomolgus monkey CD163. Signal sequence. The sequence below the black bar indicates the nine SRCR domains, and the gray line above the sequence indicates the consensus sequence. Protected and exposed regions are indicated based on the AB101-binding epitope, as determined by the protection observed in HDX-MS. The solid line below the sequence indicates a nepenthesin II-protected region. The open box below the sequence indicates a pepsin-protected region. The hatched box below the sequence indicates a pepsin-exposed region. The box with a vertical line below the sequence indicates a nepenthesin II-exposed region. The lysine (K) at position 323 of human CD163 and the glutamic acid (E) of cynomolgus monkey CD163 are indicated by boxes. [Figure 52] FIG. 1 shows that AB101 binds to human and cynomolgus E323K mutants but not to wild-type cynomolgus CD163 ECD. [Figure 53] Figure 1 shows SPR detection of AB101 binding to human CD163. AB101 was serially diluted to various concentrations using (A) EDTA or (B) calcium-containing running buffer. GHI / 61 was then injected into flow cell 2 at concentrations of 6.25 / 12.5 / 25 / 50 / 100 / 200 μg / ml with a flow rate of 30 μl / min, a contact time of 300 s, and a dissociation time of 600 s. [Figure 54]Figure 1 shows SPR detection of GHI / 61 binding to human CD163. The anti-CD163 clone GHI / 61 was serially diluted to various concentrations using (A) EDTA or (B) calcium-containing running buffer. GHI / 61 was then injected into flow cell 2 at concentrations of 3.125 / 6.25 / 12.5 / 25 / 50 / 100 μg / ml at a flow rate of 30 μl / min, with a contact time of 300 s and a dissociation time of 600 s. [Figure 55] Figure 1 shows SPR detection of CD163 binding to AB101. Human CD163 protein was serially diluted to various concentrations using calcium-containing running buffer. CD163 protein was then injected into flow cell 2 at a flow rate of 30 μl / min, concentrations of 1.25 / 2.5 / 5.0 / 10.0 / 20.0 / 40.0 μg / ml, a contact time of 300 s, and a dissociation time of 600 s. [Figure 56] Figure 1 shows the binding of AB101 to soluble CD163 in an AlphaLisa assay. AB101 (circles) or isotype control (triangles) were incubated with 750 nM CD163-His at the indicated concentrations for 1 hour. Binding was quantified by AlphaLisa using biotinylated anti-hIgG1 mAb, streptavidin receptor, and nickel donor beads. Symbols represent the mean ± standard error of five independent measurements. Curve fitting was performed with one- and two-site saturation binding models (Graphpad Prism). (R2 = 0.92). (A) Linear and (B) logarithmic x-axis scales. [Figure 57]Figure 1 shows AB101 binding to M1c macrophages. M2c macrophages were blocked with stringent FACS blocking buffer containing 0.5 mg / ml human IgG1 and then stained with AB101 (circles) or isotype control (triangles) at the indicated concentrations for 30 minutes. Binding of AlexaFluor647-labeled AB101 and isotype control to M2c macrophages was quantified by fluorescence intensity and reported as gMFI. Symbols represent the mean ± standard error of four test subjects. Curve fitting was performed with a two-site saturation binding model (Graphpad Prism). (R2=0.99). (A) Linear and (B) logarithmic x-axis scale. DETAILED DESCRIPTION OF THE INVENTION

[0067] As used herein, CD163 + Disclosed are antibodies that specifically bind to cells. In some embodiments, the antibodies specifically bind to CD163 + In some embodiments, the cells are immunosuppressive myeloid cells. + The cells are myeloid expressing human CD163. In some embodiments, CD163 + In some embodiments, the cell is a tumor cell. + The immunosuppressive myeloid cells are human macrophages. In some embodiments, human CD163 + The immunosuppressive macrophages are M2 or M2-like macrophages. In some embodiments, the immunosuppressive myeloid cells are myeloid-derived suppressor cells (MDSCs). In some embodiments, human macrophages express high levels of CD163 (CD163 Hi In contrast, other human hematopoietic cells or primary non-immune human cells do not express CD163. For example, M1 and M1-like macrophages do not express CD163.

[0068] Monocytes and macrophages exposed to specific inflammatory cytokines or microbe-associated molecular patterns differentiate into pro-inflammatory (M1 or M1-like) or anti-inflammatory M2 or M2-like macrophages. M1 and M2 are classifications used to define in vitro activated macrophages as pro-inflammatory (when classically activated with IFN-γ and lipopolysaccharide) or anti-inflammatory (when alternatively activated with IL-4 or IL-10), respectively; in vivo or ex vivo macrophages with an M1 or M2 phenotype are defined as M1-like or M2-like macrophages. In some embodiments, M2 macrophages are generated by exposing them to specific cytokines. In some embodiments, M2 macrophages are differentiated with IL-4, IL-10, IL-13, or a combination thereof.

[0069] M2 macrophage subtypes include M2a, M2b, M2c, and M2d. M2a macrophages are induced by upregulated expression of CD163, arginase-1, mannose receptor MRC1 (CD206), antigen presentation via the MHC II system, and IL-4 and IL-13, which induce the production of IL-10 and TGF-β, resulting in tissue regeneration and inhibition of pro-inflammatory molecules, preventing inflammatory responses. M2b macrophages produce IL-1, IL-6, IL-10, and TNF-α in response to immune complexes. M2c macrophages are induced by IL-10, transforming growth factor beta (TGF-β), and glucocorticoid exposure, producing IL-10 and TGF-β to suppress inflammatory responses. The M2d subtype is activated in response to IL-6 and adenosine.

[0070] M2 macrophages have functions and phenotypes corresponding to M2 macrophages and their subtypes. M2-like macrophages are in vivo or ex vivo macrophages that possess a subset of functional or phenotypic characteristics of M2 macrophages.

[0071] In some embodiments, the antibodies disclosed herein have high avidity and specific binding to immunosuppressive myeloid cells, specifically tumor-associated macrophages such as M2 and M2-like macrophages. In some embodiments, the antibodies specifically bind to M2 and M2-like TAMs derived from human primary lung tumors. In some embodiments, the antibodies disclosed herein do not significantly bind to M1 or M1-like macrophages. M1-activated macrophages express transcription factors such as interferon regulatory factor 5 (IRF5), nuclear factor of kappa light polypeptide gene enhancer (NF-κB), activator protein 1 (AP-1), and STAT1. M1 macrophages secrete pro-inflammatory cytokines such as IFN-γ, IL-1, IL-6, IL-12, IL-23, and TNFα. M1 macrophages have functions and phenotypes corresponding to M1 macrophages. M1-like macrophages are in vivo or ex vivo macrophages that possess a subset of functional or phenotypic characteristics of M1 macrophages.

[0072] In some embodiments, the antibodies of the present disclosure do not bind to primary human cells, hi some embodiments, the antibodies of the present disclosure do not bind to hematopoietic stem cells, leukocytes, T cells, B cells, NK cells, and granulocytes.

[0073] Tumor-associated macrophages (TAMs) are a heterogeneous class of macrophage cells present in high numbers in the microenvironment of solid tumors. Most evidence suggests that TAMs possess a tumor-promoting phenotype and are clearly involved in tumor cell proliferation, tumor angiogenesis, motility, invasion, metastasis, anticancer drug resistance, and tumor immune evasion.

[0074] Direct tumor cell killing by cytotoxic T cells among tumor-infiltrating lymphocytes (TILs) plays a major role in the antitumor function of the immune system. However, TAMs in the tumor microenvironment (TME) suppress T cell-mediated antitumor immune responses. TAMs have an immunosuppressive transcriptional profile and express factors including IL-10 and transforming growth factor β (TGFβ). In humans, TAMs have been shown to directly suppress T cell function through the surface expression of programmed cell death ligand 1 (PD-L1) in hepatocellular carcinoma and B7-homolog in ovarian cancer, which activate programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4), respectively, on T cells. Inhibitory signals to PD-1 and CTLA4 are immune checkpoints, and binding of these inhibitory receptors by their ligands inhibits T cell receptor signaling and T cytotoxicity, promoting T cell apoptosis. HIF-1α induces TAMs to suppress T cell function. CD163 has been identified as an immunosuppressive molecule expressed exclusively on TAMs and may be a potential therapeutic target for cancer immunotherapy.

[0075] TAMs generally consist of Th1 and Th2 type T helper cells (CD4 + Based on functional characteristics, including their relationship to Th1 cells, macrophages are classified into two categories: M1-like antitumor and M2-like immunosuppressive macrophages. M1 macrophages are the "classical" model and can be generated using IFN-γ along with pathogen-associated molecular patterns (PAMPs) (e.g., lipopolysaccharide (LPS)) or damage-associated molecular patterns (DAMPs), as well as innate immune activators such as inflammatory cytokines (e.g., tumor necrosis factor-α (TNF-α)). Furthermore, T cell-dependent macrophage activation via the CD40-CD40 ligand pathway induces M1 differentiation. M1 macrophages possess inflammatory, bactericidal, and cytotoxic functions. These macrophages are involved in the antigen-dependent induction of Th1 cells, as well as the activation of Th1 and CD8 +Enhanced T cell activation. Enhanced T cell cytotoxicity by M1-like anti-tumor macrophages is important for tumor cell elimination. In some embodiments, M1-like anti-tumor macrophages are characterized by surface marker expression measured by flow cytometry, including CD80. + , CD86 + , CD163 Lo / - , or CD206 Lo / - M1 macrophages secrete IL-12 and low levels of IL-10 and / or TGF-γ.

[0076] In contrast, M2-like immunosuppressive macrophages are a model of "alternative" or "non-classical" activation that can be generated in vitro by IL-4 or IL-10, are anti-inflammatory, and promote wound healing and tissue repair. In some embodiments, M2-like immunosuppressive macrophages are polarized from monocyte-derived macrophages and recruited by factors secreted by tumors. M2-like immunosuppressive macrophages are the primary macrophage cell type involved in the tumor-promoting functions of TAMs, including promoting tumor growth, metastasis, and immune evasion. M2-like macrophages express surface markers CD15, CD23, CD64, CD68, CD163, and CD165 as determined by flow cytometry. Hi , CD204 Hi , CD206 Hi , and / or other M2 macrophage markers. M2 macrophages secrete high levels of IL-10 and TGF-beta1, and low levels of IL-12.

[0077] In many tumor types, the level of TAM infiltration has significant prognostic value. TAMs have been associated with poor prognosis in a wide variety of tumors. For example, breast cancer patients with higher levels of M2-like tumor-associated macrophages have been found to have more aggressive tumors, greater microvascular density, and lower overall survival rates. Patients with higher levels of M1-like antitumor TAMs showed the opposite effect.

[0078] Thus, there remains a need to identify compounds and methods for improving immunotherapeutic treatments for cancer treatment.

[0079] CD163 (scavenger receptor cysteine-rich type 1 protein M130, hemoglobin scavenger receptor) is a cell surface protein that acts as a scavenger receptor for hemoglobin-haptoglobin complexes to protect tissues from free hemoglobin-mediated oxidative damage. Four isoforms of the CD163 protein have been reported, with molecular weights of 125, 451, 125,982, 121,609, and 124,958 Da. Isoform 1 is the most common isoform of CD163, with a molecular weight of 125,451 Da. It consists of a 1115 amino acid residue polypeptide, including an extracellular domain, a transmembrane segment, and a cytoplasmic tail. The extracellular domain consists of nine cysteine-rich repeat domains. Isoform 1 of the CD163 protein has four N-linked glycosylation sites, and the CD163 protein in M2 macrophages exhibits two distinct bands at maximal sizes of 150 kDa and 130 kDa on SDS-PAGE under reducing conditions.

[0080] CD163 mRNA expression is generally restricted to myeloid cells but is also expressed by certain human cancers. CD163 expression on TAMs is associated with an immunosuppressive M2-like phenotype, which has been shown to correlate with poor clinical outcomes in cancer. CD163 is required for the tumor-induced activation of macrophages in human and mouse sarcomas. CD163 has also been reported to be a macrophage scavenger receptor and promote immunosuppression. In some embodiments, interaction of hemoglobin-haptoglobin complexes with CD163 induces the secretion of the immunosuppressive cytokine IL-10 and expression of heme oxygenase-1 (HO-1). HO-1 promotes the production of the anti-inflammatory metabolite Fe. 2+ , CO, and biliverdin.

[0081] Soluble CD163, which arises in humans via ectodomain shedding, has been reported to have anti-inflammatory properties, including downregulating T cell responses, including lymphocyte proliferation stimulated by phytohemagglutinin (PHA) or 12-O-tetradecanoylphorbol-13-acetate (TPA).

[0082] Antibodies targeting CD163 have been shown to regulate the innate immune response of macrophages expressing CD163. For example, the RM3 / 1 antibody, a mouse monoclonal IgG1 (κ light chain) antibody against CD163, was developed against human monocytes. The RM3 / 1 antibody binds to the cysteine-rich domain 9 of human CD163, reducing LPS-induced TNFα and enhancing IL-10 secretion by macrophages.

[0083] Specific Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. Generally, the nomenclature utilized in connection with, and techniques of, immunology, oncology, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claimed subject matter. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0084] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and in some embodiments, reference to "an antibody" includes a plurality of antibodies, and so forth.

[0085] As used herein, all numerical values ​​or ranges of values ​​include whole integers and fractions within or including such ranges, or integers within or including such ranges, unless the context clearly indicates otherwise. Thus, for example, a reference to a range of 90-100% includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc. In another example, a reference to a range of 1 to 5,000 times includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5 times, etc., 2.1, 2.2, 2.3, 2.4, 2.5 times, etc.

[0086] As used herein, a number preceded by "about" refers to an inclusive range that extends from 10% below to 10% above that number. A range preceded by "about" refers to 10% below the lower limit of the range and 10% above the upper limit of the range.

[0087] "Percent identity" and "% identity" refer to the degree to which two sequences (nucleotides or amino acids) have the same residues at the same positions in an alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" refers to the % identity of the amino acid sequence to SEQ ID NO: Y, specifically, that X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in SEQ ID NO: Y. Generally, computer programs are used for such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(1):11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci USA. 1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98), and Gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387-95).

[0088] As used herein, "antibody" refers to a protein that binds to an antigen. Antibodies often contain variable and constant domains in each of the heavy and light chains. Thus, most antibodies have a heavy chain variable domain (VH) and a light chain variable domain (VL) that together form the portion of the antibody that binds to the antigen. Within each variable domain are three complementarity-determining regions (CDRs), which form loops in the heavy chain variable domain (VH) and the light chain variable domain (VL) and contact the surface of the antigen. "Antibodies" include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific (e.g., bispecific), natural, humanized, human, chimeric, synthetic, recombinant, hybrid, mutated, grafted, antibody fragments (e.g., portions of full-length antibodies, generally antigen-binding or variable regions thereof, e.g., Fab, Fab', F(ab')2, and Fv fragments), and in vitro-generated antibodies that have antigen-binding activity. The term also includes single chain antibodies, such as single chain Fv (sFv or scFv) antibodies in which a variable heavy chain and a variable light chain are joined together (directly or via a peptide linker) to form a continuous polypeptide.

[0089] As used herein, "complementarity-determining region (CDR)" refers to the portion of the variable chain of an antibody that binds to a specific antigen. Multiple methods may be used to define CDRs. Current technology utilizes various numbering schemes with varying definitions of CDR length and position. For example, the Kabat numbering scheme is based on sequence alignment and predicts CDRs using the "variability parameter" (the number of different amino acids at a given position divided by the frequency of the most frequently occurring amino acid at that position) for a given amino acid position. The Chothia numbering scheme, on the other hand, is a structure-based numbering scheme in which antibody crystal structures are aligned to define loop structures as CDRs. The Martin numbering scheme focuses on the structural alignment of framework regions of different lengths with atypical structure. The IMGT numbering scheme is a standardized numbering system based on the alignment of sequences from a complete reference gene database encompassing the entire immunoglobulin superfamily. The Honneger numbering scheme (AHo's) is based on the structural alignment of the 3D structures of the variable regions and uses structurally conserved Cα positions to estimate the framework and CDR lengths. Those skilled in the art should note that the definition of a CDR varies based on the method used. Any method of defining a CDR is contemplated using the sequences disclosed herein.

[0090] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to a mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. For purposes of treatment, "mammal" refers to animals classified as mammals, including humans, domestic animals, and farm animals, as well as laboratory, zoo, sport, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and monkeys. In some embodiments, the mammal is a human.

[0091] As used herein, the terms "treatment," "treating," and the like refer to administering an agent or performing a procedure, as the case may be, for the purpose of achieving an effect. In some embodiments, the effect is preventative, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or its symptoms are partially or completely cured. As used herein, "treatment" includes treatment of mammalian, particularly human, diseases or disorders (e.g., cancer), including (a) preventing the disease or its symptoms from occurring in a subject who is predisposed to the disease (including, for example, diseases associated with or caused by a primary disease) but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., halting the progression of the disease; and (c) palliating the disease, i.e., causing regression of the disease. In some embodiments, treating refers to successful treatment, amelioration, or prevention of cancer, including objective or subjective parameters such as relief, remission, a decrease in symptoms, or making the condition more tolerable to the patient, a slower rate of degeneration or decline, or a more debilitating end point of degeneration. The treatment or improvement of symptoms is based on one or more objective or subjective parameters, including the results of a medical examination. Thus, the term "treating" includes administering a compound or agent of the present disclosure to prevent, delay, or alleviate symptoms or conditions associated with a disease (e.g., cancer), or to prevent or inhibit the onset of the preceding symptoms or conditions. The term "therapeutic effect" refers to the reduction, eradication, or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject.A subject is "treated" for a disease or disorder if, after administration of a therapeutic amount of an antibody of the present disclosure, the patient exhibits an observable and / or measurable change in a parameter or symptom of the disease or disorder, e.g., in the case of cancer treatment, an increase in tumor cell killing activity as assessed ex vivo, a decrease in the level of secreted immunosuppressive factors in the blood, a decrease in tumor volume or mass, an increase in the number of cytotoxic lymphocytes and Th1-like T cells in a tumor biopsy, a decrease in morbidity or mortality, an improvement in quality of life factors, or an improvement in any objective indication related to a parameter or symptom of the disease or disorder. In some embodiments, the parameter includes converting a cold immune tumor to a warm immune tumor, for example, by increasing the number of cytotoxic lymphocyte cells and markers of T cell activation (such as CD69, ICOS, OX40) in a tumor biopsy, or by decreasing the expression of CD16, CD64, TLR2, Siglec-15 on TAMs in a tumor biopsy.

[0092] In some embodiments, "inducing a response" refers to the alleviation or reduction of signs or symptoms of disease in a subject, specifically including, but not limited to, prolonging survival.

[0093] The term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution.

[0094] In some embodiments, antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, which vary with the antibody isotype.

[0095] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody.

[0096] "Human effector cells," as used herein, refer to leukocytes that express one or more FcRs and perform effector function. For example, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include, but are not limited to, peripheral blood mononuclear cells (PBMCs), NK cells, monocytes, macrophages, cytotoxic T cells, and neutrophils.

[0097] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to their cognate antigen. To assess complement activation, for example, a CDC assay is performed.

[0098] An "internalizing" antibody is one that is taken up by (i.e., enters) a cell upon binding to an antigen (e.g., a cell surface polypeptide or receptor) on a mammalian cell. Internalizing antibodies include antibody fragments, human or chimeric antibodies, and antibody conjugates. In some cases, internalizing antibodies (e.g., such as those disclosed herein) modify the biology of the cell, altering its function.

[0099] An "antigen-binding domain," "antigen-binding region," or "antigen-binding site" is the part of an antibody that comprises the amino acid residues (or other moieties) that interact with an antigen and contribute to the antibody's specificity and affinity for the antigen. In the case of an antibody that specifically binds to its antigen, this includes at least a portion of at least one of its CDR domains.

[0100] The antigen-binding region of an antibody is called the "epitope" of the antigen, which is the antigenic determinant, or "paratope," that binds to the portion of the antigen molecule that can be bound by an antibody. In some embodiments, an antigenic substance has more than one portion recognizable by an antibody, i.e., more than one epitope, and thus a single antigenic substance can be specifically bound by various antibodies, each with specificity for a different epitope. In some embodiments, an epitope comprises non-contiguous portions of an antigen. For example, in a polypeptide, amino acid residues that are not contiguous in the primary sequence of the polypeptide but are close enough to each other in the context of the polypeptide's tertiary and quaternary structure to be bound by an antigen-binding protein constitute an epitope.

[0101] An "antibody fragment" comprises a portion of an intact antibody. In some embodiments, an antibody fragment comprises the antigen-binding or variable region of the intact antibody.

[0102] The terms "antigen-binding portion of an antibody," "antigen-binding fragment," "antigen-binding domain," and "antibody fragment" are used interchangeably herein and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Non-limiting examples of antibody fragments encompassed within these terms include, but are not limited to, (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment comprising the VL and VH domains of a single arm of an antibody; (v) a dAb fragment comprising the VH domain (Ward et al., Nature 341(6242):544-6(1989)); and (vi) an isolated CDR. "Half" antibodies comprising a single heavy chain and a single light chain are also included. Other single-chain antibody forms, such as diabodies, are also encompassed herein.

[0103] "Functional antibody fragment," as used herein, refers in context to an antibody fragment that not only binds to the antibody's antigen but also possesses functional attributes that characterize an intact antibody. For example, if the function of the antibody depends on having an Fc domain that enables an effector function, such as ADCC, then the functional fragment possesses such a function. In some embodiments, antibodies of the present disclosure are hypothesized to be effective in modulating the functional state of tumor-associated macrophages or redirecting or attenuating M2-state macrophages when they contain an Fc portion that binds to a macrophage Fc receptor, such as CD16 (FcγRIIIa) or CD64 (FcγRI).

[0104] The phrase "functional fragment or analog" of an antibody refers to a compound that has qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody binds to IgE immunoglobulins and prevents or substantially reduces the ability of such molecules to bind to the high affinity receptor, FcγRI.

[0105] An "antigen-binding protein" is a protein that comprises a portion that comprises the antigen-binding portion of an antibody, and optionally also includes a scaffold or framework portion that enables the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to an antigen.

[0106] An "intact" antibody is one that contains the antigen-binding site as well as the C L , and at least a heavy chain constant domain C H 1. C H 2, and C H 3. In some embodiments, the constant domains are native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.

[0107] As used herein, the term "recombinant antibody" refers to an antibody that comprises an antigen-binding domain of a first antibody, such as, for example, a CDR, a VH region, or an intact light chain, and domains from one or more other antibodies or proteins. Chimeric antibodies, hybrid antibodies, and humanized antibodies are examples of recombinant antibodies.

[0108] A "CDR-grafted antibody" is an antibody that contains one or more CDRs from an antibody of one species or isotype and the framework of another antibody of the same or different species or isotype.

[0109] The term "human antibody" includes all antibodies having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains of the antibody are derived from human immunoglobulin sequences (referred to as a "fully human antibody").

[0110] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two drugs, K D In one embodiment, the antibody or antigen-binding fragment thereof is -6 Range below M, or 10 -16 Range up to M or less (e.g., about 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 M or less) and K against CD163 D In certain embodiments, the antibodies described herein exhibit binding affinities as measured by the equilibrium dissociation constant (Eq. 10). -4 M or less, about 10 -5 M or less, about 10 -6 M or less, 10 -7 M or less, or 10 -8 K below M D and specifically binds to the huCD163 polypeptide.

[0111] The terms "preferentially binds" or "specifically binds" mean that an antibody or fragment thereof binds to an epitope with higher affinity than it binds to an unrelated amino acid sequence, and is not toxic at levels formulated for administration to humans if it cross-reacts with other polypeptides containing this epitope. In some embodiments, such affinity is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1000-fold greater than the affinity of the antibody or fragment thereof for an unrelated amino acid sequence.

[0112] The term "specific" refers to the situation where an antibody binds preferentially to a molecule other than the antigen containing the epitope recognized by the antibody. The term also applies to the situation where, for example, an antigen-binding domain is specific for a particular epitope carried by multiple antigens, in which case an antibody or antigen-binding fragment thereof carrying the antigen-binding domain will be able to bind to a variety of antigens carrying the epitope.

[0113] As used herein, an antibody is a molecule that binds to an antigen at a detectable level, preferably at about 10 4 M -1 That's about 10 5 M -1 That's about 10 6 M -1 That's about 10 7 M -1 or more, or 10 9 M -1 The affinity constant K a When an antibody reacts with an antigen, it is said to be "immunospecific" or "specific for" or "specifically binds" to the antigen.

[0114] The term "monospecific" as used herein refers to an antibody composition containing antibodies that exhibit preferential affinity for one particular epitope. In some embodiments, a monospecific antibody preparation is composed of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% antibodies with specific binding activity for a particular antigen.

[0115] The term "polypeptide" is used in its conventional sense, i.e., as a sequence of amino acids. A polypeptide is not limited to a particular length of the product. Peptides, oligopeptides, and proteins are included within the definition of polypeptide; these terms are used interchangeably herein unless otherwise specified. The term also does not refer to or exclude post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like, as well as other modifications, both naturally occurring and non-naturally occurring, known in the art. In some embodiments, a polypeptide is an entire protein or a subsequence thereof. A particular polypeptide of interest in the context of the antibodies of the present disclosure is an amino acid subsequence that comprises the CDRs and is capable of binding to human M2 macrophage or CD163 protein expressed by such cells.

[0116] As used herein, "substantially pure" and "substantially free" refer to, for example, a solution or suspension containing less than about 20% foreign matter, less than about 10% foreign matter, less than about 5% foreign matter, less than about 4% foreign matter, less than about 3% foreign matter, less than about 2% foreign matter, or less than about 1% foreign matter.

[0117] The term "isolated" refers to a protein (e.g., an antibody), nucleic acid, or other substance that is substantially free of other cellular material and / or chemicals. In some embodiments, the antibodies or antigen-binding fragments thereof, and nucleic acids of the present disclosure are isolated. In some embodiments, the antibodies or antigen-binding fragments thereof, and nucleic acids of the present disclosure are substantially pure.

[0118] "Isolated," when applied to a polypeptide, generally refers to a polypeptide that is separated from other proteins and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, such as antibodies or gel matrices (polyacrylamide), used to purify it. In some cases, the term refers to a polypeptide or portion thereof that, due to its origin or manipulation, (i) is present in a host cell as an expression product of part of an expression vector; (ii) is bound to proteins or other chemical moieties other than those with which it is naturally bound; or (iii) is not naturally occurring, e.g., is chemically engineered by appending or adding at least one hydrophobic moiety to the protein so that the protein is in a form not found in nature. "Isolated" also refers to a protein that is (i) chemically synthesized or (ii) expressed in a host cell and purified away from related and contaminating proteins.

[0119] The term "effective amount," as used herein, refers to an amount of an antibody or antigen-binding portion thereof described herein that is sufficient to induce a response, e.g., to achieve treatment, prognosis, or diagnosis of a disease associated with macrophage activity or TAM activity, as described herein, when administered to a subject. Therapeutically effective amounts of antibodies provided herein will vary depending on the relative activities of the antibodies and combinations (e.g., in inhibiting tumor cell growth), when used alone or in combination, as well as the subject and condition being treated, the subject's weight and age, the severity of the condition, and, optionally, the mode of administration, as readily determined by one of ordinary skill in the art.

[0120] The term "therapeutically effective amount" generally refers to an amount of an antibody or drug effective to "treat" a disease or disorder in a subject or mammal. In some embodiments, the compositions described herein are administered to a subject in an amount effective to produce some desired therapeutic effect by inhibiting a disease or disorder described herein at a reasonable benefit-risk ratio applicable to any medical treatment. A therapeutically effective amount is an amount that achieves at least a partial desired therapeutic or prophylactic effect in an organ or tissue. The amount of antibody required to prevent and / or treat a disease or disorder is not fixed per se. In some embodiments, the amount of antibody administered varies depending on the type of disease, the extent of the disease, and the size of the mammal suffering from the disease or disorder. When used in conjunction with a treatment method requiring administration of a therapeutic agent after a subject has developed symptoms of the disease or disorder, the term "therapeutically effective" means that one or more signs or symptoms of the disease or disorder are improved or eliminated following treatment.

[0121] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that, when administered to humans, are physiologically tolerable and generally do not produce allergic or similar adverse reactions, such as stomach upset or dizziness.

[0122] The term "contacting" is defined herein as a means of bringing the compositions provided herein into physical proximity with a cell, organ, tissue, or fluid as described herein.

[0123] immunomodulatory antibodies In certain embodiments herein, human CD163 + Disclosed are antibodies that specifically bind to CD163 protein expressed on cells. In some embodiments, CD163 + The cells are immunosuppressive myeloid cells. In some embodiments, the immunosuppressive human myeloid cells are human macrophages. In some embodiments, binding of an antibody disclosed herein modifies the expression of at least one marker on human macrophages.

[0124] In some embodiments, the antibodies disclosed herein bind to human CD163 (huCD163) protein expressed on human M2 or M2-like immunosuppressive macrophages. In some embodiments, the antibodies specifically bind to the approximately 140 kDa glycoform of CD163 protein. In some embodiments, the antibodies specifically bind to extracellular domain 3 of huCD163. In some embodiments, the antibodies specifically bind to extracellular domain 4 of huCD163. In some embodiments, the antibodies specifically bind to extracellular domains 3 and 4 of huCD163. In some embodiments, the antibodies specifically bind to huCD163, resulting in a conformational change in huCD163. In some embodiments, the conformational change to huCD163 exposes extracellular domains 2, 5, and 9 of humCD163. In some embodiments, the antibodies do not specifically bind to the low molecular weight (approximately 115 kDa) glycoform of huCD163.

[0125] In some embodiments, the antibodies disclosed herein target human CD163 + The antibodies disclosed herein bind to immunosuppressive myeloid cells and cause changes in the expression of certain cell markers that characterize M2 or M2-like immunosuppressive macrophages (such as M2c macrophages), indicating functional differentiation of the macrophages to a non- or hypo-immunosuppressive, as well as more anti-tumor state. In some embodiments, the antibodies disclosed herein bind to M2 or M2-like immunosuppressive macrophages and reduce the expression of certain cell markers that characterize M2 or M2-like immunosuppressive macrophages, indicating functional differentiation of the macrophages to an altered differentiation state. In some embodiments, the antibodies disclosed herein bind to CD163 + Reduces expression of one or more of CD16, CD64, TLR2, and Siglec-15 by immunosuppressive myeloid cells.

[0126] In some embodiments, the antibodies disclosed herein inhibit CD163+ Binding to immunosuppressive myeloid cells is mediated by CD163 + In some embodiments, the antibodies disclosed herein inhibit CD163. + Binding to immunosuppressive myeloid cells results in a shift to M2 or M2-like immunosuppressive macrophages. In some embodiments, CD163 bound to the antibody + Functional changes in immunosuppressive myeloid cells result in modified interactions with other cells, such as effector T cells, which subsequently modify their effects and interactions with target tumor cells.

[0127] In some embodiments, antibodies of the present disclosure reduce immunosuppression caused by tumor-associated macrophages in the tumor microenvironment. In some embodiments, reduced immunosuppression by tumor-associated macrophages in the tumor microenvironment corresponds to increased immune stimulation, e.g., enhanced production of T cell activation, T cell proliferation, NK cell activation, NK cell proliferation, or any combination thereof. In some embodiments, T cell activation and / or NK cell activation results in increased production of IFN-γ, TNF-α, perforin, or a combination thereof by T cells and / or NK cells. In some embodiments, antibodies of the present disclosure increase immune stimulation, e.g., enhanced production of T cell activation, T cell proliferation, NK cell activation, NK cell proliferation, or any combination thereof. In some embodiments, T cell activation and / or NK cell activation results in increased production of IFN-γ, TNF-α, perforin, or a combination thereof by T cells and / or NK cells. In some embodiments, an antibody of the present disclosure specifically binds to CD163 protein expressed on human macrophages, and the human macrophages have a first immunosuppressive activity before binding with the antibody and a second immunosuppressive activity after binding with the antibody, the second immunosuppressive activity being lower than the first immunosuppressive activity. In various embodiments, the first and second immunosuppressive activities are each non-zero.

[0128] In some embodiments, the antibodies of the present disclosure promote T cell activation and proliferation. In some embodiments, the antibodies skew T cell populations toward an anti-tumor T cell phenotype. In some embodiments, the antibodies reduce or block myeloid suppression of T cell activation. In some embodiments, the antibodies reduce the ability of TAMs to suppress T cell activation, resulting in greater T cell stimulation and IL-2 production. In some embodiments, the antibodies block the ability of TAMs to suppress T cell activation, resulting in greater T cell stimulation and IL-2 production.

[0129] In some embodiments, the antibodies disclosed herein reduce myelosuppression of T cell proliferation. In some embodiments, the antibodies reduce myelosuppression of T cell proliferation. + and CD8 + In some embodiments, the antibody reduces TAM suppression of Th1 cell proliferation. The expanded T cells are CD4 + Enhanced expression of activation markers on T cells is shown.

[0130] In some embodiments, the antibodies of the present disclosure modify M2-polarized macrophages such that they assume an M1-like phenotype that alleviates the immunosuppressive effects of M2 macrophages. In some embodiments, the antibodies described herein influence monocyte-derived macrophages to differentiate into a less immunosuppressive, more anti-tumor differentiation state.

[0131] In some embodiments, provided herein is a method for specifically binding to human CD163 protein (huCD163), which is expressed on human macrophages and reduces the expression of at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophages. In some embodiments, the human macrophages are tumor-associated immunosuppressive macrophages. In some embodiments, the human macrophages are M2-like immunosuppressive macrophages.

[0132] In some embodiments, the antibodies disclosed herein bind to CD163 protein expressed by macrophages as a component of a complex that includes at least one other protein expressed by macrophages. In some embodiments, the complex is a cell surface complex. In some embodiments, the complex includes at least one other protein selected from galectin-1 protein, LILRB2 protein, and casein kinase II protein.

[0133] In some embodiments, the antibodies disclosed herein bind to CD163 protein on macrophages and are internalized by macrophages.

[0134] In some embodiments, the antibodies disclosed herein are not cytotoxic to the macrophages to which they bind.

[0135] In some embodiments, the antibodies disclosed herein inhibit CD4 + In some embodiments, the antibody stimulates CD4 T cell activity or proliferation. + Promoting expression of CD69, ICOS, OX40, PD1, LAG3, or CTLA4 by T cells.

[0136] In some embodiments, the antibodies disclosed herein inhibit CD8 + In some embodiments, the antibody stimulates CD8 + Promoting the expression of ICOS, OX40, PD1, LAG3, or CTLA4 by T cells.

[0137] In some embodiments, the antibodies disclosed herein inhibit CD8 + By promoting T cell activity or proliferation, the antibody promotes tumor cell killing in the tumor microenvironment. In some embodiments, the antibody promotes cytotoxic lymphocyte-mediated cancer cell killing. In some embodiments, the antibody promotes NK cell-mediated tumor cell killing.

[0138] In some embodiments, the antibodies disclosed herein promote the expression of IL-2 by T cells. In some embodiments, binding of the antibodies of the present disclosure to the CD163 protein promotes the expression of IL-2 by T cells. + T cells, CD196 - T cells, CXCR3 + T cells, CCR4 - In some embodiments, the antibody increases CD4 T cells, or any combination thereof. + CD196 - CXCR3 + CCR4 - Increases T cells.

[0139] In some embodiments, the antibodies disclosed herein have a constant domain that binds to an Fc receptor expressed on macrophages. In some embodiments, the antibody specifically binds to huCD163 and has a constant domain that binds to an Fc receptor. In some embodiments, the antibody specifically binds to CD163, such as CD16 (FcγRIIIa) or CD64 (FcγRI). + The antibody variable domain has a constant domain that binds to an Fc receptor expressed on an immunosuppressive myeloid cell. In some embodiments, huCD163 and an Fc receptor are expressed on the same cell. In some embodiments, huCD163 and an Fc receptor are expressed on different cells. In some embodiments, the antibody variable domain specifically binds to huCD163 and an antibody constant domain that simultaneously binds to an Fc receptor.

[0140] In certain embodiments herein, antibodies are disclosed that specifically bind to CD163 protein expressed on human M2 and M2-like macrophages, said binding having the following effects: (a) a decrease in the expression by human macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) antibody internalization by human macrophages; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; (d) CD4 + T cells, CD8 + Expansion of T cells, NK cells, or any combination thereof; and (e) Promoting tumor cell death in the tumor microenvironment This results in at least one of the following:

[0141] In certain embodiments herein, antibodies are disclosed that specifically bind to CD163 protein expressed on human M2 and M2-like macrophages, said binding having the following effects: (a) a decrease in the expression by human macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) antibody internalization by human macrophages; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; (d) CD4 + T cells, CD8 + Expansion of T cells, NK cells, or any combination thereof; and (e) Promoting tumor cell death in the tumor microenvironment This will result in at least two of the following:

[0142] In certain embodiments herein, antibodies are disclosed that specifically bind to CD163 protein expressed on human M2 and M2-like macrophages, said binding having the following effects: (a) a decrease in the expression by human macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) antibody internalization by human macrophages; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; (d) CD4 + T cells, CD8 + Expansion of T cells, NK cells, or any combination thereof; and (e) Promoting tumor cell death in the tumor microenvironment This brings about at least three of the following:

[0143] In certain embodiments herein, antibodies are disclosed that specifically bind to CD163 protein expressed on human M2 and M2-like macrophages, said binding having the following effects: (a) a decrease in the expression by human macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) antibody internalization by human macrophages; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; (d) CD4 + T cells, CD8 + Expansion of T cells, NK cells, or any combination thereof; and (e) Promoting tumor cell death in the tumor microenvironment This brings at least four of the following:

[0144] In certain embodiments herein, antibodies are disclosed that specifically bind to CD163 protein expressed on human M2 and M2-like macrophages, said binding having the following effects: (a) a decrease in the expression by human macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) antibody internalization by human macrophages; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; (d) CD4 + T cells, CD8 + Expansion of T cells, NK cells, or any combination thereof; and (e) Promoting tumor cell death in the tumor microenvironment Bring at least five of them.

[0145] In some embodiments, the antibodies disclosed herein inhibit human CD163 in tumor-associated macrophage (TAM) populations. + Selectively binds to immunosuppressive myeloid cells, where the antibody specifically binds to the CD163 protein expressed on M2 macrophages, reducing the immunosuppressive activity of the TAM population.

[0146] In some embodiments, the antibodies disclosed herein inhibit human CD163 in the tumor microenvironment. + The antibody selectively binds to immunosuppressive myeloid cells, specifically binding to the CD163 protein expressed on M2 macrophages and reducing M2 macrophage-mediated suppression. In some embodiments, the antibody disclosed herein is human, humanized, or chimeric. In some embodiments, the antibody disclosed herein is an antigen-binding fragment thereof that binds as described.

[0147] In some embodiments, antibodies of the present disclosure are portions of intact immunoglobulin molecules, such as human antibodies, and humanized Ig molecules containing an antigen-binding site (i.e., paratope) or a single heavy chain and a single light chain, including Fab, Fab', F(ab)', F(ab')2, Fd, scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, bispecific Fab2, trispecific Fab3, single-chain binding polypeptide, dAb fragment, diabody, and other portions known in the art as referred to as antigen-binding fragments. In constructing immunoglobulin molecules or fragments thereof, various regions or portions thereof are, in some embodiments, fused, joined, or otherwise attached to one or more constant regions or portions thereof to produce any of the antibodies or fragments thereof described herein. Thus, in some embodiments, the antigen-binding fragment of any one of the above-described antibodies is a Fab, Fab', Fd, F(ab')2, Fv, scFv, a single-chain binding polypeptide (e.g., an scFv having an Fc portion), or other functional fragment as described herein.

[0148] In some embodiments, antibodies of the present disclosure are of any immunoglobulin class, and thus in some embodiments have a gamma, mu, alpha, delta, or epsilon heavy chain. In some embodiments, the gamma chain is gamma 1, gamma 2, gamma 3, or gamma 4. In some embodiments, the alpha chain is alpha 1 or alpha 2.

[0149] In some embodiments, the antibodies of the present disclosure are IgG immunoglobulins. In some embodiments, the antibodies of the present disclosure are antibodies of any IgG subclass. In some embodiments, the antibodies are IgG1.

[0150] In some embodiments, antibodies of the present disclosure comprise a variable light chain that is either kappa or lambda. In some embodiments, the lambda chain is of any of the subtypes, including, for example, lambda 1, lambda 2, lambda 3, and lambda 4. In some embodiments, the light chain is kappa.

[0151] In some embodiments, the antibodies disclosed herein comprise a human variable framework region and a human constant region. In some embodiments, the antibodies comprise a human light chain variable framework region and a human light chain constant region. In some embodiments, the antibodies comprise a human heavy chain variable framework region and a human heavy chain constant region. In some embodiments, the antibodies comprise a human light chain variable framework region, a human light chain constant region, a human heavy chain variable framework region, and a human heavy chain constant region.

[0152] In some embodiments, the human heavy chain constant region is IgG1 or IgG4, or a fragment thereof. In some embodiments, the heavy chain constant region is human IgG1. An example of an antibody having IgG1 is AB101. AB101 comprises a light chain comprising SEQ ID NO:9 and a heavy chain comprising SEQ ID NO:10, as described in Example 1 below.

[0153] In some embodiments, the heavy chain constant region is a human IgG1 with reduced ADCC function (i.e., an Fc-null antibody). An exemplary Fc-null antibody of the present disclosure is AB102. AB102 comprises a light chain comprising SEQ ID NO:9 and a heavy chain containing the variable regions of AB101, the heavy chain constant region comprising SEQ ID NO:11, an Fc-null form of human IgG1. AB102 is further described in the Examples below.

[0154] In some embodiments, the heavy chain constant region is a human IgG1 modified to enhance ADCC function. An exemplary antibody of the present disclosure with enhanced ADCC function is AB103. AB103 comprises a light chain comprising SEQ ID NO:9 and a heavy chain containing the variable regions of AB101, the heavy chain constant region of which comprises SEQ ID NO:12, an enhanced ADCC form of human IgG1.

[0155] In some embodiments, the heavy chain constant region is human IgG4. An exemplary antibody of the present disclosure having IgG4 is AB104. AB104 comprises a light chain comprising SEQ ID NO: 9 and a heavy chain containing the variable region of AB101 and the heavy chain constant region comprising SEQ ID NO: 13, which is human IgG4.

[0156] In some embodiments, an antibody of the disclosure comprises a human variable framework region and a murine constant region. In some embodiments, an antibody of the disclosure comprises a human heavy chain variable framework region and a murine heavy chain constant region. In some embodiments, an antibody of the disclosure comprises a human light chain variable framework region, a murine light chain constant region, a human heavy chain variable framework region, and a murine heavy chain constant region.

[0157] In some embodiments, the heavy chain constant region is mouse IgG2A. An example of an antibody having mouse IgG2A is AB211. AB211 comprises a light chain comprising SEQ ID NO: 14 and a heavy chain comprising the human variable region of AB101, the heavy chain constant region of which is mouse IgG1 Fc-null and the light chain constant region of which is mouse κ, comprising SEQ ID NO: 15. AB211 is further described in the Examples below.

[0158] In some embodiments, the heavy chain constant region is mouse IgG2A. An example of an antibody having a mouse IgG2A heavy chain is AB212. AB211 comprises a light chain comprising SEQ ID NO: 14 and a heavy chain comprising SEQ ID NO: 16, which contains the human variable region of AB101 and in which the heavy chain constant region is mouse IgG2a and the light chain constant region is mouse kappa. AB212 is further described in the Examples below.

[0159] Binding of the antibody or antigen-binding fragment to the CD163 protein expressed on M2 macrophages partially (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or any number therein) or fully modulates the biological function of M2 macrophages in some embodiments. The activity of the antibody or antigen-binding fragment is determined using in vitro and / or in vivo assays, for example, using art-recognized assays such as those described herein or otherwise known in the art.

[0160] In some embodiments, antibodies of the present disclosure are further modified, if necessary, to alter specific properties of the antibody while retaining desired functionality. For example, in one embodiment, an antibody of the present disclosure is modified to alter the pharmacokinetic properties of the antibody, including, but not limited to, in vivo stability, solubility, bioavailability, or half-life.

[0161] In some embodiments, the dissociation constant (Kd) of the antibodies described herein is about 1 to about 10 pM, about 10 to about 20 pM, about 1 to about 29 pM, about 30 to about 40 pM, about 10 to about 100 pM, or about 20 to about 500 pM.

[0162] In some embodiments, the dissociation constant (Kd) of the antibodies described herein is less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 75 pM, less than about 50 pM, less than about 30 pM, less than about 25 pM, less than about 20 pM, less than about 18 pM, less than about 15 pM, less than about 10 pM, less than about 75 pM, less than about 5 pM, less than about 2.5 pM, or less than about 1 pM.

[0163] In some embodiments, the affinity of the antibodies described herein for a huCD163 protein or peptide is about 10 -9 ~about 10 -14 , about 10 -10 ~about 10 -14 , about 10 -11 ~about 10 -14 , about 10 -12 ~about 10 -14 , about 10 -13 ~about 10 -14 , about 10 -10 ~about 10 -11 , about 10 -11 ~about 10 -12 , about 10 -12 ~about 10 -13 , or 10 -13 ~about 10 -14 is.

[0164] In some embodiments, the antibodies described herein have more than one binding site. In some embodiments, the multiple binding sites are identical to one another. In some embodiments, the multiple binding sites are different from one another. Naturally occurring human immunoglobulins generally have two identical binding sites, while engineered antibodies, for example, have two or more different binding sites.

[0165] In some embodiments, the antibodies of the present disclosure are bispecific or multispecific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies, in some embodiments, bind to two different epitopes of a single antigen. Other such antibodies, in some embodiments, combine a first antigen-binding site with a binding site for a second antigen. In some embodiments, bispecific antibodies bind to at least two different epitopes and have a constant domain that binds to an Fc receptor. In some embodiments, binding of one or more epitopes of the bispecific antibody occurs simultaneously with binding of the constant domain of the bispecific antibody to an Fc receptor.

[0166] In some embodiments, antibodies of the present disclosure have two or more valencies, also referred to as multivalent. In some embodiments, antibodies of the present disclosure are trispecific. In some embodiments, multivalent antibodies are internalized (and / or catabolized) more rapidly than bivalent antibodies by cells expressing the antigen to which the antibody binds. In some embodiments, antibodies of the present disclosure are multivalent antibodies (e.g., tetravalent antibodies) having three or more antigen-binding sites. In some embodiments, multivalent antibodies of the present disclosure are produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. In some embodiments, multivalent antibodies comprise a dimerization domain and three or more antigen-binding sites. In some embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen-binding sites amino-terminal to the Fc region. In some embodiments, multivalent antibodies of the present disclosure comprise about three to about eight, preferably four, antigen-binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain comprises two or more variable regions. For example, the polypeptide chain may comprise VD1-(X1) n -VD2-(X2) n-Fc, where VD1 is a first variable region, VD2 is a second variable region, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. In some embodiments, the polypeptide chains each independently comprise V H -C H 1-Flexible Linker-V H -C H 1 - Fc region chain, or V H -C H 1-V H -C H In some embodiments, the multivalent antibodies herein further comprise at least two (preferably four) light chain variable region polypeptides. In some embodiments, the multivalent antibodies herein comprise from about two to about eight light chain variable region polypeptides. In some embodiments, the light chain variable region polypeptides described herein comprise a light chain variable region. In some embodiments, the light chain variable region polypeptides described herein comprise a C L Further includes domains.

[0167] In some embodiments, antibodies of the present disclosure are constructed to fold into multivalent forms, which in some embodiments improve binding affinity, specificity, and / or serum half-life. Multivalent forms of antibodies are prepared, for example, by techniques known in the art.

[0168] In some embodiments, antibodies of the disclosure are target protein-specific SMIPs or binding domain immunoglobulin fusion proteins. These constructs are single-chain polypeptides comprising an antigen-binding domain fused to immunoglobulin domains necessary to carry out antibody effector functions.

[0169] In some embodiments, antibodies of the present disclosure comprise a heavy chain variable region and / or a single-chain binding polypeptide comprising a light chain variable region that binds to an epitope disclosed herein and optionally comprises an immunoglobulin Fc region. Such molecules are single-chain variable fragments (scFv), which optionally have effector function or increased half-life due to the presence of the immunoglobulin Fc region.

[0170] Anti-CD163 antibody Specific embodiments herein provide antibodies that specifically bind to CD163 protein. In some embodiments, the CD163-binding antibody comprises at least one heavy chain and at least one light chain. In some embodiments, the CD163-binding antibody comprises at least one heavy chain comprising a heavy chain variable domain (VH) and at least one light chain comprising a light chain variable domain (VL). Each VH and VL comprises three complementarity-determining regions (CDRs). The amino acid sequences of the VH and VL and the CDRs determine the antigen-binding specificity and antigen-binding strength of the antibody. The VH and VL domains are summarized in Table 1. The light and heavy chains are summarized in Table 2. The amino acid sequences of the CDRs are summarized in Table 3.

[0171] In some embodiments, the antibodies disclosed herein are monoclonal antibodies. In some embodiments, the antibodies disclosed herein are antigen-binding fragments. In some embodiments, the antibodies disclosed herein are selected from whole immunoglobulins, scFvs, Fabs, F(ab')2s, or disulfide-linked Fvs. In some embodiments, the antibodies disclosed herein are IgGs or IgMs. In some embodiments, the antibodies disclosed herein are humanized. In some embodiments, the antibodies disclosed herein are human, humanized, or chimeric.

[0172] [Table 1]

[0173] In certain embodiments, disclosed herein are antibodies comprising a light chain variable domain (VL) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 7. In some embodiments, the VL has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 7. In some embodiments, the VL has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 7.

[0174] In certain embodiments, disclosed herein are antibodies comprising a heavy chain variable domain (VH) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 8.

[0175] In certain embodiments, disclosed herein are antibodies comprising a light chain variable domain (VL) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:7 and a heavy chain variable domain (VH) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:8. In some embodiments, the VL has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:7, and the VH has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:8. In some embodiments, the VL has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:7, and the VH has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:8.

[0176] [Table 2-1]

[0177] [Table 2-2]

[0178] In certain embodiments, disclosed herein are antibodies comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 9. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 9. In some embodiments, the light chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 9.

[0179] In certain embodiments disclosed herein are antibodies comprising a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 10. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 10. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 10.

[0180] In certain embodiments, disclosed herein are antibodies comprising a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 11. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 11. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 11.

[0181] In certain embodiments, disclosed herein are antibodies comprising a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 12. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 12. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 12.

[0182] In certain embodiments, disclosed herein are antibodies comprising a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 13. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 13. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 13.

[0183] In certain embodiments herein, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:9 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:10. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:10. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:10.

[0184] In certain embodiments herein, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:9 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:11. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:11. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:11.

[0185] In certain embodiments herein, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:9 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:12. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:12. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:12.

[0186] In certain embodiments herein, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:9 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:13. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:13. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:13.

[0187] In certain embodiments, disclosed herein are antibodies comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 14. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 14. In some embodiments, the light chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 14.

[0188] In certain embodiments, disclosed herein are antibodies comprising a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 15. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 15. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 15.

[0189] In certain embodiments, disclosed herein are antibodies comprising a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 16. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 16. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 16.

[0190] In certain embodiments herein, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 14 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 15. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 14, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 15. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO: 14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO: 15.

[0191] In certain embodiments herein, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 14 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 16. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 14, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 16. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO: 14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO: 16.

[0192] [Table 3]

[0193] In certain embodiments, antibodies are disclosed herein that comprise at least one of a light chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 2, and a light chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 3. In some embodiments, antibodies that bind to CD163 include a light chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 2, and a light chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 3. , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:3; and at least one light chain CDR3 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:3. In some embodiments, an antibody that binds to CD163 comprises at least one of a light chain CDR1 having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO: 2, and a light chain CDR3 having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO: 3.

[0194] Disclosed herein in certain embodiments are antibodies comprising at least one of a heavy chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 5, and a heavy chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 6. In some embodiments, an antibody that binds to CD163 comprises a heavy chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 5, and a heavy chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 6. , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:6, and at least one heavy chain CDR3 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:6. In some embodiments, an antibody that binds to CD163 comprises at least one of a heavy chain CDR1 having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:4, a heavy chain CDR2 having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:5, and a heavy chain CDR3 having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:6.

[0195] In certain embodiments of the present specification, an antibody is disclosed that comprises at least one of: a light chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 1; a light chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 2; a light chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 3; a heavy chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 4; a heavy chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 5; and a heavy chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 6.In some embodiments, an antibody that binds CD163 comprises a light chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 1; a light chain CDR2 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 2; a light chain CDR2 having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:3; a light chain having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:3; CDR3, a heavy chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:4; and at least one heavy chain CDR3 having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:6.In some embodiments, an antibody that binds to CD163 comprises at least one of a light chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 2, a light chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 3, a heavy chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 5, and a heavy chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 6.

[0196] Binding affinity and immunoreactivity The binding affinity and / or binding activity of an antibody or its antigen-binding fragment can be improved by modifying the framework region. Any suitable method for modifying a framework region is known in the art and is contemplated herein. The selection of one or more relevant framework amino acid positions for modification depends on various criteria. For example, the relative difference between the amino acid framework residues of donor and acceptor molecules is one of the criteria for selecting relevant framework amino acids for modification. The selection of relevant framework positions for modification using this method has the advantage of avoiding subjective bias in residue determination or any bias in the contribution of residues to CDR binding affinity.

[0197] Binding interactions are manifested as intermolecular contacts with one or more amino acid residues of one or more CDRs in some embodiments. Antigen binding can occur, for example, between a CDR or CDR pair, or optionally, a V H Chain and V L It requires the interaction of up to all six CDRs of a chain.

[0198] The binding affinity and avidity of antibodies or antigen-binding fragments can be measured using surface plasmon resonance (SPR) measurements, AlphaLisa assays, or the equilibrium dissociation constant (KD ) can be measured by flow cytometry.

[0199] As used herein, K D The present specification discloses antibodies that specifically bind to human CD163 having a binding affinity of 0.1 to about 500 nM, about 0.1 to about 100 nM, about 0.1 to about 50 nM, about 0.1 to about 20 nM, about 0.1 to about 10 nM, about 0.1 to about 5 nM, about 0.1 to about 2 nM, about 0.1 to about 1 nM, about 0.1 to about 0.5 nM, about 0.5 to about 1000 nM, about 0.5 to about 500 nM, about 0.5 to about 100 nM, about 0.5 to about 50nM, about 0.5 to about 20nM, about 0.5 to about 10nM, about 0.5 to about 5nM, about 0.5 to about 2nM, about 0.5 to about 1nM, about 1 to about 1000nM , about 1 to about 500 nM, about 1 to about 100 nM, about 1 to about 50 nM, about 1 to about 20 nM, about 1 to about 10 nM, about 1 to about 5 nM, about 1 to about 2 nM, about 2 to about 1000 nM, about 2 to about 500 nM, about 2 to about 100 nM, about 2 to about 50 nM, about 2 to about 20 nM, about 2 to about 10 nM, about 2 to about 5 nM, about 5 to about 1000 nM, about 5 ~about 500nM, about 5 to about 100nM, about 5 to about 50nM, about 5 to about 20nM, about 5 to about 10nM, about 10 to about 1000nM, about 10 to about 500nM, about 10 to about K of about 100 nM, about 10 to about 50 nM, about 10 to about 20 nM, about 20 to about 1000 nM, about 20 to about 500 nM, about 20 to about 100 nM, about 20 to about 50 nM, about 50 to about 1000 nM, about 50 to about 500 nM, about 50 to about 100 nM, about 100 to about 500 nM, about 100 to about 1000 nM, about 500 to about 1000 nM D Disclosed are antibodies that specifically bind to human CD163, in some embodiments, the antibodies have a K of 1.8 nM, 12 nM, 45 nM, or 89 nM. D It specifically binds to human CD163.

[0200] The antibodies disclosed herein bind to the myeloid scavenger receptor CD163, which is highly expressed on tumor-associated macrophages (TAMs) and detected on a variety of tumor cells of different origins. Expression of CD163 in tumor tissue is associated with poor prognosis. The binding affinity of the antibodies disclosed herein to IL-10-polarized M2c macrophages is measured by flow cytometry assay.

[0201] As used herein, K D The present specification discloses antibodies that specifically bind to M2c macrophages having a specific binding affinity of 0.1 to about 500 nM, about 0.1 to about 100 nM, about 0.1 to about 50 nM, about 0.1 to about 20 nM, about 0.1 to about 10 nM, about 0.1 to about 5 nM, about 0.1 to about 2 nM, about 0.1 to about 1 nM, about 0.1 to about 0.5 nM, about 0.5 to about 1000 nM, about 0.5 to about 500 nM, about 0.5 to about 100 nM, about 0.5 to about 50nM, about 0.5 to about 20nM, about 0.5 to about 10nM, about 0.5 to about 5nM, about 0.5 to about 2nM, about 0.5 to about 1nM, about 1 to about 1000nM , about 1 to about 500 nM, about 1 to about 100 nM, about 1 to about 50 nM, about 1 to about 20 nM, about 1 to about 10 nM, about 1 to about 5 nM, about 1 to about 2 nM, about 2 to about 1000 nM, about 2 to about 500 nM, about 2 to about 100 nM, about 2 to about 50 nM, about 2 to about 20 nM, about 2 to about 10 nM, about 2 to about 5 nM, about 5 to about 1000 nM, about 5 ~about 500nM, about 5 to about 100nM, about 5 to about 50nM, about 5 to about 20nM, about 5 to about 10nM, about 10 to about 1000nM, about 10 to about 500nM, about 10 to about K of about 100 nM, about 10 to about 50 nM, about 10 to about 20 nM, about 20 to about 1000 nM, about 20 to about 500 nM, about 20 to about 100 nM, about 20 to about 50 nM, about 50 to about 1000 nM, about 50 to about 500 nM, about 50 to about 100 nM, about 100 to about 500 nM, about 100 to about 1000 nM, about 500 to about 1000 nM D Disclosed is an antibody that specifically binds to M2c macrophages. In some embodiments, the antibody has a K D It specifically binds to M2c macrophages.

[0202] Binding epitope An antibody epitope may be a linear peptide sequence (i.e., "continuous") or may be composed of non-contiguous amino acid sequences (i.e., "conformational" or "discontinuous"). In some embodiments, an antibody recognizes more than one amino acid sequence, and thus an epitope defines more than one distinct amino acid sequence. Epitopes recognized by antibodies are determined, for example, by peptide mapping and sequence analysis techniques well known to those skilled in the art. Binding interactions are manifested as intermolecular contacts with one or more amino acid residues of the CDRs.

[0203]

[0204] Disclosed herein are antibodies that specifically bind to an epitope in human CD163. In some embodiments, the antibodies disclosed herein bind to an epitope comprising a non-contiguous amino acid sequence. In some embodiments, the antibody binds to an epitope in human CD163 comprising the amino acid sequence IGRVNASKGFGHIWLDSVSCQGHEPAI (SEQ ID NO: 18). In some embodiments, the antibody binds to an epitope in human CD163 comprising the amino acid sequence VVCRQLGCGSA (SEQ ID NO: 19). In some embodiments, the antibody binds to an epitope in human CD163 comprising the amino acid sequence WDCKNWQWGGLTCD (SEQ ID NO: 20). In some embodiments, the antibody binds to an epitope in human CD163 comprising the amino acid sequence of SEQ ID NOs: 18-20.

[0205] Also disclosed herein are additional antibodies that specifically bind to the epitopes disclosed herein. These additional antibodies, or antigen-binding fragments thereof, that specifically bind to the epitopes disclosed herein can be identified using techniques known in the art. For example, computational methods for designing epitope-specific antibodies can be used. Nimrod et al., Computational Design of Epitope-Specific Functional Antibodies, Cell Reports 25, 2121-2131, November 20, 2018 (incorporated herein by reference). Another method for identifying antibodies that bind to specific epitopes from a library of antigen-binding antibodies can also be used, such as first incorporating non-standard amino acids (ncAAs) p-benzoyl-L-phenylalanine (pBpa) and p-azido-L-phenylalanine (pAzF) into the target epitope, and then selecting antibodies that cross-react with the incorporated ncAA epitopes after UV irradiation. Because cross-linking occurs only when the antibody and epitope are sufficiently close to each other, this method can efficiently select antibodies that specifically bind to the target epitope. Chen et al., Epitope-directed antibody selection by site-specific photocrosslinking, Science Advances, Vol. 6, no. 14, eaaz7825, 01 Apr 2020 (incorporated herein by reference).

[0206] Antibody Modification Antibodies or antigen-binding fragments thereof are optionally modified using techniques known in the art for various purposes, such as the addition of polyethylene glycol (PEG). In some embodiments, PEG modification (PEGylation) results in one or more of improved circulation time, improved solubility, improved tolerance to proteolysis, reduced antigenicity and immunogenicity, improved bioavailability, reduced toxicity, improved stability, and easier formulation.

[0207] Optionally, if the antigen-binding fragment does not contain an Fc portion, an Fc portion is added (recombinantly) to the fragment to, for example, increase the half-life of the antigen-binding fragment in blood circulation when administered to a subject. Selection of an appropriate Fc region and methods for incorporating such a fragment are known in the art. Incorporation of an IgG Fc region into a polypeptide of interest to increase its circulating half-life without losing its biological activity can be achieved, for example, using conventional techniques in the art. In some embodiments, the Fc portion of the antibody is further modified to increase the half-life of the antigen-binding fragment in blood circulation when administered to a subject. Modifications can be determined, for example, using conventional means in the art.

[0208] Additionally, in some embodiments, antibodies and antigen-binding fragments thereof are produced or expressed so that they do not contain fucose on their complex N-glycoside-linked sugar chains. Removal of fucose from complex N-glycoside-linked sugar chains is known to increase the effector functions of antibodies and antigen-binding fragments, including, but not limited to, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Similarly, an epitope-binding antibody or antigen-binding fragment thereof is optionally attached at its C-terminus to all or a portion of an immunoglobulin heavy chain derived from any of the antibody isotypes, e.g., IgG, IgA, IgE, IgD, and IgM, and isotype subclasses, specifically IgG1, IgG2, IgG3, and IgG4.

[0209] In addition, in some embodiments, the antibodies or antigen-binding fragments described herein are also modified to enable them to cross the blood-brain barrier. Such modifications of the antibodies or antigen-binding fragments described herein enable the treatment of brain diseases such as glioblastoma multiforme (GBM). Exemplary modifications that enable proteins such as antibodies or antigen-binding fragments to cross the blood-brain barrier are described in U.S. Patent Application Publication No. 2007 / 0082380.

[0210] Glycosylation of immunoglobulins is recognized to have profound effects on their effector functions, structural stability, and secretion rate from antibody-producing cells. The carbohydrate groups responsible for these properties are generally attached to the constant (C) regions of antibodies. For example, C H Glycosylation of IgG at asparagine 297 in the C domain is required for the full ability of IgG to activate the classical pathway of complement-dependent cytolysis (Tao and Morrison, J Immunol 143:2595 (1989)). H Glycosylation of IgM at asparagine 402 in the C3 domain is required for proper assembly and cytolytic activity of the antibody (Muraoka and Shulman, J Immunol 142:695 (1989)). H 1 and C HRemoval of glycosylation sites at positions 162 and 419 in the three domains resulted in at least 90% inhibition of intracellular degradation and secretion (Taylor and Wall, Mol Cell Biol 8:4197 (1988)). Additionally, in some embodiments, antibodies and antigen-binding fragments thereof are produced or expressed so that they do not contain fucose on their complex N-glycoside-linked sugar chains. Removal of fucose from complex N-glycoside-linked sugar chains is known to increase the effector functions of antibodies and antigen-binding fragments, including, but not limited to, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). These "defucosylated" antibodies and antigen-binding fragments are produced in some embodiments by a variety of systems utilizing molecular cloning techniques known in the art, including transgenic animals, plants, or cell lines that have been genetically engineered so that they no longer contain the enzymes and biochemical pathways necessary to include fucose in complex N-glycosidically linked sugar chains (also known as fucosyltransferase knockout animals, plants, or cells). Non-limiting examples of cells engineered to be fucosyltransferase knockout cells include CHO cells, SP2 / 0 cells, NS0 cells, and YB2 / 0 cells.

[0211] Glycosylation of immunoglobulins in the variable (V) region has also been observed. Sox and Hod reported that approximately 20% of human antibodies are glycosylated in the V region (Proc Natl Acad Sci USA 66:975 (1970)). Glycosylation of the V domain is thought to result from the occasional occurrence of the N-linked glycosylation signal Asn-Xaa-Ser / Thr in the V region sequence, and is not recognized in the art as playing a role in immunoglobulin function.

[0212] Glycosylation at variable domain framework residues can potentially modify the binding interactions of an antibody with an antigen. The present disclosure includes criteria by which a limited number of amino acids in the framework or CDRs of a humanized immunoglobulin chain are selected to be mutated (e.g., by substitution, deletion, or addition of residues) to increase the affinity of the antibody.

[0213] In some embodiments, cysteine ​​residues are removed or introduced into the Fc region of an antibody or Fc-containing polypeptide, thereby eliminating or increasing interchain disulfide bond formation in this region. Homodimeric specific binding agents or antibodies produced using such methods, in some embodiments, exhibit improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).

[0214] It has been recognized that sequences within the CDRs allow antibodies to bind to MHC class II and, in some cases, induce unwanted helper T cell responses. In some embodiments, conservative substitutions allow the antibody to retain binding activity but reduce its ability to induce unwanted T cell responses. In one embodiment, one or more of the N-terminal 20 amino acids of the heavy or light chain are deleted.

[0215] In some embodiments, antibody molecules are produced with modified carbohydrate structures that result in improved effector activity, including antibody molecules with no or reduced fucosylation that exhibit improved ADCC activity. Various methods for achieving this are known in the art. For example, ADCC effector activity is mediated by binding of antibody molecules to the FcγRIII receptor, which is involved in the binding of C HIt has been shown that the activity of FcγRIII depends on the carbohydrate structure of the N-linked glycosylation at Asn-297 in the 2 domain. Non-fucosylated antibodies bind to this receptor with increased affinity and induce FcγRIII-mediated effector function more efficiently than naturally fucosylated antibodies. Some host cell lines, such as the Lec13 or rat hybridoma YB2 / 0 cell lines, naturally produce antibodies with reduced levels of fucosylation. Increasing the concentration of bisecting sugars, for example, by recombinantly producing antibodies in cells overexpressing the GnTIII enzyme, has also been shown to increase ADCC activity. In some embodiments, the absence of only one of the two fucose residues is sufficient to increase ADCC activity.

[0216] Covalent modifications of antibodies are also included herein. In some embodiments, covalent modifications are made by chemical synthesis, or by enzymatic or chemical cleavage of antibodies, if applicable. In some embodiments, other types of covalent modifications are introduced by reacting targeted amino acid residues with organic derivatizing agents that can react with selected side chains or N- or C-terminal residues.

[0217] Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0218] In some embodiments, histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful in some embodiments. In some embodiments, the reaction is performed in 0.1 M sodium cacodylate at pH 6.0.

[0219] In some embodiments, lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing α-amino-containing residues include imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, 0-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylic acid.

[0220] In some embodiments, arginyl residues are modified by reaction with one or more conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires the reaction to be carried out under alkaline conditions due to the high pKa of the guanidine functional group. Furthermore, these reagents, in some embodiments, react with lysine groups as well as the arginine ε-amino group.

[0221] In some embodiments, the specific modification of tyrosyl residues is carried out for the purpose of introducing spectral labels into tyrosyl residues, particularly by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used in some embodiments to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues can also be reacted with tyrosyl groups to prepare labeled proteins for use in radioimmunoassays. 125 I or 131 It is iodinated using I.

[0222] Carboxyl side groups (aspartyl or glutamyl) are specifically modified by reaction with carbodiimides (RN=C=N-R'), where R and R' are various alkyl groups such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0223] In some embodiments, glutaminyl and asparaginyl residues are deamidated to the corresponding glutamyl and aspartyl residues, respectively, which are deamidated under neutral or basic conditions.

[0224] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0225] Another type of covalent modification involves chemically or enzymatically attaching glycosides to specific binding agents or antibodies. These procedures do not require production of the polypeptide or antibody in a host cell with glycosylation capabilities for N- or O-linked glycosylation. Depending on the attachment mode used, in some embodiments, sugars are attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.

[0226] Removal of any carbohydrate moieties present on a polypeptide or antibody is, in some embodiments, accomplished chemically or enzymatically. Chemical deglycosylation involves exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. In some embodiments, enzymatic cleavage of carbohydrate moieties on antibodies is achieved through the use of various endo- and exoglycosidases.

[0227] Another type of covalent modification involves conjugating the antibody to one of a variety of nonproteinaceous polymers, e.g., polysaccharide polymers such as polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylenes, or dextran. Such methods are known in the art.

[0228] The binding affinity of a predetermined polypeptide to an antigen is generally adjusted by introducing one or more mutations into the V region framework, typically in one or more CDR adjacent regions and / or one or more framework regions.Typically, such mutations involve the introduction of conservative amino acid substitutions that disrupt or create glycosylation site sequences but do not substantially affect the hydrophobic structural characteristics of the polypeptide.Typically, mutations that introduce proline residues are avoided.

[0229] Effector function Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Generally, Fc-mediated functions involve the binding of the Fc portion of an antibody by specialized receptor molecules, "Fc receptors," or "FcRs," expressed by the cells whose functions are affected.

[0230] IgG is considered the most versatile immunoglobulin because, in some embodiments, it performs all the functions of immunoglobulin molecules. IgG is the predominant Ig in serum and the only Ig class that crosses the placenta. IgG also fixes complement, but the IgG4 subclass does not. Macrophages, monocytes, polymorphonuclear leukocytes (PMNs), and some lymphocytes have receptors for the Fc region of IgG. Not all subclasses bind equally. IgG2 and IgG4 do not bind to Fc receptors. Binding to Fc receptors on PMNs, monocytes, and macrophages results in the cells sometimes better internalizing antigens. IgG is an opsonin that enhances phagocytosis. Binding of IgG to Fc receptors on other cell types results in the activation of other functions.

[0231] In one embodiment, the FcR is a native-sequence human FcR. Furthermore, a preferred FcR is one that binds an IgG antibody (a gamma ("γ") receptor) and includes receptors of the FcγI, FcγII, and FcγIII subclasses, as well as allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic domain.

[0232] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" the cytotoxic cells and are required for such killing. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. To assess ADCC activity of a molecule of interest, in some embodiments, an in vitro ADCC assay is performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0233] Alternatively, or additionally, in some embodiments, ADCC activity of the molecule of interest is assessed in vivo, e.g., in an animal model.

[0234] In some embodiments, the antibodies of the present disclosure bind to surface membrane proteins of M2-like macrophages and are internalized by them. This internalization process is thought to be responsible for the observed changes in the functional immunosuppressive characteristics of the cells, i.e., their differentiation from an M2 state to a subtly activated state, without killing these cells or inhibiting their proliferation. In some embodiments, upon internalization, the antibodies reduce the expression of immunosuppressive soluble factors, while increasing the expression of CD4 + Increases the expression of availability factors that stimulate or promote the activity or proliferation of T cells, including helper T cells and cytotoxic lymphocytes.

[0235] In some therapeutic applications, the internalization process is utilized to kill or reduce the activity or proliferation of target cells that express CD163 protein.The number of antibody molecules that are internalized is sufficient or appropriate to kill or inhibit the proliferation of cells, particularly cancer cells.Depending on the potency of antibody or antibody conjugate, in some cases, the uptake of a single antibody molecule into cells is sufficient to kill the target cells to which the antibody binds.For example, certain toxins are so potent in killing that the internalization of one molecule of the toxin conjugated to antibody is sufficient to kill the target cells.

[0236] In some embodiments, the antibody or antigen-binding fragment provided herein is conjugated or bound to a therapeutic moiety, an imaging or detectable moiety, or an affinity tag. Methods for conjugating or binding polypeptides are well known in the art. The association (binding) of a compound with a label includes any means known in the art, including, but not limited to, covalent and non-covalent interactions, chemical bonds, and recombinant techniques. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to or recombinantly engineered with an affinity tag (e.g., a purification tag). For example, an affinity tag, such as a polyhistidine (e.g., His6) tag, is a conventional tag in the art.

[0237] In some embodiments, the antibody or antigen-binding fragment further comprises a detectable moiety. Detection is accomplished, for example, in vitro, in vivo, or ex vivo. For example, in vitro assays for detecting and / or determining (quantitating, qualifying, etc.) huCD163 protein expressed by macrophages using an antibody or its antigen-binding fragment include, but are not limited to, ELISA, RIA, and Western blot. In some embodiments, in vitro detection, diagnosis, or monitoring of an antibody's antigen is performed, for example, by obtaining a sample (e.g., a blood sample) from a subject and testing the sample in a standard ELISA assay.

[0238] Also disclosed herein in certain embodiments are compositions comprising an antibody disclosed herein and a carrier.

[0239] Pharmaceutical Composition In certain embodiments, disclosed herein are pharmaceutical compositions comprising an antibody disclosed herein and a pharmaceutically acceptable excipient.

[0240] Such compositions are useful for in vitro or in vivo analysis or, in the case of pharmaceutical compositions, for administration to a subject in vivo or ex vivo for treatment of the subject with the disclosed antibodies.

[0241] In some embodiments, the excipient is a carrier, buffer, stabilizer, or other suitable material known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration.

[0242] Pharmaceutical formulations comprising antibodies or antigen-binding fragments identified by the methods described herein can, in some embodiments, be prepared for storage by mixing the protein with a desired purity with optional physiologically acceptable carriers, excipients, or stabilizers (see, e.g., Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. Ed. (1980)), frozen preparations, or aqueous solutions. Acceptable carriers, or stabilizers, are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl and propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; gelatin; or immunoglobulins. The pharmaceutical composition may comprise a protein such as globulin, a hydrophilic polymer such as polyvinylpyrrolidone, an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine, a monosaccharide, a disaccharide, and other carbohydrates including glucose, mannose, or dextrin, a chelating agent such as EDTA, a sugar such as sucrose, mannitol, trehalose, or sorbitol, a salt-forming counterion such as sodium, a metal complex (e.g., a Zn-protein complex), and / or a non-ionic surfactant such as TWEEN®, PLURONIC®, or polyethylene glycol (PEG). In one embodiment, the pharmaceutical composition comprises an antibody at a concentration of 5 to 200 mg / mL, preferably 10 to 100 mg / mL.

[0243] An acceptable carrier is physiologically acceptable to the subject to which it is administered and maintains the therapeutic properties of the administered compound. Acceptable carriers and their formulations are generally described, for example, in Remington's Pharmaceutical Sciences, supra. One exemplary carrier is physiological saline. As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, required for the delivery or transport of the subject compound from the administration site in one organ or body part to another, or for an in vitro assay system. Each carrier is acceptable in the sense that it is compatible with the other components of the formulation and is not harmful to the subject to which it is administered. An acceptable carrier also does not modify the specific activity of the subject compound.

[0244] In another embodiment, the pharmaceutical compositions disclosed herein further comprise an acceptable additive to improve the stability of the compound in the composition and / or to control the release rate of the composition. An acceptable additive does not alter the specific activity of the compound of interest. Exemplary acceptable additives include, but are not limited to, mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, and mixtures thereof. In some embodiments, the acceptable additive is combined with an acceptable carrier and / or excipient, such as dextrose. Alternatively, exemplary acceptable additives include, but are not limited to, surfactants, such as polysorbate 20 and polysorbate 80, to increase peptide stability and reduce solution gelation. In some embodiments, surfactants are added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

[0245] In one embodiment, the pharmaceutical compositions disclosed herein contain an isotonic buffer, such as phosphate, acetate, or TRIS, in combination with a tonicity-stabilizing and tonicity-adjusting agent, such as a polyol, sorbitol, sucrose, or sodium chloride. In some embodiments, the tonicity-adjusting agent is present in the composition in an amount of about 5%.

[0246] In another embodiment, the pharmaceutical compositions disclosed herein contain a surfactant to prevent aggregation and stabilize at 0.01-0.02% wt / vol.

[0247] In another embodiment, the pH of the pharmaceutical compositions disclosed herein ranges from 4.5 to 6.5 or 4.5 to 5.5.

[0248] In some embodiments, the pharmaceutical compositions disclosed herein also contain more than one active compound as needed for the indication being treated, such as compounds with complementary activities that do not adversely affect each other. For example, the treatment method further provides an immunosuppressant. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0249] In some embodiments, the active ingredient is encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, respectively, prepared, for example, by coacervation techniques or interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

[0250] Suspension and crystalline forms of antibodies are also contemplated herein. Methods of making suspension and crystalline forms are known to those of skill in the art.

[0251] In some embodiments, the pharmaceutical compositions disclosed herein are sterile.In some embodiments, the pharmaceutical compositions disclosed herein are sterilized by conventional well-known sterilization techniques.For example, sterilization can be easily achieved by filtration through a sterile filtration membrane.In some embodiments, the resulting solution is packaged for use or filtered under aseptic conditions and lyophilized.The lyophilized preparation is combined with a sterile solution before administration.

[0252] In some embodiments, lyophilization is utilized to stabilize the polypeptide for long-term storage, such as when the polypeptide is relatively unstable in a liquid composition.

[0253] In some embodiments, some excipients, such as polyols (including mannitol, sorbitol, and glycerol), sugars (including glucose and sucrose), and amino acids (including alanine, glycine, and glutamic acid), act as stabilizers for lyophilized products. In some embodiments, polyols and sugars are also used to protect polypeptides from damage caused by freezing and drying, and to enhance stability during storage in a dry state. In some embodiments, sugars are effective both during the lyophilization process and during storage. Other classes of molecules, including monosaccharides, disaccharides, and polymers such as PVP, have also been reported as stabilizers for lyophilized products.

[0254] In some embodiments, for injection, the pharmaceutical compositions disclosed herein are powders suitable for reconstitution with an appropriate solution as described above. Examples include, but are not limited to, freeze-dried, rotary-dried, or spray-dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the compositions optionally contain stabilizers, pH adjusters, surfactants, bioavailability adjusters, and combinations thereof.

[0255] In some embodiments, sustained-release preparations are prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (see, e.g., U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods. In some embodiments, encapsulated antibodies persist in the body for extended periods but denature or aggregate upon exposure to moisture at 37°C, resulting in loss of biological activity and possibly altered immunogenicity. Rational strategies devised for stabilization may depend on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular S—S bond formation via thio-disulfide exchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solution, adjusting water content, using appropriate additives, and developing specific polymer matrix compositions.

[0256] In some embodiments, the pharmaceutical compositions disclosed herein are designed to be short-acting, fast-releasing, long-acting, or sustained-releasing as described herein, hi one embodiment, the pharmaceutical compositions disclosed herein are formulated for controlled or sustained release.

[0257] Pharmaceutical compositions are administered by injection, including, but not limited to, subcutaneous, intravitreal, intradermal, intravenous, intraarterial, intraperitoneal, intracerebrospinal, or intramuscular injection. Excipients and carriers used in formulating each type of injectable composition are contemplated herein. The following descriptions are merely exemplary and are not intended to limit the scope of the compositions. Injectable compositions include, but are not limited to, aqueous solutions (where water soluble) or dispersions, as well as sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In some embodiments, the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In some embodiments, isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, are included in the composition. In some embodiments, the resulting solution is packaged for immediate use or lyophilized. In some embodiments, the lyophilized preparation is subsequently combined with a sterile solution before administration. For intravenous injection or injection at the site of pain, the active ingredient is in the form of a pyrogen-free, parenterally acceptable aqueous solution having appropriate pH, isotonicity, and stability. One skilled in the art can prepare an appropriate solution using, for example, an isotonic vehicle such as sodium chloride injection, Ringer's injection, and lactated Ringer's injection. In some embodiments, preservatives, stabilizers, buffers, antioxidants, and / or other additives are included as needed.In some embodiments, sterile injection solution is prepared by incorporating the required amount of active ingredient in suitable solvent with one or combination of above-listed components, and then optionally sterilizing by filtering.Generally, dispersion is prepared by incorporating active ingredient into sterile solvent that contains basic dispersion medium and other components that are required from above-listed components.For the sterile powder that is used to prepare sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying, thereby obtaining the powder of active ingredient that is added with desired additional components from the solution that has been previously sterilized and filtered.

[0258] In some embodiments, the composition is conventionally administered intravenously, for example, by injection of a unit dose. For injection, in some embodiments, the active ingredient is in the form of a parenterally acceptable aqueous solution that is substantially pyrogen-free and has appropriate pH, isotonicity, and stability. In some embodiments, an appropriate solution is prepared using an isotonic solvent, such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. In some embodiments, preservatives, stabilizers, buffers, antioxidants, and / or other additives are included as needed. In addition, in some embodiments, the composition is administered via aerosolization (Lahn et al., Int Arch Allergy Immunol 134:49-55(2004)).

[0259] For parenteral administration, antibodies are formulated in unit-dose injectable forms (solutions, suspensions, emulsions) with pharmaceutically acceptable parenteral solvents. Examples of such solvents include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous solvents such as fixed oils and ethyl oleate are also used. Liposomes are used as carriers. The solvent contains small amounts of additives such as buffers and preservatives to enhance isotonicity and chemical stability. Antibodies are generally formulated in such solvents at a concentration of about 1 mg / mL to 10 mg / mL.

[0260] In one embodiment, the pharmaceutical compositions disclosed herein are lyophilized, for example, to increase shelf life during storage. When the compositions are contemplated for use in the medicaments or methods provided herein, in some embodiments, the compositions are substantially pyrogen-free so as not to cause inflammatory or dangerous allergic reactions when administered to a human subject. Testing compositions for pyrogens and preparing substantially pyrogen-free compositions are well understood by those skilled in the art and, in some embodiments, are accomplished using commercially available kits.

[0261] In some embodiments, acceptable carriers contain compounds that stabilize, increase, or delay absorption or clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose, and dextrans; compositions that reduce the clearance or hydrolysis of low-molecular-weight proteins and peptides; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. In some embodiments, detergents are also used to stabilize, increase, or decrease absorption of pharmaceutical compositions containing liposomal carriers. In some embodiments, to protect against digestion, the compound is complexed with the composition to confer resistance to acidic and enzymatic hydrolysis, or in some embodiments, the compound is complexed in a carrier that exhibits appropriate resistance, such as a liposome. Means for protecting compounds from digestion are known in the art.

[0262] In some embodiments, the composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.The amount administered depends on the subject being treated, the ability of the subject's immune system to utilize the active ingredient, and the desired degree of binding capacity.The exact amount of active ingredient required for administration depends on the judgment of the medical professional and is unique to each individual.Appropriate regimens for initial administration and booster shots are also variable, but feature an initial administration, followed by repeated administrations at intervals of one hour or more by subsequent injections or other administrations.Alternatively, continuous intravenous infusion sufficient to maintain blood concentration is contemplated.

[0263] In some embodiments, the present disclosure provides use of a composition described herein to make a medicament for treating a disease, condition, or disorder described herein. In some embodiments, the medicament is formulated based on the physical characteristics of the subject requiring treatment and in single or multiple formulations based on the stage of the disease, condition, or disorder. In some embodiments, the medicament is packaged in suitable packaging with suitable labeling for distribution to hospitals and clinics, the labeling relating to instructions for treating subjects with a disease described herein. In some embodiments, the medicament is packaged as a single or multiple unit. Instructions for dosage and administration of the composition are, in some embodiments, included with the package, as described below. The present disclosure is further directed to a medicament comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0264] In some embodiments, the composition (antibody or antigen-binding fragment described herein) is administered alone or in combination with a second composition, either simultaneously or sequentially, depending on the disease being treated. In one embodiment, the second therapeutic treatment is an anti-cancer therapy or anti-cancer therapeutic. When two or more compositions are administered, the compositions are administered, for example, in combination (sequentially or simultaneously). In some embodiments, the compositions are administered in a single dose or multiple doses.

[0265] In some embodiments, when formulated for administration to a human subject, the compositions are formulated to be pyrogen-free. Testing of compositions for pyrogens and preparation of pyrogen-free pharmaceutical compositions are well understood to those of skill in the art.

[0266] In some embodiments, the antibody or antigen-binding fragment thereof is formulated for any suitable route of administration to a subject, including, but not limited to, injection. Injection includes, for example, subcutaneous injection, peritoneal injection, intravenous injection, intramuscular injection, or spinal injection into cerebrospinal fluid (CSF). In some embodiments, administration is performed at 1, 2, 3, 4, 5, 6, 7, or more injection sites. In one embodiment, administration is performed via 6 injection sites.

[0267] In in vivo applications, contacting occurs, for example, via administration of a composition (as described herein) to a subject by any suitable means. In some embodiments, the antibodies described herein are administered by suitable means, such as systemic or local administration, including parenteral, subcutaneous, intraperitoneal, intracerebrospinal, intrapulmonary, and intranasal administration, and, where necessary, local treatment, intralesional administration. Parenteral routes include, for example, intravenous, intraarterial, intraperitoneal, epidural, intramuscular, and intrathecal administration. In some embodiments, such administration is performed as a bolus injection, continuous infusion, or pulse infusion. In some embodiments, the composition is administered by injection, depending in part on whether the administration is brief or chronic. Other methods of administration are contemplated, including local administration, particularly transdermal, transmucosal, rectal, oral, or topical administration, e.g., via a catheter placed near the desired site.

[0268] Methods of Treatment and Use In certain embodiments, disclosed herein are methods for treating cancer in an individual in need thereof, the methods comprising administering to the individual an antibody disclosed herein. In some embodiments, the present disclosure provides the use of an antibody as disclosed herein in the manufacture of a medicament for treating cancer in a human subject. In some embodiments, the antibody specifically binds to CD163 protein expressed on human tumor-associated macrophages and reduces the expression of at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophages.

[0269] In certain embodiments, disclosed herein are methods of modulating immune activity in a subject in need thereof, the method comprising administering to the subject an antibody described herein. In some embodiments, the antibody specifically binds to CD163 protein expressed on human tumor-associated macrophages and reduces expression of at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophages.

[0270] In certain embodiments, disclosed herein are methods of treating a subject with pathologically or inappropriately elevated levels of M2 macrophages (e.g., inappropriately elevated levels relative to levels useful for promoting immune-mediated tumor cell killing in the subject), the methods comprising administering to the subject an antibody described herein. In some embodiments, the antibody specifically binds to the CD163 protein expressed on human tumor-associated macrophages and reduces expression of at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophages.

[0271] In some embodiments, the antibody comprises at least one of a light chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 2, and a light chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 3. In some embodiments, the antibody comprises at least one of a light chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 2, and a light chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 3. and at least one light chain CDR3 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 3. In some embodiments, the antibody comprises at least one light chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 2, and a light chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 3.

[0272] In some embodiments, the antibody comprises at least one of a heavy chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 5, and a heavy chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 6. In some embodiments, the antibody comprises at least one of a heavy chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 5, and a heavy chain CDR3 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 4, a heavy chain CDR3 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 5, and at least one heavy chain CDR3 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 6. In some embodiments, the antibody comprises at least one heavy chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 5, and a heavy chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 6.

[0273] In some embodiments, the antibody disclosed comprises at least one of a light chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 2, a light chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 3, a heavy chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 5, and a heavy chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 6.In some embodiments, the antibody comprises a light chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:1, a light chain CDR2 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:2, , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:3; a heavy chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:4; a heavy chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, to the amino acid sequence set forth as SEQ ID NO:5; and at least one heavy chain CDR3 having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:6.In some embodiments, the antibody comprises at least one of a light chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 2, a light chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 3, a heavy chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 5, and a heavy chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 6.

[0274] In some embodiments, the antibody comprises a light chain variable domain (VL) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 7. In some embodiments, the VL has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 7. In some embodiments, the VL has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 7.

[0275] In some embodiments, the antibody comprises a heavy chain variable domain (VH) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 8.

[0276] In some embodiments, the antibody comprises a light chain variable domain (VL) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:7, and a heavy chain variable domain (VH) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:8. In some embodiments, the VL has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:7, and the VH has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:8. In some embodiments, the VL has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:7, and the VH has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:8.

[0277] In some embodiments, the antibody comprises a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 9. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 9. In some embodiments, the light chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 9.

[0278] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 10. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 10. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 10.

[0279] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 11. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 11. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 11.

[0280] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 12. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 12. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 12.

[0281] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 13. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 13. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 13.

[0282] In some embodiments, the antibody comprises a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:9 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:10. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:10. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:10.

[0283] In some embodiments, the antibody comprises a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:9 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:11. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:11. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:11.

[0284] In some embodiments, the antibody comprises a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:9 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:12. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:12. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:12.

[0285] In some embodiments, the antibody comprises a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:9 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO:13. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:13. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:13.

[0286] In some embodiments, the antibody comprises a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 14. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 14. In some embodiments, the light chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 14.

[0287] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 15. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 15. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 15.

[0288] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 16. In some embodiments, the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 16. In some embodiments, the heavy chain has an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 16.

[0289] Also in some embodiments, the antibody comprises a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 14 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 15. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 14, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 15. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO: 14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO: 15.

[0290] In some embodiments, the antibody comprises a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 14 and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth as SEQ ID NO: 16. In some embodiments, the light chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 14, and the heavy chain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 16. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO: 14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO: 16.

[0291] In some embodiments, the disclosure provides for the use of an antibody described herein in the manufacture of a medicament for reducing immunosuppression by tumor-associated macrophages in a human subject with cancer.

[0292] In some embodiments, the disclosure provides for the use of an antibody described herein in the manufacture of a medicament for promoting T cell-mediated tumor cell killing in a human subject with cancer.

[0293] In some embodiments, the disclosure provides a method of treating a human subject with cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody described herein, thereby reducing immunosuppression by tumor-associated macrophages in the subject.

[0294] In some embodiments, the present disclosure provides a method of treating a human subject with cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody described herein, thereby increasing T cell-mediated tumor cell killing in the subject.

[0295] In certain embodiments, disclosed herein are methods for functionally redirecting tumor-associated macrophages to reduce immunosuppression in cancer patients, the methods comprising: + or CD8 + The method includes administering to the patient an amount of a pharmaceutical composition comprising an antibody described herein effective to improve T cell activity or proliferation.

[0296] In certain embodiments, disclosed herein are methods of promoting lymphocyte-mediated tumor cell killing in a human subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody described herein.

[0297] In certain embodiments, disclosed herein are methods for modulating the activity of tumor-associated macrophages in a tumor microenvironment, the methods comprising contacting tumor-associated macrophages with an antibody disclosed herein, wherein the method has the following effects: (a) a decrease in the expression by human macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) antibody internalization by human macrophages; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; (d) CD4 + T cells, CD8 + Expansion of T cells, NK cells, or any combination thereof; and (e) Promoting tumor cell death in the tumor microenvironment This results in at least one of the following:

[0298] In certain embodiments, disclosed herein are methods for modulating the activity of tumor-associated macrophages in a tumor microenvironment, the methods comprising contacting tumor-associated macrophages with an antibody disclosed herein, wherein the method has the following effects: (a) a decrease in the expression by human macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) antibody internalization by human macrophages; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; (d) CD4 + T cells, CD8 + Expansion of T cells, NK cells, or any combination thereof; and (e) Promoting tumor cell death in the tumor microenvironment This will result in at least two of the following:

[0299] In certain embodiments, disclosed herein are methods for modulating the activity of tumor-associated macrophages in a tumor microenvironment, the methods comprising contacting tumor-associated macrophages with an antibody disclosed herein, wherein the method has the following effects: (a) a decrease in the expression by human macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) antibody internalization by human macrophages; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; (d) CD4 + T cells, CD8 + Expansion of T cells, NK cells, or any combination thereof; and (e) Promoting tumor cell death in the tumor microenvironment This brings about at least three of the following:

[0300] In certain embodiments, disclosed herein are methods for modulating the activity of tumor-associated macrophages in a tumor microenvironment, the methods comprising contacting tumor-associated macrophages with an antibody disclosed herein, wherein the method has the following effects: (a) a decrease in the expression by human macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) antibody internalization by human macrophages; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; (d) CD4 + T cells, CD8 + Expansion of T cells, NK cells, or any combination thereof; and (e) Promoting tumor cell death in the tumor microenvironment This brings at least four of the following:

[0301] In certain embodiments, disclosed herein are methods for modulating the activity of tumor-associated macrophages in a tumor microenvironment, the methods comprising contacting tumor-associated macrophages with an antibody disclosed herein, wherein the method has the following effects: (a) a decrease in the expression by human macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) antibody internalization by human macrophages; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; (d) CD4 + T cells, CD8 + Expansion of T cells, NK cells, or any combination thereof; and (e) Promoting tumor cell death in the tumor microenvironment Bring at least five of them.

[0302] In some embodiments, the methods disclosed herein reduce myeloid suppression of CD8 T cell activation and proliferation.

[0303] M2-to-M1 macrophage polarization refers to the process by which M2 or M2-like macrophages of the present disclosure are modified such that the resulting macrophages have specific functional or phenotypic characteristics associated with M1 or M1-like macrophages. In some embodiments, M2 or M2-like macrophages are polarized when they no longer express CD163.

[0304] In some embodiments, the antibody reduces myeloid suppression of CD19-CD3 BiTE-mediated killing of Raji cells by CD8 T cells.

[0305] In some embodiments, the antibody reduces myeloid suppression of CAR T cell-mediated killing of cancer cells.

[0306] In some embodiments, the antibody reduces myeloid cell suppression of NK cell-mediated cancer cell killing by ADCC.

[0307] Any of the methods disclosed herein, in some examples, further comprise administering to the subject an additional anti-cancer therapy, including, but not limited to, surgery, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, and cytokine therapy, and combinations thereof. In one embodiment, the antibody or antigen-binding fragment thereof and the anti-cancer therapy are administered simultaneously or sequentially.

[0308] In some embodiments, the additional anti-cancer therapy is an immunotherapy. In some embodiments, the immunotherapy is a composition comprising a checkpoint inhibitor. In some embodiments, the additional anti-cancer therapy is an immune checkpoint inhibitor.

[0309] In some embodiments, the present disclosure provides an in vitro or ex vivo method for identifying huCD163-expressing macrophages in a cell sample suspected of containing huCD163-expressing macrophages, the method comprising contacting the cell sample with an antibody described herein and measuring binding to cells in the sample, which is a positive signal indicating the presence of huCD163-expressing macrophages in the sample.

[0310] In some embodiments, the present disclosure provides methods for identifying human M2c macrophages in a cell sample, the methods comprising contacting a cell sample containing blood cells suspected of containing human M2c macrophages with an antibody described herein and measuring binding to cells in the sample, which is a positive signal indicating the presence of human M2c-expressing macrophages in the sample. In some embodiments, the cell sample comprises cells obtained from a human subject. In some embodiments, the cell sample comprises cultured cells. In some embodiments, the methods for detecting M2 cells in a sample comprise using an antibody or fragment that is not internalized by the cells upon binding but remains bound to the exterior of the cells to facilitate detection.

[0311] In some embodiments, the present disclosure provides methods for identifying huCD163-expressing cancer cells in a cell sample, the method comprising contacting a cell sample containing cells suspected of containing huCD163-expressing cancer cells with an antibody described herein and measuring binding to cells in the sample, which is a positive signal indicating the presence of huCD163-expressing cancer cells in the sample. In some embodiments, the cell sample comprises cells obtained from a human subject. In some embodiments, the cell sample comprises cultured cells.

[0312] In some embodiments, the present disclosure provides methods for modulating the expression of cell surface markers on M2c macrophages. In some embodiments, the expression of at least one of CD16 (FcγRIIIa), CD64 (FcγRI), Siglec-15, and TLR2 is reduced. In some embodiments, the expression of CD16, CD64, Siglec-15, and TLR2 is reduced.

[0313] In some embodiments, the disclosure provides a method for reducing myeloid suppression of T cell activation. In some embodiments, the method induces increased IL-2 production by T cells. In some embodiments, the disclosure provides a method for increasing Th1 cell proliferation. In some embodiments, the disclosure provides a method for increasing the proportion of Th1 cells in proliferating T cells. In each of these aspects, the method comprises administering an antibody described herein in vivo or contacting the antibody with a combined immune cell system in vivo or ex vivo, wherein macrophages, effector T cells, and optional tumor target cells are allowed to interact in a manner that recapitulates or models their interactions in vivo.

[0314] In some embodiments, the present disclosure provides methods for reducing myeloid suppression of T cell proliferation. + Cell proliferation or CD8 + and / or increasing T cell proliferation.

[0315] In some embodiments, the present disclosure provides methods for increasing T cell expression of at least one of CD69, ICOS, OX40, PD-1, LAG3, and CTLA4 in a patient in need thereof. + In some embodiments, the method increases expression of at least one of CD69, ICOS, OX40, PD-1, LAG3, and CTLA4 by T cells. +Increases expression of at least one of ICOS, OX40, PD-1, LAG3, and CTLA4 by T cells.

[0316] In some embodiments, the present disclosure provides methods for increasing cancer cell killing. In some embodiments, cytotoxic lymphocyte (CTL)-mediated cancer cell killing is increased. In some embodiments, T cell-mediated killing of MHC-mismatched cancer cells is increased.

[0317] In some embodiments, the present disclosure provides a method for detecting CD8 + T cell activation or CD8 + Methods are provided for contacting bone marrow cells with the antibodies described herein to reduce macrophage-mediated suppression of T cell proliferation. In some embodiments, the antibody is contacted with bone marrow cells comprising M0 macrophages. In some embodiments, the antibody is contacted with bone marrow cells comprising M2 macrophages. In some embodiments, the antibody is contacted with bone marrow cells comprising M0 and M2 macrophages.

[0318] In some embodiments, the present disclosure provides a method of treating a human patient with lung cancer, the method comprising administering to the patient an effective amount of an antibody described herein. In some embodiments, the lung cancer is a carcinoma or adenocarcinoma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0319] An effective response of the present disclosure is achieved when a subject experiences partial or complete alleviation or reduction of signs or symptoms of the disease, and in the case of cancer treatment, specifically includes, but is not limited to, cure, remission, prolonged survival, or other objective response. In some embodiments, expected progression-free survival is measured in months to years, depending on prognostic factors, including the number of recurrences, the stage of the disease, and other factors. Prolonged survival includes, but is not limited to, at least 1 month (mo), at least about 2 mos, at least about 3 mos, at least about 4 mos, at least about 6 mos, at least 1 year, at least 2 years, at least 3 years, etc. Overall survival is also measured, for example, in months to years. Alternatively, in some embodiments, an effective response is when the subject's symptoms remain static. Further indications for treatment of an indication are described in detail below.

[0320] In some embodiments, administration of a therapeutic agent in a prophylactic method occurs before symptoms of the undesired disease or disorder appear, thereby preventing, or alternatively slowing, the progression of, the disease or disorder. Thus, when used in conjunction with a prophylactic method, the term "therapeutically effective" means that, following treatment, fewer subjects (on average) will develop the undesired disease or disorder or progress to a more severe form of the symptom.

[0321] In some embodiments, the amount of active ingredient in the composition, the composition formulation, and the mode of administration are among the factors that are varied to provide an amount of the active ingredient effective to achieve a desired therapeutic response for each subject without being excessively toxic to the subject. The selected dosage value will depend on a variety of factors, including the activity of the particular compound employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, diet, and medical history of the subject being treated, and similar factors well known in the medical field.

[0322] In some embodiments, the antibodies and antigen-binding fragments described herein are administered to subjects at various dosages and over various time periods. Non-limiting dosages include about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, or any integer therebetween. In addition, in some embodiments, the antibodies or antigen-binding fragments are administered twice a week, every week, every two weeks, every three weeks, every four weeks, every six weeks, every eight weeks, every twelve weeks, or any combination thereof. For example, administration cycles such as administering an antibody or antigen-binding fragment thereof once or twice weekly for four weeks, followed by two weeks without treatment, are also contemplated. Additional administration cycles, including, for example, different combinations of the doses and weekly cycles described herein, are also contemplated within the scope of this disclosure.

[0323] In some embodiments, the therapeutically effective amount of the composition will vary and depend on the severity of the disease and the weight and general condition of the subject being treated, but will generally be in the range of about 1.0 μg / kg to about 100 mg / kg, about 10 μg / kg to about 30 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 10 mg / kg per application. Administration may be daily, every other day, weekly, twice monthly, monthly, or more or less frequently, as needed, depending on the response to the disorder or condition and the subject's tolerance to treatment. In some embodiments, extended maintenance administration over a period of 4, 5, 6, 7, 8, 10, 12 weeks or more is required until the desired suppression of disorder symptoms occurs, with dosage adjustments made as necessary. The progress of this therapy is easily monitored by conventional techniques and assays.

[0324] In some embodiments, antibodies of the disclosure are administered intravenously in physiological solution at a frequency of daily to weekly to monthly (e.g., daily, every other day, every three days, or 2, 3, 4, 5, or 6 times per week) at a dose ranging from 0.01 mg / kg to 100 mg / kg, preferably 2 or 3 times per week at a dose ranging from 0.1 to 45 mg / kg, 0.1 to 15 mg / kg, or 0.1 to 10 mg / kg, or up to 45 mg / kg once per month.

[0325] A response is achieved when a subject experiences partial or complete alleviation or reduction in signs or symptoms of the disease, including, but not limited to, prolonged survival. Expected progression-free survival is measured in months to years, depending on prognostic factors, including, for example, the number of recurrences, the stage of the disease, and other factors. Prolonged survival includes, but is not limited to, periods of at least 1 month (mo), at least about 2 months (mos), at least about 3 mos, at least about 4 mos, at least about 6 mos, at least 1 year, at least 2 years, at least 3 years, or longer. In some embodiments, overall survival is also measured in months to years. In some embodiments, the subject's symptoms remain static or decrease.

[0326] In some cases, a physician or veterinarian, including those skilled in the art, will determine the effective amount (ED) of the composition required. 50 ) can be easily determined and prescribed. For example, a physician or veterinarian can start administering a compound utilized in a composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Alternatively, in some embodiments, the dose remains constant.

[0327] In some embodiments, other antibodies, small molecule therapeutic agents, and / or other agents are combined in separate compositions for simultaneous or sequential administration. In one embodiment, simultaneous administration includes one or more compositions administered simultaneously or within 30 minutes of each other. In some embodiments, administration occurs at the same site or at different sites.

[0328] In some embodiments, the toxicity and therapeutic efficacy of such components can be measured, e.g., by LD 50 (lethal dose for 50% of the population) and ED 50 The LD (the dose therapeutically effective in 50% of the population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. In some embodiments, the dose ratio between toxic and therapeutic effects is the therapeutic index, which is defined as the LD 50 / ED 50 In some embodiments, compounds that exhibit toxic side effects are used, but care must be taken to design delivery systems that target such compounds to the affected tissue site in order to minimize potential damage to healthy cells, thereby reducing side effects.

[0329] The data obtained from cell culture assays and animal studies are, in some embodiments, used to formulate a range of dosages for use in humans. The dose of such compounds can be adjusted to achieve the ED400 with little or no toxicity. 50 In some embodiments, the dosage will vary within this range depending on the dosage form utilized and the route of administration used. For any compound used in the methods of the present disclosure, the therapeutically effective dose is, in some embodiments, initially estimated from cell culture assays. In some embodiments, the dose is determined to be greater than or equal to the IC50 or IC60 as determined in cell culture. 50 The compound is formulated in an animal model to achieve a circulating plasma concentration range (i.e., the concentration of the test compound that achieves half-maximal inhibition) comprising: Plasma levels are measured, for example, by high performance liquid chromatography. In some cases, such information is used to more accurately determine useful doses in humans.

[0330] In some embodiments, the present disclosure provides a method of treating a cancer patient, the method comprising administering to the patient a therapeutically effective amount of an antibody described herein, and further comprising treating the subject with an anti-cancer therapy selected from surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. In some embodiments, the antibody or antigen-binding fragment thereof and another anti-cancer therapy are administered simultaneously or sequentially.

[0331] Diagnostic Products and Methods In some embodiments, disclosed herein are methods for detecting huCD163 protein or M2 macrophages in a sample or subject to assess a patient's treatment status or to diagnose a disease or disorder associated with or correlated with M2 macrophage or TAM activity, such as the diseases and disorders described herein.

[0332] For in vivo detection, diagnosis, or monitoring of soluble huCD163 protein, expression of huCD163 protein by cells or tissues, or the presence or activity of M2 macrophages, a subject is administered an antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment is conjugated to a detectable moiety. In some embodiments, the detectable moiety is visualized using art-recognized methods, including, but not limited to, magnetic resonance imaging (MRI), fluorescence, radioimaging, a light source provided by an endoscope, laparoscope, or intravascular catheter (i.e., via detection of a photoactive agent), optical scanning, positron emission tomography (PET) scanning, whole-body nuclear magnetic resonance (NMR), radioscintigraphy, single-photon emission computed tomography (SPECT), targeted near-infrared (NIR) scanning, X-ray, ultrasound, and the like. Labels for detecting compounds using such methods are also known in the art. In some embodiments, visualization of the detectable moiety enables detection, diagnosis, and / or monitoring of a disease or disorder associated with M2 macrophage activity or the activity of another cell expressing huCD163 protein. Additional diagnostic assays utilizing antibodies specific for the desired target protein, ie, huCD163 protein, are known in the art and are also contemplated herein.

[0333] For in vitro detection methods, samples obtained from a subject include, but are not limited to, blood, tissue biopsies, and fluids derived therefrom.

[0334] Thus, the present disclosure provides antibodies and antigen-binding fragments thereof that are useful for detecting or diagnosing levels of M2 or TAM macrophages associated with a disease or disorder and may indicate the need for therapeutic treatment. In other embodiments, the antibodies further comprise a second agent. In some embodiments, such an agent is a molecule or moiety, such as a reporter molecule or a detectable label. Detectable labels / moieties for such detection methods are known in the art and are described in more detail below. A reporter molecule is any moiety that can be detected, for example, using an assay. Non-limiting examples of reporter molecules conjugated to a polypeptide include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands such as biotin, etc. In some embodiments, detectable labels comprise compounds and / or elements that are detectable due to their specific functional and / or chemical properties, allowing the polypeptide to which they are attached to be detected and / or further quantified, if desired. Many suitable detectable (imaging) agents are known in the art, as are methods for attaching them to polypeptides.

[0335] In some embodiments, the polypeptides are conjugated to a wide range of fluorescent dyes, quenchers, and haptens, such as fluorescein, R-phycoerythrin, and biotin, hi some embodiments, conjugation occurs either during polypeptide synthesis or after the polypeptide has been synthesized and purified.

[0336] Alternatively, in some embodiments, antibodies, antigen-binding fragments, or binding proteins are conjugated with fluorescent moieties.Conjugating polypeptides with fluorescent moieties (e.g., R-phycoerythrin, fluorescein isothiocyanate (FITC) and the like) can be achieved, for example, by using art-recognized techniques.Many commercially available fluorescent dyes and dye conjugation kits are available for specific applications, such as fluorescence microscopy, flow cytometry, and fluorescence-activated cell sorting (FACS).

[0337] In one non-limiting embodiment, the antibody or antigen-binding fragment is conjugated to a detectable label, such as a radionuclide, dye, contrast agent, or fluorescent agent, for immunodetection of binding to the antigen used to visualize antibody binding to M2 macrophages, or soluble or bound huCD163, in vitro and / or in vivo.

[0338] Non-limiting examples of radiolabels include, for example: 32 P, 33 P, 43 K. 52 Fe, 57 Co, 64 Cu, 67 Ga, 67 Cu, 68 Ga, 71 Ge, 75 Br, 76 Br, 77 Br, 77 As, 77 Br, 81 Rb / 81m Kr, 87m Sr, 90 Y, 97 Ru, 99 Tc, 99m Tc, 100 Pd, 101 Rh, 103 Pb, 105 Rh, 109 Pd, 111 Ag, 111 In, 113 In, 119 Sb, 121Sn, 123 I, 125 I, 127 Cs, 128 Ba, 129 Cs, 131 I, 131 Cs, 143 Pr, 153 Sm, 161 Tb, 166 Ho, 169 EU, 177 Lu, 186 Re, 188 Re, 189 Re, 191 Os, 193 Pt, 194 Ir, 197 Hg, 199 Au, 203 Pb, 211 At, 212 Pb, 212 Bi, and 213 In some embodiments, the radiolabel is attached to the compound using conventional chemistry known in the art of antibody imaging. Radiolabeled compounds are useful in in vitro diagnostic techniques, in vivo radioimaging techniques, and radioimmunotherapy.

[0339] The antibody and antigen-binding fragment compositions described herein are also used, in some embodiments, as non-therapeutic agents (eg, affinity purification agents).

[0340] antibody technology As will be appreciated by those skilled in the art, the general description herein of antibodies and methods of preparing and using same also applies to the individual antibody polypeptide components and antibody fragments.

[0341] The antibodies of the present disclosure may be polyclonal or monoclonal. However, in preferred embodiments, they are monoclonal. In certain embodiments, the antibodies of the present disclosure are human antibodies. Methods for producing polyclonal and monoclonal antibodies are known in the art.

[0342] Antibodies, antigen-binding fragments, and other proteins that bind to huCD163 expressed by M2 macrophages are generated using such methods and, in some embodiments, tested for one or more of their binding affinity, binding activity, and modulatory ability.

[0343] In some embodiments, conventional methods are used to identify antibodies or antigen-binding fragments thereof that bind to huCD163 protein. In some embodiments, antibodies and antigen-binding fragments are evaluated for one or more of binding affinity, association rate, dissociation rate, and binding activity. Measurement of such parameters is achieved using binding assays, competitive binding assays, and the like, including, but not limited to, enzyme-linked immunosorbent assay (ELISA), ELISpot assay, Scatchard analysis, surface plasmon resonance (e.g., BIACORE) analysis, and the like. In one non-limiting embodiment, an ELISA assay is used to measure the binding ability of specific antibodies or antigen-binding fragments that bind to huCD163 protein. Surface plasmon resonance technology is described in Liljeblad et al., Glyco J 17:323-9 (2000).

[0344] In some embodiments, antibodies according to the present disclosure are recombinantly produced using vectors and methods available in the art, as detailed below. In some embodiments, human antibodies are also produced by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275).

[0345] In some embodiments, human antibodies are produced in transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, chimeric and germ-line mutant mouse antibody heavy chain joining regions (J HIt has been described that homozygous deletion of the IL-16A / IL-16B gene results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc Natl Acad Sci USA, 90:2551 (1993); Jakobovits et al., Nature 362:255-58 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807, and WO 97 / 17852. In some embodiments, such animals are genetically engineered to produce human antibodies comprising a polypeptide of the present disclosure.

[0346] Antibodies are isolated and purified from culture supernatants or ascites fluid (if produced in animals) using methods known to those skilled in the art, such as, for example, saturated ammonium sulfate precipitation, euglobulin precipitation, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52), or affinity chromatography using an anti-Ig column or a protein A, G, or L column.

[0347] As mentioned above, the present disclosure further provides antibody fragments. In certain situations, there are advantages to using antibody fragments rather than whole antibodies. For example, the smaller size of fragments allows for rapid clearance, which in some embodiments improves access to certain tissues, such as solid tumors. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments.

[0348] Various techniques have been developed to produce antibody fragments. Traditionally, these fragments were obtained via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J Biochem Biophys Methods 1992 24(1-2):107-17, and Brennan et al., Science 1985 229:81). However, these fragments, in some embodiments, have been produced directly by recombinant host cells. In some embodiments, Fab, Fv, and Scfv antibody fragments are all expressed in and secreted from E. coli, allowing for the facile production of large amounts of these fragments. In some embodiments, Fab'-SH fragments are directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167(1992)). According to another approach, in some embodiments, F(ab')2 fragments are directly isolated from recombinant host cell culture. The C of the Fc region of IgG is H Fab and F(ab')2 fragments containing salvage receptor binding epitopes derived from two loops of two domains and having increased in vivo half-lives are described in U.S. Patent Nos. 5,869,046 and 6,121,022. Techniques for producing other antibody fragments will be apparent to those skilled in the art.

[0349] In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 93 / 16185, U.S. Patent Nos. 5,571,894, and 5,587,458. Fv and sFv are the only species with intact binding sites that lack constant regions. This makes them suitable for reduced nonspecific binding during in vivo use. In some embodiments, sFv fusion proteins are constructed to provide fusion of an effector protein at either the amino or carboxy terminus of the sFv. See Antibody Engineering, ed. Borrebaeck, supra. In some embodiments, the antibody fragment is also a "linear antibody," e.g., as described in U.S. Patent No. 5,641,870. In some embodiments, such linear antibody fragments are monospecific or bispecific.

[0350] Methods for producing bispecific or other multispecific antibodies are known in the art and include chemical cross-linking, use of leucine zippers (Kostelny et al., J Immunol 148:1547-53 (1992)), diabody technology (Hollinger et al., Proc Natl Acad Sci USA 90:6444-8 (1993)), scFv dimers (Gruber et al., J Immunol 152:5368 (1994)), linear antibodies (Zapata et al., Protein Eng 8:1057-62 (1995)), and chelating recombinant antibodies (Neri et al., J Mol Biol 246:367-73 (1995)).

[0351] Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy and light chain pairs, where the specificities of the two chains differ (Millstein et al., Nature 305:537-9 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a mixture of potentially 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is achieved, for example, by affinity chromatography. Similar procedures are disclosed in WO93 / 08829 and Traunecker et al., EMBO J 10:3655-9 (1991).

[0352] According to another approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion includes at least part of the hinge, C H 2, and C H The Ig heavy chain constant domain contains three regions: the first heavy chain constant region (C) with the site necessary for light chain binding; H Preferably, 1) is present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, optionally, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host cell. This allows for greater flexibility in adjusting the ratio of the four polypeptide fragments to each other in embodiments where unequal ratios of the four polypeptide chains used in the construct result in optimal yields of the desired bispecific antibody. However, when expression of at least two polypeptide chains in equal ratios results in high yields, or when the ratio has no significant effect on the yield of the desired chain combination, it is possible to insert the coding sequences for two or all four polypeptide chains into a single expression vector.

[0353] Bispecific antibodies, for example, are composed of a hybrid immunoglobulin heavy chain with a first bispecificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only half of the bispecific molecule provides a facile separation method. This technique is disclosed in WO 94 / 04690. For further details on bispecific antibody production, see, for example, Suresh et al., Methods Enzymol 121:210 (1986).

[0354] According to another approach described in U.S. Patent No. 5,731,168, in some embodiments, the interface between a pair of antibody molecules is engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. A preferred interface is C H The antibody comprises at least a portion of the three domains. In this method, one or more small amino acid side chains at the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large amino acid side chains are created at the interface of a second antibody molecule by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products, such as homodimers.

[0355] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies, for example, where one of the antibodies in the heteroconjugate is conjugated to avidin and the other to biotin. Such antibodies have been proposed, for example, for targeting immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and for treating HIV infection (WO 91 / 00360, WO 92 / 20373, and EP 03089). In some embodiments, heteroconjugate antibodies are made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques. Another method is designed to create tetramers by adding a streptavidin-encoding sequence at the C-terminus of the scFv. Since streptavidin is composed of four subunits, when scFv-streptavidin is folded, the four subunits associate to form a tetramer (Kipriyanov et al., Hum Antibodies Hybridomas 6(3):93-101(1995)).

[0356] According to another approach to making bispecific antibodies, in some embodiments, the interface between a pair of antibody molecules is engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. One interface is the C H The antibody comprises at least a portion of three domains. In this method, one or more small amino acid side chains at the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created at the interface of a second antibody molecule by replacing the large amino acid side chain(s) with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. See WO 96 / 27011.

[0357] Techniques for generating bispecific antibodies from antibody fragments are also described in this literature. For example, bispecific antibodies are prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNF derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNF derivative to form the bispecific antibody. In some embodiments, the generated bispecific antibodies are used as agents for the selective immobilization of enzymes.

[0358] Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can then be chemically coupled to form bispecific antibodies. Shalaby et al., J Exp Med 175:217-25 (1992) describe the production of humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was capable of binding to cells overexpressing the ErbB2 receptor and normal human T cells, as well as inducing the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0359] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See Kostelny et al., J Immunol 148(5):1547-53 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method is used in some embodiments to produce antibody homodimers.

[0360] Antibody identification and preparation In some embodiments, polynucleotide sequences encoding antibodies, their variable regions, or antigen-binding fragments are determined using conventional sequencing techniques and subcloned into expression vectors for recombinant production of the antibodies. This is accomplished by obtaining mononuclear cells from the blood of a subject, e.g., a cancer patient, generating B cell clones from the mononuclear cells, inducing the B cells to become antibody-producing plasma cells, and screening the supernatant produced by the plasma cells to determine whether they contain the antibody. Identification of other antibodies with the specificity of the antibodies of the present disclosure is, in some embodiments, achieved using similar methods. For example, once an antibody-producing B cell clone is identified, reverse transcription polymerase chain reaction (RT-PCR) is performed to clone DNA encoding the antibody variable region or portions thereof. These sequences are then subcloned into expression vectors suitable for recombinant production of human antibodies. In some embodiments, binding specificity is confirmed by determining the ability of the antibody or recombinant antibody to bind to M2 cells or other cells expressing the human CD163 polypeptide expressed by M2 cells.

[0361] In certain embodiments of the methods described herein, B cells isolated from peripheral blood or lymph nodes are sorted, e.g., based on CD19 positivity, and seeded, e.g., in a 96-, 384-, or 1536-well format, e.g., at as low a density as a single cell per well. The cells are induced to differentiate into antibody-producing cells, e.g., plasma cells, and the culture supernatant is harvested and tested for binding to cells expressing the target polypeptide on their surface, e.g., using FMAT or FACS analysis. Positive wells are then subjected to whole-well RT-PCR to amplify the heavy and light chain variable regions of IgG molecules expressed by clonal daughter plasma cells. The resulting PCR products, encoding the heavy and light chain variable regions, or portions thereof, are subcloned into human antibody expression vectors for recombinant expression. The resulting recombinant antibodies are then tested to confirm their original binding specificity and, in some embodiments, for cross-reactivity to other cells or proteins.

[0362] Thus, in one embodiment, a method for identifying antibodies is performed as follows: First, a full-length or near-full-length CD163 cDNA is transfected into a cell line for expression of the CD163 polypeptide. Next, individual human plasma or serum samples are tested for antibodies that bind to the cell-expressed polypeptide. Finally, MAbs obtained from plasma or seropositive individuals are characterized for binding to the same cell-expressed CD163 polypeptide. In some embodiments, further definition of the fine specificity of the MAbs is performed at this point.

[0363] Polynucleotides encoding antibodies or portions thereof of the present disclosure are, in some embodiments, isolated from antibody-expressing cells by methods available in the art and described herein. These methods include polymerase chain reaction amplification using primers specific to conserved regions of human antibody polypeptides. For example, light and heavy chain variable regions are cloned from B cells by molecular biology techniques described in WO 92 / 02551, U.S. Patent No. 5,627,052, or Babcook et al., Proc Natl Acad Sci USA 93:7843-48 (1996). In certain embodiments, polynucleotides encoding all or part of the heavy and light chain variable regions of IgG molecules expressed by antibody-expressing clonal daughter plasma cells are subcloned and sequenced. In some embodiments, the sequence of the encoded polypeptide is readily determined from the polynucleotide sequence.

[0364] Isolated polynucleotides encoding polypeptides of the disclosure are subcloned into expression vectors to recombinantly produce the antibodies and polynucleotides of the disclosure using procedures known in the art and described herein.

[0365] In some embodiments, the binding properties of an antibody (or fragment thereof) to a CD163 polypeptide or M2 cells are generally determined and evaluated using immunodetection methods, including, for example, immunofluorescence-based assays such as immunohistochemistry (IHC) and / or fluorescence-activated cell sorting (FACS). In some embodiments, the immunoassay includes controls and procedures for determining whether the antibody specifically binds to a CD163 polypeptide or M2 macrophages, but does not recognize or cross-react with control cells, e.g., M1 cells, or host cells transfected to express a control protein.

[0366] Following pre-screening of serum to identify patients producing antibodies against CD163 polypeptides or M2 macrophages, the methods of the present disclosure generally involve the isolation or purification of B cells from a biological sample previously obtained from the patient or subject. In some embodiments, the patient or subject is suspected of having, currently diagnosed with, or already diagnosed with a cancer or particular disease of interest, or is believed to be cancer or disease-free. Typically, the patient or subject is a mammal, and in certain embodiments, a human. In some embodiments, the biological sample is a sample containing B cells, including, but not limited to, lymph nodes or lymph node tissue, pleural effusion, peripheral blood, ascites, tumor tissue, or cerebrospinal fluid (CSF). In some embodiments, B cells are isolated from different types of biological samples, such as tumor biopsies or other biological samples affected by a particular disease. However, it is understood that in some embodiments, any biological sample containing B cells may be used in any of the embodiments of the present disclosure.

[0367] Once isolated, the B cells are induced to produce antibodies, e.g., by culturing the B cells under conditions that support the proliferation or development of B cells into plasma cells, plasmablasts, or plasma cells. The antibodies are then screened, typically using high-throughput techniques, to identify antibodies that specifically bind to a target antigen, e.g., a particular tissue, cell, or polypeptide. In some embodiments, the specific antigen, e.g., the cell surface polypeptide bound by the antibody, is not known, while in other embodiments, the antigen specifically bound by the antibody is known.

[0368] According to the present disclosure, in some embodiments, B cells are isolated from biological samples, such as tumor, tissue, peripheral blood, or lymph node samples, by any means known and available in the art. B cells are typically sorted by FACS based on the presence of B cell-specific markers on their surface, such as CD19, CD138, and / or surface IgG. However, in some embodiments, other methods known in the art are utilized, such as column purification using CD19 magnetic beads or IgG-specific magnetic beads, followed by elution from the column. However, in some embodiments, magnetic isolation of B cells using any marker results in the loss of specific B cells. Therefore, in certain embodiments, the isolated cells are not sorted; instead, Ficoll-purified mononuclear cells isolated from the tumor are directly seeded into wells at an appropriate or desired number of specificities.

[0369] To identify antibody-producing B cells, B cells are typically seeded at low density (e.g., single-cell specificity per well, 1-10 cells per well, 10-100 cells per well, 1-100 cells per well, 1-100 cells per well, less than 10 cells per well, or less than 100 cells per well) in multiwell or microtiter plates, e.g., in a 96-, 384-, or 1536-well configuration. When B cells are initially seeded at a density of greater than 1 cell per well, methods of the present disclosure include successively diluting the cells in wells identified as producing antigen-specific antibodies until single-cell specificity per well is achieved, thereby, in some embodiments, facilitating the identification of antigen-specific antibody-producing B cells. In some embodiments, cell supernatants or portions thereof, and / or cells are cryopreserved for future testing and subsequent recovery of antibody polynucleotides.

[0370] In certain embodiments, B cells are cultured under conditions that favor antibody production by the B cells. For example, B cells are cultured under conditions that favor B cell proliferation and differentiation to yield antibody-producing plasmablasts, plasma cells, or plasma cells. In certain embodiments, B cells are cultured in the presence of a B cell mitogen, such as lipopolysaccharide (LPS) or CD40 ligand. In one specific embodiment, B cells are differentiated into antibody-producing cells by culturing with feed cells and / or other B cell activators, such as CD40 ligand.

[0371] In some embodiments, cell culture supernatants, or antibodies obtained therefrom, are tested for their ability to bind to the target antigen using conventional methods available in the art, including those described herein. In certain embodiments, culture supernatants are tested for the presence of antibodies that bind to the target antigen using high-throughput methods. For example, B cells are cultured in multiwell microtiter dishes, such that a robotic plate handler is used to simultaneously sample multiple cell supernatants and test for the presence of antibodies that bind to the target antigen. In certain embodiments, the antigen is coupled to beads, such as paramagnetic or latex beads, to facilitate capture of the antibody / antigen complex. In other embodiments, the antigen and antibody are fluorescently labeled (with different labels), and FACS analysis is performed to identify the presence of antibodies that bind to the target antigen. In one embodiment, antibody binding is determined using FMAT™ analysis and instrumentation (Applied Biosystems, Foster City, Calif.). FMAT is a fluorescent macro-confocal platform for high-throughput screening, allowing for mix-and-read, non-radioactive assays using live cells or beads.

[0372] When the binding of an antibody to a particular target antigen (e.g., a biological sample such as cancerous tissue or cells, an infectious agent, etc.) is compared to the binding of the antibody to a control sample (e.g., a biological sample such as normal cells, control cells from another species, a different cancerous tissue or cells, a different infectious agent, etc.), in some embodiments, an antibody is considered to preferentially bind to a particular target antigen if at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold more of the antibody binds to the particular target antigen compared to the amount that binds in the control sample.

[0373] Polynucleotides encoding antibody chains, variable regions, or fragments thereof are isolated from cells, in some embodiments, using any means available in the art. In one embodiment, polynucleotides are isolated using polymerase chain reaction (PCR), for example, reverse transcription PCR (RT-PCR) using oligonucleotide primers that specifically bind to heavy or light chain encoding polymerase sequences or their complements, using routine procedures available in the art. In one embodiment, positive wells are subjected to whole-well RT-PCR to amplify the heavy and light chain variable regions of IgG molecules expressed by clonal daughter plasma cells. In some embodiments, these PCR products are sequenced, and then products encoding the heavy and light chain variable regions, or portions thereof, are subcloned into human antibody expression vectors and recombinantly expressed using routine procedures in the art (see, e.g., U.S. Patent No. 7,112,439). In some embodiments, nucleic acid molecules encoding antibodies or fragments thereof specific for M2 macrophages, as described herein, are propagated and expressed using a variety of well-known procedures for nucleic acid cleavage, ligation, transformation, and transfection. For this reason, in certain embodiments, expression of antibody fragments is preferred in prokaryotic host cells such as E. coli (see, e.g., Pluckthun et al., Methods Enzymol 178:497-515 (1989)). In other specific embodiments, expression of antibodies or antigen-binding fragments thereof is preferred in eukaryotic host cells such as yeast (e.g., Saccharomyces cerevisiae, S. pombe, Pichia pastoris), animal cells (including mammalian cells), or plant cells. Examples of suitable animal cells include, but are not limited to, myeloma, COS, CHO, or hybridoma cells. Examples of plant cells include tobacco, corn, soybean, and rice cells.In some embodiments, by methods known to those of skill in the art and based on the present disclosure, a nucleic acid vector is designed for expression of a heterologous sequence in a particular host system, and then a polynucleotide sequence encoding a tumor-specific antibody (or fragment thereof) is inserted. Regulatory elements will vary depending on the particular host.

[0374] In some embodiments, one or more replicable expression vectors containing polynucleotides encoding the variable and / or constant regions are prepared and used to transform a suitable cell line, e.g., a non-producing myeloid cell line such as the mouse NSO strain, or a bacterium such as E. coli, in which antibody production occurs. For efficient transcription and translation, the polynucleotide sequence in each vector should contain appropriate regulatory sequences, specifically a promoter and leader sequence operably linked to the variable region sequence.

[0375] Specific methods for producing antibodies in this way are generally well known and routinely used. For example, molecular biology procedures are described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989. See also Sambrook et al., 3rd ed., Cold Spring Harbor Laboratory, New York, (2001)). Although not required, in certain embodiments, the region of the polynucleotide encoding recombinant antibody is sequenced. DNA sequencing is carried out, for example, by any method or using any system known in the art. Basic sequencing techniques are described, for example, in Sanger et al., Proc Natl Acad Sci USA 74:5463(1977)) and the Amersham International plc sequencing handbook and its revised editions.

[0376] In certain embodiments, the resulting recombinant antibody or fragment thereof is then tested to confirm its original specificity, and in some embodiments is further tested, for example, for cross-reactivity with related polypeptides. In certain embodiments, antibodies identified or produced according to the methods described herein are tested for their ability to internalize other effector functions using conventional methods.

[0377] Packages, Kits, and Pre-filled Containers Also provided herein are kits containing one or more of the compounds described above, hi some embodiments, the kits comprise an antibody or antigen-binding fragment thereof described herein in suitable container means.

[0378] In some embodiments, a container means is provided that contains a composition described herein. In some embodiments, the container means is any suitable container for containing, for example, a liquid or lyophilized composition, including, but not limited to, a vial, a syringe, a bottle, an intravenous (IV) bag, or an ampoule. In some embodiments, the syringe holds a liquid suitable for injection into a subject in any volume, including, but not limited to, 0.5 cc, 1 cc, 2 cc, 5 cc, 10 cc, or more.

[0379] Provided herein are kits comprising one or more compositions described herein. In some embodiments, provided herein are kits for treating a subject with cancer, comprising an antibody described herein and an anti-cancer therapeutic agent.

[0380] In some embodiments, provided herein is a kit for treating cancer comprising an antibody described herein and a label attached to or packaged with a container, the label describing the use of the antibody in combination with an anti-cancer therapeutic agent.

[0381] In some embodiments, provided herein are kits for treating cancer that include an anti-cancer therapeutic and a label attached to or packaged with a container, the label describing the use of the anti-cancer therapeutic in combination with an antibody described herein.

[0382] In some embodiments, the container means of the kits will generally include at least one vial, test tube, flask, bottle, ampoule, syringe, intravenous (IV) bag, and / or other container means, into which at least one polypeptide may be disposed, and / or preferably suitably aliquoted. Provided herein are container means which comprise the compositions described herein.

[0383] In some embodiments, the kit contains at least one fusion protein, detectable moiety, reporter molecule, and / or any other reagents in close confinement for commercial sale. In some embodiments, such containers include syringe bottles and / or spray-molded plastic containers into which the desired vials are held. In some embodiments, the kit also includes printed materials regarding the use of the materials in the kit.

[0384] In some embodiments, the package and kit further comprises a buffering agent, a preservative, and / or a stabilizer in the pharmaceutical formulation. In some embodiments, each component of the kit is enclosed in an individual container, and all of the various containers may be in one package. In some embodiments, the kit of the present disclosure is designed for cold storage or room temperature storage.

[0385] In addition, in some embodiments, the preparation contains a stabilizer that increases the shelf life of the kit, such as bovine serum albumin (BSA). If the composition is lyophilized, the kit in some embodiments contains an additional solution preparation for reconstituting the lyophilized preparation. Acceptable reconstitution solutions are well known in the art, including, for example, pharmaceutically acceptable phosphate-buffered saline (PBS).

[0386] In some embodiments, the packages and kits further include one or more components for an assay, such as an ELISA assay. Samples to be tested in this application include, for example, blood, plasma, tissue sections, and secretions, urine, lymph, and products thereof. In some embodiments, the packages and kits further include one or more components for sample collection (e.g., syringes, cups, swabs, etc.).

[0387] In some embodiments, the packages and kits contain information required by the US FDA or similar regulatory authority, such as labeling identifying product description, dosage, administration form, and / or indication for treatment. The packages provided herein may contain any of the compositions described herein.

[0388] The term "packaging material" refers to a physical structure that houses the components of the kit. In some embodiments, the packaging material maintains the components in a sterile condition and is made of materials commonly used for such purposes (e.g., paper, cardboard, glass, plastic, foil, ampoules, etc.). In some embodiments, the label or package insert contains appropriate written instructions. Thus, the kit, in some embodiments, further includes a label or instructions for using the kit components in any of the methods of the present disclosure. In some embodiments, the kit contains the compound in a pack or dispenser along with instructions for administering the compound in the methods described herein.

[0389] In yet a further embodiment, the kit further comprises container means for an anti-cancer therapeutic agent.

[0390] The instructions, in some embodiments, include instructions for carrying out any of the methods described herein, including treatment methods, and may further include indications for successful clinical endpoints or any adverse symptoms that occur, or, in some embodiments, additional information required by regulatory agencies, such as the U.S. Food and Drug Administration, for use in human subjects.

[0391] In some embodiments, the instructions are present on "printed material," e.g., on paper or cardboard within or affixed to the kit, on a label affixed to the kit or packaging material, or attached to a vial or tube containing a component of the kit. In some embodiments, the instructions are further contained on a computer-readable medium, such as, for example, a CD-ROM, a DVD, a flash memory device, a solid-state memory, a magnetic disk and disk device, a magnetic tape, a cloud computing device and service, or the like. In some cases, the program and instructions are encoded permanently, nearly permanently, semi-permanently, or non-transiently on the medium.

[0392] Provided herein is a container means containing a composition described herein. In some embodiments, the container means is any suitable container for containing a liquid or lyophilized composition, including, but not limited to, a vial, a syringe, a bottle, an intravenous (IV) bag, or an ampoule. The syringe holds a liquid suitable for injection into a subject in any volume, including, but not limited to, 0.5 cc, 1 cc, 2 cc, 5 cc, 10 cc, or more.

[0393] Provided herein are kits comprising the compositions described herein. In some embodiments, provided herein are kits for treating cancer comprising an antibody described herein in combination with an anti-cancer therapeutic.

[0394] In some embodiments, provided herein is a kit for treating cancer comprising an antibody described herein and a label attached to or packaged with a container, the label describing use of the antibody or antigen-binding fragment thereof with an anti-cancer therapeutic.

[0395] In some embodiments, provided herein are kits for treating cancer that include an anti-cancer therapeutic and a label attached to or packaged with a container, the label describing the use of the anti-cancer therapeutic in combination with an antibody described herein. [Example]

[0396] The present disclosure is further illustrated in the following examples, which are provided for illustrative purposes only and are not intended to limit the disclosure in any way.

[0397] Example 1 - Identification and cloning We isolated and cloned antibodies that specifically bind to human myeloid-derived suppressor cells (MDSCs), which are produced by patients responding to anti-PD-1 checkpoint inhibitor treatment. We further investigated the immunomodulatory properties of these monoclonal antibodies with the aim of identifying antibodies with therapeutic potential to target and reverse the immunosuppressive effects of MDSCs, thereby improving tumor clearance.

[0398] Cancer patients who achieved a partial or complete response to immune checkpoint inhibitors for at least 6 months were identified and selected for memory B cell repertoire analysis via the I-STAR platform. This platform utilizes a short-term B cell culture system to examine the memory B cell repertoire. Based on CD19 and IgG surface expression, over 15,000 memory B cells were isolated from 10 million peripheral blood mononuclear cells (PBMCs) from each donor patient. These memory B cells were then plated at approximately 1 cell / well into 40 384-well microtiter plates under conditions that promoted B cell activation, proliferation, terminal differentiation, and antibody secretion. The seeding density of 1 cell / well allowed for the expansion of single B cell clones, allowing authentic antibody heavy and light chain pairs to be reconstituted from each culture well. Using a high-throughput, miniaturized multiplexed flow cytometry assay, secreted IgG antibodies in each well were screened for binding to MDSCs. Forty-nine positive B cell clones were identified. A selected subset of antibodies, prioritized based on MDSC binding profile and antibody variable region sequence, were sequenced, cloned, and expressed as recombinant IgG1 for further in vitro characterization.

[0399] The heavy (VH) and light (VL) variable regions of immunoglobulin genes from B cell clones producing MDSC-specific antibodies were amplified by RT-PCR using family-specific primer sets. From positive family-specific PCR reactions, pools of VH or VL region clones were cloned upstream of human IgG1 constant domain sequences into expression vectors, resulting in functional antibodies with the same binding characteristics as those produced by the B cell clones. DNA plasmids were designed and ordered for gene synthesis in the constant regions at GenScript, NJ, USA. These plasmids were combined in all possible family-specific heavy and light chain pairs and used to transiently transfect HEK293 cells. All transfectant supernatants containing secreted recombinant antibodies were screened in a flow-based MDSC-binding assay. In wells containing more than one B cell clone per well, multiple VH and VL domain sequences were amplified and expressed as described above. An MDSC screen was then used to identify pools of heavy and light chain combinations that recapitulated the binding activity observed with antibodies found in mixed cultures. The DNA sequences of the VH and VL variable regions for all binding mAbs were confirmed by multiple sequencing reactions using DNA purified from maxipreps (Genscript-derived and Macherey Nagel-amplified plasmids).

[0400] One B cell clone (Germline ID for heavy chain VH3.30-3 / IGHG1 and light chain VK1.012) was identified from the MDSC screen and designated AB101, containing a light chain comprising SEQ ID NO:9 and a heavy chain comprising SEQ ID NO:10. The donor from whom the clone was obtained was a patient diagnosed with non-small cell lung cancer (NSCLC). The patient had progressive disease in response to chemotherapy and was in complete remission upon anti-PD-1 treatment, but was still receiving treatment at the time of blood collection. Sequencing confirmed that the B cell clone well contained only one heavy chain and one light chain. As observed with IgG antibodies secreted from single B cell clones, the recapitulated antibody also had distinct bimodal binding to MDSCs, confirming that the antibody target was highly expressed on select subpopulations of MDSCs. See Figure 1. Results from replicate screens on two MDSC donors under relaxed block (recombinant Fc block 10 μg / mL (Abcam)) and strict block (recombinant Fc block 10 μg / mL + 1 μg / mL anti-CD16, anti-CD32, and anti-CD64) conditions showed dose-dependent saturable binding of AB101 to human MDSCs with an IC50 of approximately 10 nM under relaxed block conditions. Under strict block conditions, overall reduced binding of AB101 was observed, as evidenced by a decrease in MFI.

[0401] CD163 is a marker for cells derived from the monocyte / macrophage lineage. It has been reported that CD163 expression on in vitro differentiated MDSCs is bimodal. We hypothesized that the bimodal binding of AB101 might correlate with CD163 expression. To test this hypothesis, in vitro MDSCs were generated (see Example 11), co-stained with anti-CD163 (BioLegend 333611) and AF647-conjugated AB101 as described in Example 8, and binding was analyzed by FACS. Cells were first gated as CD163 high or low, and then examined for binding with various concentrations of AB101 or a human IgG1 isotype control. The subpopulation of cells to which the AB101 antibody bound showed CD163 expression. Hi cells. See Figure 2.

[0402] Example 2 - Isolation of autologous monocytes and T cells This example demonstrates the isolation of autologous monocytes and T cells. Human monocytes and T cells were obtained using techniques commonly used in the art. During the platelet apheresis collection process, white blood cells (WBCs) were captured in an integrated chamber known as the LeukoReduction System (LRS) chamber, from which human monocytes and T cells were isolated. Peripheral blood mononuclear cells (PBMCs) were purified from the LRS sample by standard density gradient centrifugation (Ficoll™ Paque Premium 1.073, GE Healthcare No. 17-5449-52). The supernatant was discarded, and the pellet was resuspended in 20 mL Easysep™ buffer (StemCell Technologies No. 20144) for enumeration of PBMCs and further isolation of monocytes and T cells.

[0403] Monocytes were isolated using the EasySep Human Monocyte Isolation Kit (Stem Cell No. 19359) according to the manufacturer's instructions.

[0404] Human CD3 + T Cell Isolation Kit (StemCell 19051), EasySep™ Human CD4 + T Cell Isolation Kit (StemCell No. 17952), EasySep™ Human CD8 + Total CD3, CD4, or CD8 T cells were isolated using a T cell isolation kit (StemCell No. 17953). These negative selection kits use antibodies to label unwanted cell types for removal, allowing the desired target cells to be isolated from the sample.

[0405] Example 3 - AB101 specific binding to immunosuppressive myeloid cells To assess specificity, the binding of antibody AB101, an antibody of the present invention conjugated to the far-red fluorescent dye AF647, was tested against different cell types, including MDSCs, immunosuppressive M2c and pro-inflammatory M1 macrophages generated as described in Example 11 and below. In addition, distinct immune populations obtained from PBMCs of healthy donors were evaluated for antibody binding. At least three individual donors were used in each of these studies. PBMCs were isolated from blood using a Ficoll gradient using standard procedures. PBMC cells were then stained with hematopoietic lineage markers (CD45-BV421 (BD642275), CD3-BV510 (BD563109), CD11c-PE-Cy7 (BD561356), CD14-FITC (BD347493), CD20-APC-Cy7 (BD562643), CD56-PE (BD347747), and CD66c (BD551478)) to differentiate immune cell populations. Distinct lineage populations were further characterized for the following expression patterns, which were assessed for binding to AB101: T cell CD45 + CD3 + , B cell CD45 + CD20 + , monocyte CD45 + CD14 + , NK cell CD45 + SSC low CD14 - CD3 - CD56 + , granulocyte CD45 + SSC Hi CD14 - CD66 + , and dendritic cell CD45 + CD14 - CD66 - CD11c +We also evaluated antibody binding to primary human non-immune cells (Lonza), including small airway epithelial cells (SAECs, #CC-2547), renal proximal tubule epithelial cells (RPTECs, #CC-2553), pulmonary microvascular endothelial cells (HMVECs, #CC-2527), umbilical vein endothelial cells (HUVECs, #C2519A), aortic smooth muscle cells (AOSMCs, #CC2571), and keratinocytes (#00192627). These cells were cultured in cell-type-specific media and conditions according to the manufacturer's instructions until they reached 60-70% confluence, at which point they were harvested and tested for antibody binding by flow cytometry (Figure 4 and Figure 5).

[0406] In vitro monocytic MDSCs were generated from monocytes isolated by standard methods. On day 0, monocytes were cultured at 1.5 × 10 in RPMI 1640 (Hyclone SH30027.02, serum-free). 5 / cm 2The cells were seeded in RPMI media and incubated at 37°C with 5% CO2 for 1 hour. Then, they were washed with prewarmed RPMI and MDSC medium (RPMI + 10% FBS (Hyclone SH30070.03) + 50 ng / mL GM-CSF (R&D Systems 215-GM-010) + 50 ng / mL IL-6 (R&D Systems 206-IL-010), 20 mL per T75 flask) was added. The cells were then cultured at 37°C with 5% CO2 for 7 days without a medium change. After 7 days, the cells were harvested by washing twice with PBS (Hyclone SH30028.03) + 2 mM EDTA and then adding 1.5 mL per T75 flask of cold macrophage detachment reagent (PromoCell C-41330), followed by incubation at 2–8°C for 40 minutes. The flask was gently tapped to dislodge the cells, which were then pooled and diluted 1:1 with PBS + 2 mM EDTA. The cells were pelleted in a conical tube by centrifugation at 450 x g for 15 minutes, washed once with PBS + 2 mM EDTA, counted, and diluted to 1 x 10 in FACS blocking buffer (PBS + 1% FBS + 0.1 μg / mL Fc block (Abcam Ab90285) for relaxed staining conditions, or PBS + 1% FBS + Fc block and + 0.01 μg / mL CDR block (antibodies against FcR CD16, CD32, and CD64, BD Biosciences 556617, 557333, and 555525, respectively) for stringent staining conditions). 7 The cells were resuspended at 1 × 10 / mL. The cells were incubated in FACS blocking buffer at room temperature (RT) for 20 minutes (min) and then at 4°C for 30 minutes. The cells were then resuspended at 1 × 10 / mL in FACS buffer + 5% BSA (Sigma A3059). 6 Dilute to 40 μL of cells (4 × 10) / mL. 4Cells (100 μg / mL) were aliquoted into wells for staining. Primary antibodies (AB101 at 20, 6, 2, 0.75, 0.25, 0.08, and 0.02 μg / mL, or human IgG1 isotype control at 6 μg / mL) were added to the cells and incubated for 90 minutes at room temperature. Cells were washed three times with 250 μL / well of FACS buffer + 5% BSA. Secondary APC goat anti-human IgG (Jackson IR109-136-097) antibody was prepared at 1:250 (at a dilution of 1:1000) in FACS buffer + BSA + e780 vital dye and added to the cells (50 μL per well). After a 45-minute incubation at 4°C, cells were washed three times with 250 μL of FACS buffer. Cells were then fixed in 100 μL of 4% PFA per well for 10–15 minutes at room temperature, washed once with 250 μL of FACS buffer, pelleted at 650 × g for 5 minutes, and then resuspended in 100 μL of FACS buffer and analyzed by flow cytometry.

[0407] As shown in Figure 3, the AB101 antibody binds to human immunosuppressive myeloid cells (M2c macrophages and monocytic MDSCs). This figure plots the MFI of AB101 or isotype staining on M2c, M1, and M0. The AB101 antibody does not bind to B cells, T cells, NKT cells, neutrophils, monocytes, and granulocytes, as shown in Figure 4, which compares staining of T cells, B cells, NKT cells, neutrophils, monocytes, and dendritic cells (black curves) with the isotype control (gray curve). Furthermore, the AB101 antibody does not bind to non-immune cells such as SAECs, RPTECs, HMVECs, HUVECs, AOSMCs, and keratinocytes, as shown in Figure 5. Thus, the AB101 antibody specifically binds to CD163-expressing immunosuppressive myeloid cells without affecting other immune or non-immune cells.

[0408] Example 4 - Immunoprecipitation of CD163 polypeptide with AB101 FcNull antibody This example demonstrates immunoprecipitation of CD163 using an FcNull antibody containing the AB101 variable domain in an IgG1 sequence modified to substantially reduce antibody binding to an Fc receptor (FcNull) designated AB102, which contains a light chain containing SEQ ID NO:9 and a heavy chain containing SEQ ID NO:11. Immunoprecipitation (IP) was performed according to Klockenbusch and Kast, J Biomed Biotechnol Article ID 927585 (2010). Antibody was added before paraformaldehyde (PFA) fixative in a more classical crosslinking IP procedure, or after bis(sulfoccinimidyl) suberate (BS3) crosslinking followed by IP.

[0409] In IP involving crosslinking using the PFA technique, monocytes were isolated from human blood and then polarized into M2 cells using the protocols in Examples 2 and 11. M2 macrophages were detached from the plate using macrophage detachment reagent (Macrophage Detachment Solution DXF, PromoCell, No. C-41330) after incubation at 37°C for up to 10 minutes, during which time the cells began to clump and detach. The flask was tapped firmly to encourage cell detachment. After detachment, the macrophage detachment reagent was quenched by adding FACS buffer to the cells. The cells were pelleted at 300 x g for 10 minutes, and the supernatant was removed. The cell pellet was resuspended in 30 mL of PBS containing 5% BSA (w / v) and 1 mM EDTA, pH 8.0, and incubated on ice for 30 minutes.

[0410] Aliquot 15 x 10 cells 6The cells were divided into six aliquots (5 mL per aliquot), and biotinylated antibodies were added to each. Two aliquots received 50 μg of mouse IgG1 anti-hCD163 (R&D, No. MAB1607), two aliquots received 100 μg of the isotype control antibody ISO2 (in an Fc-null framework), and two aliquots received 100 μg of the test antibody AB102 (containing the AB101 variable domain in an IgG1 sequence modified to significantly reduce binding to Fc receptors (Fc-null)). The cells were incubated at 4°C for 1 hour (hr), with occasional gentle mixing by inverting the tube. The cells were pelleted at 300 × g for 5 minutes, washed three times with PBS-EDTA, and then resuspended in 5 mL of PBS (without magnesium or calcium, HyClone, No. SH30028.02). Paraformaldehyde (PFA, 5 mL of 0.8%) was added to each tube to achieve a final PFA concentration of 0.4%. Cells were incubated in PFA for 5 minutes at room temperature with gentle rocking. Cells were pelleted by centrifugation at 800 × g for 5 minutes, and the supernatant was removed. Cells were resuspended and snap-chilled in 10 mL of ice-cold PBS containing 1.25 M glycine. Cells were pelleted at 800 × g for 5 minutes and resuspended in ice-cold PBS. Cells were pelleted at 800 × g for 5 minutes and resuspended in 1.0 mL of RIPA buffer (ThermoFisher Scientific, No. 89900) containing 1× protease inhibitors. Cells were incubated on ice for 2 hours and then passed 15 times through a 2 mL Dounce homogenizer.

[0411] The cell lysates were spun in a hanging bucket centrifuge to pellet nuclei, and the supernatant was used for IP. Protein lysate (50 μL) was set aside as the input fraction, and 2.0 mL of cold PBS containing 1x protease inhibitors was added to the remaining supernatant. Dynabeads Myone Streptavidin (250 μL) (ThermoFisher Scientific, No. 65601) was added to each sample, and these were rotated overnight at 4°C. The next day, the beads were collected with a StemCell magnet, and the supernatant was removed. The beads were washed successively with 5 mL of Paro Buffer I, 5 mL of Paro Buffer II, and 5 mL of Paro Buffer III for 5 minutes at 4°C (Oncotarget. 2017;8(7):11105-11113). The beads were then washed three times with cold PBS and finally resuspended in 100 μL of PBS and frozen at -80°C.

[0412] The beads were analyzed by mass spectrometry. This analysis was generally performed according to the method described by Yan et al., Mol Cell Proteomics 10(3):M110.005611 (2011). In this method, formaldehyde cross-linking was reversed and proteins were eluted from the streptavidin beads using 6 M guanidine, 150 mM Tris buffer (pH 8.3) at 60°C for 3 hours with constant stirring. The supernatant was denatured, reduced, and alkylated in the same buffer with 10 mM tris(2-carboxyethyl)phosphine (TCEP) and 50 mM chloroacetamide (CAA) at 95°C for 10 minutes. The sample was then diluted 10-fold and digested with 1.3 μg of trypsin overnight at 37°C. Peptides were purified using a C18 cartridge. One replicate of AB102 IP and one replicate of anti-CD163 IP were eluted from the C18 cartridge for direct MS / MS analysis. One replicate of AB102 IP and one replicate of anti-CD163 were labeled with heavy dimethylation (d4) by incubation with 0.1 M formaldehyde-d2, 0.4 M sodium cyanohydride in PBS buffer, pH 7.5. Both replicates of ISO2 (Fc null) IP were labeled with light dimethylation (d0) on the C18 cartridge by incubation with 0.1 M formaldehyde, 0.4 M sodium cyanohydride in PBS buffer, pH 7.5 for 1 hour. The C18 cartridge was then washed with 0.1% trifluoroacetic acid (TFA) and eluted with 80% acetonitrile (ACN). The d0 / d4 dimethylated peptides were resuspended and mixed in buffer A (20% ACN, 0.1% TFA). Peptides were fractionated using a microcapillary HPLC strong cation exchange column (SCX) (200 mm × 20 cm, Proteomix SCX 3 μm, Sepax Technologies) prepared in-house. Peptides were loaded onto a microcapillary column equilibrated in buffer A and washed with buffer A. Bound peptides were eluted with 20 μL of buffer A containing 30% and 50% of buffer B (800 mM ammonium formate, 20% ACN, pH 2.8), followed by 20 μL of buffer D (0.5 M ammonium acetate, 50% ACN).All samples were dried in a Spede-Vac and analyzed directly using a Thermo-Oritrap-Fusion. Spectra were searched against the human UniProt database using the Comet search engine (https: / / sourceforge.net / projects / comet-ms / ). For demethylation labeling, differential modifications of 28.03 Da (for d0 dimethylation) and 32.06 Da (for d4 dimethylation) on the N-terminus and Lys side chain were used.

[0413] Heavy and light samples were passed through an in-line HPLC column using a strong cation exchange column (200 mm x 20 cm, Proteomix SCX 3 μm, Sepax Technologies) and subjected to MS / MS using Thermo-Oritrap-Fusion. For heavy and light labeled samples, only fully methylated spectra were included. Eighty-nine proteins were identified when all peptides contained heavy isotopes. This confirmed that the proteins were identified in AB102IP and not in the negative control. Twelve of the 89 proteins unique to AB102IP, namely CD163, RIPK1, NEUA, SLC31, LRP8, SLIT1, RAF1, ILK, ATRN1, MCA32, FNBP2, and LRRN3, were considered plausibly present on the cell surface. One additional protein, TNR5, was found to have two heavily methylated peptides and one unmethylated peptide (origin unknown), supporting the possibility that it may be exclusive to AB102IP.

[0414] Peptides from one replicate of AB102IP and one replicate of the control anti-CD163IP were analyzed separately by mass spectrometry. Of the 360 ​​proteins identified in AB102IP, 45 were selected as potentially membrane-bound or secreted. Among proteins identified in other datasets, CD163, galectin-1, galectin-3, and peptidyl-prolyl cis-trans isomerase A (PPIA) were found in AB102IP. Casein kinase IIb, which has been reported to directly interact with CD163, was also identified in this dataset.

[0415] For IP by crosslinking using the BS3 method, macrophages were harvested by collecting the supernatant from the flask into a 250 mL centrifuge tube. Cold macrophage detachment reagent (30 mL) was added to each flask and incubated at 4°C for 45 minutes. The flask was then scraped with a scraper, and the cells were collected into a 250 mL centrifuge tube and centrifuged at 650 x g for 10 minutes. The medium was aspirated, leaving the cell pellet in the tube. The cells were then resuspended in 20 mL of cold PBS + 2 mM EDTA.

[0416] The cells were then diluted to 1 × 10 in PBS + 2 mM EDTA. 7The cells / mL were diluted and divided into three volumes: 40%, 40%, and 20% total volume. AB102 (2.5 mg) was added to one of the 40% fractions. Anti-PDL1 antibody in an FcNull framework (2.5 mg) was added to the other 40% fraction, which served as a positive control. An isotype control (ISO2 in an FcNull framework, 1.25 mg) was added to the 20% fraction, which served as a negative control. Each fraction was incubated at 4°C for 2 hours with gentle mixing. They were then centrifuged at 650 x g for 10 minutes, and the supernatant was carefully decanted. The pellets were each resuspended in 15 mL PBS + EDTA and then centrifuged at 650 x g for 10 minutes. This washing step was then repeated. The wash buffer was then carefully removed without disrupting the pellet. The pellets from the 40% fraction were resuspended in 2 mL of crosslinking buffer. The pellet from the 40% fraction was resuspended in 1 mL of crosslinking buffer. A stock concentration of 50 mM BS3 was dissolved in 70 μL of ultrapure water for each 8 mg vial. BS3 (60 μl / mL cells) was added to each resuspended cell fraction to a final concentration of 3 mM BS3, and each cell fraction was mixed by gently swirling. The cell fractions were then incubated on ice for 1 hour, swirling and mixing every 10 minutes. After the BS3 incubation, 15 mL of quenching solution was added directly to the cells and incubated at room temperature for 15 minutes. The cells were centrifuged at 1200 x g for 15 minutes, and the quenching buffer was carefully decanted. The pellet was washed once with PBS + EDTA as previously described. Cells were then lysed by adding 20 mL of lysis buffer (Pierce™ IP Lysis Buffer #87788) to each 40% fraction and 10 mL to the 20% fraction, followed by incubation on ice for 15 minutes. The cell lysates were then centrifuged at 13,000 x g for 10 minutes at 4°C.

[0417] MabSelect SuRe re...

Claims

1. A heavy chain variable region (V) having at least 80% identity to the amino acid sequence of SEQ ID NO: 8 Ｈ ) and a light chain variable region (V) having at least 80% identity to the amino acid sequence of SEQ ID NO:

7. Ｌ ) and an antibody or recombinant antibody comprising the same.

2. A light chain variable region (V) having at least 85% identity to the amino acid sequence of SEQ ID NO:7 Ｌ 2. The antibody or recombinant antibody of claim 1, comprising:

3. A light chain variable region (V) having at least 90% identity to the amino acid sequence of SEQ ID NO:

7. Ｌ 2. The antibody or recombinant antibody of claim 1, comprising:

4. A light chain variable region (V) having at least 95% identity to the amino acid sequence of SEQ ID NO:7 Ｌ 2. The antibody or recombinant antibody of claim 1, comprising:

5. A light chain variable region (V) having at least 99% identity to the amino acid sequence of SEQ ID NO:

7. Ｌ 2. The antibody or recombinant antibody of claim 1, comprising:

6. A light chain variable region (V) having at least 100% identity to the amino acid sequence of SEQ ID NO:

7. Ｌ 2. The antibody or recombinant antibody of claim 1, comprising:

7. A heavy chain variable region (V) having at least 85% identity to the amino acid sequence of SEQ ID NO:8 Ｈ 7. The antibody or recombinant antibody of claim 1 , comprising:

8. A heavy chain variable region (V) having at least 90% identity to the amino acid sequence of SEQ ID NO:8 Ｈ 7. The antibody or recombinant antibody of claim 1 , comprising:

9. A heavy chain variable region (V) having at least 95% identity to the amino acid sequence of SEQ ID NO:8 Ｈ 7. The antibody or recombinant antibody of claim 1 , comprising:

10. A heavy chain variable region (V) having at least 99% identity to the amino acid sequence of SEQ ID NO:8 Ｈ 7. The antibody or recombinant antibody of claim 1 , comprising:

11. A heavy chain variable region (V) having at least 100% identity to the amino acid sequence of SEQ ID NO:

8. Ｈ 7. The antibody or recombinant antibody of claim 1 , comprising:

12. An antibody or recombinant antibody comprising a heavy chain sequence comprising a complementarity determining region (CDR) H1 having at least 80% identity to the amino acid sequence of SEQ ID NO:4, a CDR H2 having at least 80% identity to the amino acid sequence of SEQ ID NO:5, and a CDR H3 having at least 80% identity to the amino acid sequence of SEQ ID NO:6, and a light chain sequence comprising a CDR L1 having at least 80% identity to the amino acid sequence of SEQ ID NO:1, a CDR L2 having at least 80% identity to the amino acid sequence of SEQ ID NO:2, and a CDR L3 having at least 80% identity to the amino acid sequence of SEQ ID NO:

3.

13. 13. The antibody or recombinant antibody of claim 12, wherein the CDR L1 has at least 85% identity to the amino acid sequence of SEQ ID NO: 1, the CDR L2 has at least 85% identity to the amino acid sequence of SEQ ID NO: 2, and the CDR L3 has at least 85% identity to the amino acid sequence of SEQ ID NO:

3.

14. 13. The antibody or recombinant antibody of claim 12, wherein the CDR L1 has at least 90% identity to the amino acid sequence of SEQ ID NO: 1, the CDR L2 has at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and the CDR L3 has at least 90% identity to the amino acid sequence of SEQ ID NO:

3.

15. 13. The antibody or recombinant antibody of claim 12, wherein the CDR L1 has at least 95% identity to the amino acid sequence of SEQ ID NO: 1, the CDR L2 has at least 95% identity to the amino acid sequence of SEQ ID NO: 2, and the CDR L3 has at least 95% identity to the amino acid sequence of SEQ ID NO:

3.

16. 13. The antibody or recombinant antibody of claim 12, wherein the CDR L1 has at least 99% identity to the amino acid sequence of SEQ ID NO: 1, the CDR L2 has at least 99% identity to the amino acid sequence of SEQ ID NO: 2, and the CDR L3 has at least 99% identity to the amino acid sequence of SEQ ID NO:

3.

17. 13. The antibody or recombinant antibody of claim 12, wherein the CDR L1 has at least 100% identity to the amino acid sequence of SEQ ID NO: 1, the CDR L2 has at least 100% identity to the amino acid sequence of SEQ ID NO: 2, and the CDR L3 has at least 100% identity to the amino acid sequence of SEQ ID NO:

3.

18. 18. The antibody or recombinant antibody of any one of claims 12 to 17, wherein the CDR H1 has at least 85% identity to the amino acid sequence of SEQ ID NO: 4, the CDR H2 has at least 85% identity to the amino acid sequence of SEQ ID NO: 5, and the CDR H3 has at least 85% identity to the amino acid sequence of SEQ ID NO:

6.

19. 18. The antibody or recombinant antibody of any one of claims 12 to 17, wherein the CDR H1 has at least 90% identity to the amino acid sequence of SEQ ID NO: 4, the CDR H2 has at least 90% identity to the amino acid sequence of SEQ ID NO: 5, and the CDR H3 has at least 90% identity to the amino acid sequence of SEQ ID NO:

6.

20. 18. The antibody or recombinant antibody of any one of claims 12 to 17, wherein the CDR H1 has at least 95% identity to the amino acid sequence of SEQ ID NO: 4, the CDR H2 has at least 95% identity to the amino acid sequence of SEQ ID NO: 5, and the CDR H3 has at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

21. 18. The antibody or recombinant antibody of any one of claims 12 to 17, wherein the CDR H1 has at least 99% identity to the amino acid sequence of SEQ ID NO: 4, the CDR H2 has at least 99% identity to the amino acid sequence of SEQ ID NO: 5, and the CDR H3 has at least 99% identity to the amino acid sequence of SEQ ID NO:

6.

22. 18. The antibody or recombinant antibody of any one of claims 12 to 17, wherein the CDR H1 has at least 100% identity to the amino acid sequence of SEQ ID NO: 4, the CDR H2 has at least 100% identity to the amino acid sequence of SEQ ID NO: 5, and the CDR H3 has at least 100% identity to the amino acid sequence of SEQ ID NO:

6.

23. A heavy chain variable region (V) having at least 80% identity to the amino acid sequence of SEQ ID NO: 8 Ｈ ) an antibody or recombinant antibody comprising:

24. The heavy chain variable region (V Ｈ 24. The antibody or recombinant antibody of claim 23, wherein the amino acid sequence of SEQ ID NO: 8 is at least 90% identical to the amino acid sequence of SEQ ID NO:

8.

25. The heavy chain variable region (V Ｈ 24. The antibody or recombinant antibody of claim 23, wherein the amino acid sequence of SEQ ID NO: 8 is at least 95% identical to the amino acid sequence of SEQ ID NO:

8.

26. The heavy chain variable region (V Ｈ 24. The antibody or recombinant antibody of claim 23, wherein the amino acid sequence of SEQ ID NO: 8 is at least 99% identical to the amino acid sequence of SEQ ID NO:

8.

27. The heavy chain variable region (V Ｈ 24. The antibody or recombinant antibody of claim 23, wherein the amino acid sequence of SEQ ID NO: 8 is at least 100% identical to the amino acid sequence of SEQ ID NO:

8.

28. A light chain variable region (V) having at least 80% identity to the amino acid sequence of SEQ ID NO:

7. Ｌ 28. The antibody or recombinant antibody of any one of claims 23 to 27, further comprising:

29. A light chain variable region (V) having at least 90% identity to the amino acid sequence of SEQ ID NO:

7. Ｌ 28. The antibody or recombinant antibody of any one of claims 23 to 27, further comprising:

30. A light chain variable region (V) having at least 95% identity to the amino acid sequence of SEQ ID NO:7 Ｌ 28. The antibody or recombinant antibody of any one of claims 23 to 27, further comprising:

31. A light chain variable region (V) having at least 99% identity to the amino acid sequence of SEQ ID NO:

7. Ｌ 28. The antibody or recombinant antibody of any one of claims 23 to 27, further comprising:

32. A light chain variable region (V) having at least 100% identity to the amino acid sequence of SEQ ID NO:

7. Ｌ 28. The antibody or recombinant antibody of any one of claims 23 to 27, further comprising:

33. A light chain variable region (V) having at least 99% identity to the amino acid sequence of SEQ ID NO:

7. Ｌ ) an antibody or recombinant antibody comprising:

34. The light chain variable region (V Ｌ 34. The antibody or recombinant antibody of claim 33, wherein the amino acid sequence of SEQ ID NO: 7 is at least 100% identical to the amino acid sequence of SEQ ID NO:

7.

35. A heavy chain variable region (V) having at least 80% identity to the amino acid sequence of SEQ ID NO: 8 Ｈ 35. The antibody or recombinant antibody of any one of claims 33 to 34, further comprising:

36. A heavy chain variable region (V) having at least 90% identity to the amino acid sequence of SEQ ID NO:8 Ｈ 35. The antibody or recombinant antibody of any one of claims 33 to 34, further comprising:

37. A heavy chain variable region (V) having at least 95% identity to the amino acid sequence of SEQ ID NO:8 Ｈ 35. The antibody or recombinant antibody of any one of claims 33 to 34, further comprising:

38. A heavy chain variable region (V) having at least 99% identity to the amino acid sequence of SEQ ID NO:8 Ｈ 35. The antibody or recombinant antibody of any one of claims 33 to 34, further comprising:

39. A heavy chain variable region (V) having at least 100% identity to the amino acid sequence of SEQ ID NO:

8. Ｈ 35. The antibody or recombinant antibody of any one of claims 33 to 34, further comprising:

40. An antibody or recombinant antibody comprising a complementarity determining region (CDR) H1 having at least 80% identity to the amino acid sequence of SEQ ID NO:4, a CDR H2 having at least 80% identity to the amino acid sequence of SEQ ID NO:5, and a CDR H3 having at least 80% identity to the amino acid sequence of SEQ ID NO:

6.

41. 41. The antibody or recombinant antibody of claim 40, comprising a complementarity determining region (CDR) H1 having at least 90% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 90% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 90% identity to the amino acid sequence of SEQ ID NO:

6.

42. 41. The antibody or recombinant antibody of claim 40, comprising a complementarity determining region (CDR) H1 having at least 95% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 95% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

43. 41. The antibody or recombinant antibody of claim 40, comprising a complementarity determining region (CDR) H1 having at least 99% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 99% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 99% identity to the amino acid sequence of SEQ ID NO:

6.

44. 41. The antibody or recombinant antibody of claim 40, comprising a complementarity determining region (CDR) H1 having at least 100% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 100% identity to the amino acid sequence of SEQ ID NO: 5, and a CDR H3 having at least 100% identity to the amino acid sequence of SEQ ID NO:

6.

45. 45. The antibody or recombinant antibody of any one of claims 40 to 44, further comprising a light chain sequence comprising at least one complementarity determining region (CDR) L1 having at least 80% identity to the amino acid sequence of SEQ ID NO: 1, CDR L2 having at least 80% identity to the amino acid sequence of SEQ ID NO: 2, and CDR L3 having at least 80% identity to the amino acid sequence of SEQ ID NO:

3.

46. 46. ​​The antibody or recombinant antibody of claim 45, wherein the CDR L1 has at least 90% identity to the amino acid sequence of SEQ ID NO: 1, the CDR L2 has at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and the CDR L3 has at least 90% identity to the amino acid sequence of SEQ ID NO:

3.

47. 46. ​​The antibody or recombinant antibody of claim 45, wherein the CDR L1 has at least 95% identity to the amino acid sequence of SEQ ID NO: 1, the CDR L2 has at least 95% identity to the amino acid sequence of SEQ ID NO: 2, and the CDR L3 has at least 95% identity to the amino acid sequence of SEQ ID NO:

3.

48. 46. ​​The antibody or recombinant antibody of claim 45, wherein the CDR L1 has at least 99% identity to the amino acid sequence of SEQ ID NO: 1, the CDR L2 has at least 99% identity to the amino acid sequence of SEQ ID NO: 2, and the CDR L3 has at least 99% identity to the amino acid sequence of SEQ ID NO:

3.

49. 46. ​​The antibody or recombinant antibody of claim 45, wherein the CDR L1 has at least 100% identity to the amino acid sequence of SEQ ID NO: 1, the CDR L2 has at least 100% identity to the amino acid sequence of SEQ ID NO: 2, and the CDR L3 has at least 100% identity to the amino acid sequence of SEQ ID NO:

3.

50. A light chain variable region (V) having at least 80% identity to the amino acid sequence of SEQ ID NO:

7. Ｌ 45. The antibody or recombinant antibody of any one of claims 40 to 44, further comprising:

51. Light chain variable region (V Ｌ 50. The antibody or recombinant antibody of any one of claims 45 to 49, wherein said antibody or recombinant antibody has at least 80% identity to the amino acid sequence of SEQ ID NO:

7.

52. A heavy chain variable region (V) having at least 80% identity to the amino acid sequence of SEQ ID NO: 8 Ｈ 52. The antibody or recombinant antibody of any one of claims 40 to 51, comprising:

53. An antibody or recombinant antibody comprising a light chain sequence comprising a complementarity determining region (CDR) L1 having at least 99% identity to the amino acid sequence of SEQ ID NO:1, a CDR L2 having at least 99% identity to the amino acid sequence of SEQ ID NO:2, and a CDR L3 having at least 99% identity to the amino acid sequence of SEQ ID NO:

3.

54. An antibody or recombinant antibody comprising a light chain sequence comprising a complementarity determining region (CDR) L1 having at least 100% identity to the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 100% identity to the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 100% identity to the amino acid sequence of SEQ ID NO:

3.

55. 55. The antibody or recombinant antibody of any one of claims 53 to 54, further comprising a heavy chain sequence comprising at least one complementarity determining region (CDR) H1 having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, CDR H2 having at least 80% identity to the amino acid sequence of SEQ ID NO: 5, and CDR H3 having at least 80% identity to the amino acid sequence of SEQ ID NO:

6.

56. 56. The antibody or recombinant antibody of claim 55, wherein the CDR H1 has at least 90% identity to the amino acid sequence of SEQ ID NO: 4, the CDR H2 has at least 90% identity to the amino acid sequence of SEQ ID NO: 5, and the CDR H3 has at least 90% identity to the amino acid sequence of SEQ ID NO:

6.

57. 56. The antibody or recombinant antibody of claim 55, wherein the CDR H1 has at least 95% identity to the amino acid sequence of SEQ ID NO: 4, the CDR H2 has at least 95% identity to the amino acid sequence of SEQ ID NO: 5, and the CDR H3 has at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

58. 56. The antibody or recombinant antibody of claim 55, wherein the CDR H1 has at least 99% identity to the amino acid sequence of SEQ ID NO: 4, the CDR H2 has at least 99% identity to the amino acid sequence of SEQ ID NO: 5, and the CDR H3 has at least 99% identity to the amino acid sequence of SEQ ID NO:

6.

59. 56. The antibody or recombinant antibody of claim 55, wherein the CDR H1 has at least 100% identity to the amino acid sequence of SEQ ID NO: 4, the CDR H2 has at least 100% identity to the amino acid sequence of SEQ ID NO: 5, and the CDR H3 has at least 100% identity to the amino acid sequence of SEQ ID NO:

6.

60. A heavy chain variable region (V) having at least 80% identity to the amino acid sequence of SEQ ID NO: 8 Ｈ 55. The antibody or recombinant antibody of any one of claims 53 to 54, further comprising:

61. The heavy chain variable region (V Ｈ 60. The antibody or recombinant antibody of any one of claims 55 to 59, wherein said antibody or recombinant antibody has at least 80% identity to the amino acid sequence of SEQ ID NO:

8.

62. The light chain sequence comprises a light chain variable region (V) having at least 80% identity to the amino acid sequence of SEQ ID NO:

7. Ｌ 60. The antibody or recombinant antibody of any one of claims 53 to 59, comprising:

63. 63. The antibody or recombinant antibody of any one of claims 1 to 62, further comprising a human heavy chain constant region or a human light chain constant region.

64. 64. The antibody or recombinant antibody of claim 63, wherein the human heavy chain constant region is IgG1 or IgG4, or a fragment thereof.

65. 65. The antibody or recombinant antibody of any one of claims 1 to 64, wherein the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO:

10.

66. 66. The antibody or recombinant antibody of any one of claims 1 to 65, wherein the light chain has at least 80% identity to the amino acid sequence of SEQ ID NO:

9.

67. 67. The antibody or recombinant antibody of any one of claims 1 to 64 or 66, wherein the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO:

12.

68. 67. The antibody or recombinant antibody of any one of claims 1 to 65 or 66, wherein the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO:

11.

69. 65. The antibody or recombinant antibody of any one of claims 1 to 64, wherein the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO:

13.

70. 70. The antibody or recombinant antibody of any one of claims 1 to 69, comprising a human variable framework region and a mouse constant region.

71. 71. The antibody or recombinant antibody of claim 70, further comprising a mouse heavy chain constant region or a mouse light chain constant region.

72. 72. The antibody or recombinant antibody of claim 71, wherein the mouse heavy chain constant region is IgG2A.

73. 73. The antibody or recombinant antibody of claim 72, wherein the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO:

15.

74. 73. The antibody or recombinant antibody of claim 72, wherein the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO:

16.

75. 73. The antibody or recombinant antibody of claim 72, wherein the light chain has at least 80% identity to the amino acid sequence of SEQ ID NO:

14.

76. Single heavy chain, single light chain, Fab, Fab', F(ab)', F(ab') ２ , Fd, scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, bispecific Fab ２ , trispecific Fab ３ 76. The antibody or recombinant antibody of any one of claims 1 to 75, which is an antibody fragment including a single-chain binding polypeptide, a dAb fragment, or a diabody.

77. and specifically binds to the CD163 protein expressed on immunosuppressive human myeloid cells, and the binding of said antibody or recombinant antibody to said myeloid cells is determined by the following parameters: (i) CD4 ＋ T cells, CD8 ＋ Activation of T cells, NK cells, or any combination thereof, and (ii) CD4 ＋ T cells, CD8 ＋ Proliferation of T cells, NK cells, or any combination thereof 77. The antibody or recombinant antibody of any one of claims 1 to 76, which enhances immune cell function as measured by one or both of the following:

78. CD4 ＋ T cells, CD8 ＋ 78. The antibody or recombinant antibody of claim 77, wherein activation of T cells, NK cells, or any combination thereof is measured as an increase in the levels of IFN-γ, TNF-α, or perforin, or any combination thereof.

79. 78. The antibody or recombinant antibody of claim 77, wherein the immunosuppressive human myeloid cells are macrophages or myeloid-derived suppressor cells.

80. 78. The antibody or recombinant antibody of claim 77, wherein the immune cell function is in a tumor microenvironment.

81. 81. The antibody or recombinant antibody of any one of claims 77 to 80, wherein the immune cell function is in vivo.

82. 77. The antibody or recombinant antibody of any one of claims 1 to 76, which specifically binds to CD163 protein expressed on human macrophages, and wherein binding of the antibody or recombinant antibody to the macrophages increases immunostimulatory activity in the tumor microenvironment.

83. 83. The antibody or recombinant antibody of claim 82, wherein the tumor microenvironment is in vivo.

84. 84. The antibody or recombinant antibody of any one of claims 79 to 83, wherein binding of the antibody or recombinant antibody to the macrophage reduces the immunosuppressive activity of the macrophage.

85. 85. The antibody or recombinant antibody of any one of claims 79 to 84, wherein binding of the antibody or recombinant antibody to the macrophage reduces the tumor-promoting activity of the macrophage.

86. 86. The antibody or recombinant antibody of any one of claims 79 to 85, wherein the macrophage is a tumor-associated macrophage.

87. 86. An antibody or recombinant antibody according to any one of claims 79 to 85, which modifies the expression of at least one marker on macrophages.

88. 87. An antibody or recombinant antibody according to any one of claims 77 to 86, wherein the CD163 protein is a glycoform of CD163.

89. 88. An antibody or recombinant antibody according to any one of claims 77 to 87, wherein the CD163 protein is the 150 kDa glycoform of CD163.

90. 90. An antibody or recombinant antibody according to any one of claims 79 to 89, which does not specifically bind to the 130 kDa glycoform of CD163 expressed by human macrophages.

91. 91. An antibody or recombinant antibody according to any one of claims 1 to 90, having a constant domain capable of binding to an Fc receptor.

92. 92. The antibody or recombinant antibody of claim 91, wherein the Fc receptor is expressed on macrophages.

93. Single heavy chain, single light chain, Fab, Fab', F(ab)', F(ab') ２ , Fd, scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, bispecific Fab ２ , trispecific Fab ３ 93. The antibody or recombinant antibody of any one of claims 1 to 92, having an antibody fragment comprising a single-chain binding polypeptide, a dAb fragment, or a diabody.

94. The antibody or recombinant antibody of any one of claims 79 to 93, wherein the at least one marker on human macrophages is CD16, CD64, TLR2, or Siglec-15.

95. 94. The antibody or recombinant antibody of any one of claims 79 to 93, wherein the human macrophage is an M2 macrophage or an M2-like macrophage.

96. 94. The antibody or recombinant antibody of any one of claims 79 to 93, wherein the human macrophage is an M2a, M2b, M2c, or M2d macrophage.

97. 97. The antibody or recombinant antibody of any one of claims 79 to 96, wherein the CD163 protein is a component of a cell surface complex comprising at least one other protein expressed by the macrophage.

98. 98. The antibody or recombinant antibody of claim 97, wherein the at least one other protein is a galectin-1 protein, a LILRB2 protein, a casein kinase II protein, or any combination thereof.

99. 99. The antibody or recombinant antibody of any one of claims 79 to 98, which is internalized by the human macrophage upon binding to the CD163 protein.

100. 100. The antibody or recombinant antibody of any one of claims 79 to 99, wherein binding to the CD163 protein is not cytotoxic to the macrophage.

101. The binding to the CD163 protein is ＋ T cell activation, CD4 ＋ T cell proliferation, or CD4 ＋ 101. The antibody or recombinant antibody of any one of claims 79 to 100, which promotes both T cell activation and proliferation.

102. The binding to the CD163 protein is ＋ 102. The antibody or recombinant antibody of any one of claims 77 to 101, which promotes expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by T cells.

103. The binding to the CD163 protein is ＋ T cell activation, CD8 ＋ T cell proliferation, or CD8 ＋ 103. The antibody or recombinant antibody of any one of claims 77 to 102, which promotes both T cell activation and proliferation.

104. The binding to the CD163 protein is ＋ 104. The antibody or recombinant antibody of any one of claims 77 to 103, which promotes expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by T cells.

105. 105. The antibody or recombinant antibody of any one of claims 77 to 104, wherein binding to the CD163 protein reduces immunosuppression in the tumor microenvironment.

106. 106. The antibody or recombinant antibody of any one of claims 77 to 105, wherein binding to the CD163 protein promotes tumor cell killing in the tumor microenvironment.

107. 107. An antibody or recombinant antibody according to any one of claims 77 to 106, wherein binding to the CD163 protein promotes cytotoxic lymphocyte-mediated cancer cell killing.

108. 108. The antibody or recombinant antibody of any one of claims 77 to 107, wherein binding to the CD163 protein promotes NK cell-mediated tumor cell killing.

109. The antibody or recombinant antibody of any one of claims 77 to 108, wherein binding to the CD163 protein promotes the expression of IL-2 by T cells.

110. The binding to the CD163 protein is ＋ T cells, CD196 － T cells, CXCR3 ＋ T cells, CCR4 － 110. The antibody or recombinant antibody of any one of claims 77 to 109, which increases T cells, or any combination thereof.

111. An antibody or recombinant antibody described in any one of claims 77 to 110, wherein binding to CD163 reduces immunosuppression caused by macrophages in the tumor microenvironment.

112. It specifically binds to the CD163 protein expressed on human macrophages, and the binding has the following effects: (a) a decrease in the expression by macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) internalization of antibodies by macrophages; (c) secretion of IFN-γ, TNF-α, and perforin; (d) CD4 ＋ T cells, CD8 ＋ Activation of T cells, NK cells, or any combination thereof; (e) CD4 ＋ T cells, CD8 ＋ Proliferation of T cells, NK cells, or any combination thereof; and (f) promoting tumor cell death in the tumor microenvironment 112. An antibody or recombinant antibody according to any one of claims 1 to 111, which results in at least one of:

113. 113. The antibody or recombinant antibody of claim 112, wherein the binding results in two or more of (a) through (e), three or more of (a) through (e), four or more of (a) through (e), or all of (a) through (e).

114. CD163 in the tumor microenvironment ＋ 114. The antibody or recombinant antibody of any one of claims 1 to 113, which specifically binds to immunosuppressive myeloid cells, and wherein this binding reduces the inhibition of cytotoxic T cell-mediated killing of tumor cells in the tumor microenvironment.

115. 115. The antibody or recombinant antibody of any one of claims 1 to 114, which specifically binds to CD163 protein expressed on human tumor-associated macrophages and reduces the expression of CD16, CD64, TLR2, Siglec-15, or any combination thereof, by said macrophages, for use in a method for treating cancer.

116. 116. The antibody or recombinant antibody of any one of claims 79 to 115, wherein binding of the antibody or recombinant antibody to the macrophage modulates cellular immune function in the tumor microenvironment.

117. 116. The antibody or recombinant antibody of any one of claims 79 to 115, wherein binding of the antibody or recombinant antibody to the macrophages promotes anti-tumor immune function.

118. An antibody or recombinant antibody described in any one of claims 1 to 117, which specifically binds to a CD163 epitope comprising the amino acid sequence of SEQ ID NO:

18.

119. An antibody or recombinant antibody described in any one of claims 1 to 117, which specifically binds to a CD163 epitope comprising the amino acid sequence of SEQ ID NO:

19.

120. An antibody or recombinant antibody described in any one of claims 1 to 117, which specifically binds to a CD163 epitope comprising the amino acid sequence of SEQ ID NO:

20.

121. An antibody or recombinant antibody described in any one of claims 1 to 117, which specifically binds to a CD163 epitope comprising the amino acid sequences of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively.

122. K between 1 nM and 100 nM Ｄ 122. An antibody or recombinant antibody according to any one of claims 1 to 121, which specifically binds to CD163 with

123. K between 1 nM and 50 nM Ｄ 123. The antibody or recombinant antibody of claim 122, which specifically binds to CD163.

124. K between 1 nM and 10 nM Ｄ 124. The antibody or recombinant antibody of claim 123, which specifically binds to CD163.

125. K between 1 nM and 100 nM Ｄ 122. The antibody or recombinant antibody of any one of claims 1 to 121, which specifically binds to human M2c macrophages with

126. K between 1 nM and 50 nM Ｄ 126. The antibody or recombinant antibody of claim 125, which specifically binds to human M2c macrophages with

127. K between 1 nM and 10 nM Ｄ 127. The antibody or recombinant antibody of claim 126, which specifically binds to human M2c macrophages.

128. 128. A composition comprising an antibody or recombinant antibody according to any one of claims 1 to 127 and an excipient.

129. 128. A pharmaceutical composition comprising an antibody or recombinant antibody according to any one of claims 1 to 127 and a pharmaceutically acceptable excipient.

130. 130. Use of an antibody or recombinant antibody according to any one of claims 1 to 127 in the manufacture of a medicament for treating cancer in a human subject.

131. 130. Use of an antibody or recombinant antibody according to any one of claims 1 to 127 in the manufacture of a medicament for reducing immunosuppression by tumor-associated macrophages in a human subject with cancer.

132. 130. Use of an antibody or recombinant antibody according to any one of claims 1 to 127 in the manufacture of a medicament for promoting T cell-mediated tumor cell killing in a human subject with cancer.

133. 1. A method of promoting immune cell function in a subject in need thereof, comprising: Administering the antibody or recombinant antibody of any one of claims 1 to 132 to an individual in need thereof; The following parameters (i) CD4 ＋ T cells, CD8 ＋ Activation of T cells, NK cells, or any combination thereof, and (ii) CD4 ＋ T cells, CD8 ＋ Proliferation of T cells, NK cells, or any combination thereof promoting immune cell function as measured by one or both of A method comprising:

134. CD4 ＋ T cells, CD8 ＋ 134. The method of embodiment 133, wherein the activation of T cells, NK cells, or any combination thereof, is measured as an increase in the levels of IFN-γ, TNF-α, or perforin, or any combination thereof.

135. 134. The method of claim 133, wherein the immunosuppressive human bone marrow cells are macrophages.

136. 136. The method of claim 135, wherein said immunosuppressive human myeloid cells are myeloid-derived suppressor cells.

137. 137. The method of claim 136, wherein the antibody is an antibody or recombinant antibody described in any one of claims 76 to 127.

138. A method for treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody or recombinant antibody described in any one of claims 1 to 127, thereby treating the individual's cancer.

139. A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody or recombinant antibody described in any one of claims 1 to 127, thereby reducing immunosuppression by tumor-associated macrophages in the individual.

140. 128. A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody or recombinant antibody of any one of claims 1 to 127, thereby increasing T cell-mediated tumor cell killing in the individual.

141. A method for reducing tumor-promoting activity of tumor-associated macrophages in an individual in need thereof, comprising administering to said individual a CD4 ＋ T cell activation, CD4 ＋ T cell proliferation, CD8 ＋ T cell activation, CD8 ＋ 128. A method comprising administering to said individual a therapeutically effective amount of an antibody or recombinant antibody of any one of claims 1 to 127 effective to regulate T cell proliferation, T cell proliferation, or any combination thereof.

142. A method for promoting lymphocyte-mediated tumor cell killing in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody or recombinant antibody described in any one of claims 1 to 127.

143. 143. The method of any one of claims 138 to 142, wherein the cancer is lung cancer or sarcoma.

144. 144. The method of claim 143, wherein the lung cancer is lung carcinoma or lung adenocarcinoma.

145. 143. The method of any one of claims 138 to 142, wherein the antibody or recombinant antibody binds to macrophages in the tumor microenvironment.

146. 143. The method of any one of claims 138 to 142, further comprising administering to the individual an additional anti-cancer therapy.

147. 147. The method of claim 146, wherein the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, and cytokine therapy, and combinations thereof.

148. 148. The method of claim 147, wherein the additional anti-cancer therapy is immunotherapy.

149. 149. The method of claim 148, wherein the immunotherapy is a composition comprising a checkpoint inhibitor.

150. A method for modulating the activity of tumor-associated macrophages in a tumor microenvironment, comprising binding to CD163-expressing human macrophages and exerting the following effects: (a) binding of the antibody reduces the expression on macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) upon binding of the antibody to CD163 protein, the antibody is internalized by human macrophages; (c) the binding of the antibody is not cytotoxic to macrophages; (d) binding of the antibody increases the levels of IFN-γ, TNF-α, and perforin; (e) the binding of the antibody is ＋ T cells, CD8 ＋ promotes activation of T cells, NK cells, or any combination thereof; (f) the binding of the antibody is detected by CD4 ＋ T cells, CD8 ＋ Promoting the proliferation of T cells, NK cells, or any combination thereof; and (g) binding of the antibody promotes tumor cell killing in the tumor microenvironment.

128. A method comprising contacting the tumor-associated macrophages with an antibody or recombinant antibody according to any one of claims 1 to 127, comprising at least one of:

151. 151. The method of claim 150, wherein the combination results in two or more of (a)-(g), three or more of (a)-(g), four or more of (a)-(g), five or more of (a)-(g), six or more of (a)-(g), or all of (a)-(g).

152. 1. An antibody that specifically binds to CD163 protein expressed on immunosuppressive human myeloid cells, wherein the binding of the antibody to the myeloid cells is determined by the following parameters: (i) CD4 ＋ T cells, CD8 ＋ Activation of T cells, NK cells, or any combination thereof, and (ii) CD4 ＋ T cells, CD8 ＋ Proliferation of T cells, NK cells, or any combination thereof The method of claim 1, wherein the method promotes immune cell function as measured by one or both of the following:

153. The antibody of claim 152, wherein the immunosuppressive human myeloid cells are macrophages or myeloid-derived suppressor cells.

154. An antibody that specifically binds to CD163 protein expressed on human macrophages, wherein the binding of the antibody to the macrophages is determined by the following parameters: (i) CD4 ＋ T cells, CD8 ＋ Activation of T cells, NK cells, or any combination thereof, and (ii) CD4 ＋ T cells, CD8 ＋ Proliferation of T cells, NK cells, or any combination thereof Antibodies that promote immune cell function as measured by one or both of the following:

155. The antibody of any one of claims 152 to 154, wherein the immune cell function is in a tumor microenvironment.

156. 156. The antibody of any one of claims 152 to 155, wherein the immune cell function is in vivo.

157. An antibody that specifically binds to CD163 protein expressed on human macrophages, wherein binding of the antibody to the macrophages increases immunostimulatory activity in the tumor microenvironment.

158. The antibody of claim 157, wherein the tumor microenvironment is in vivo.

159. 159. The antibody of any one of claims 153 to 158, wherein binding of the antibody to the macrophage reduces the immunosuppressive activity of the macrophage.

160. 160. The antibody of any one of claims 153 to 159, wherein binding of the antibody to the macrophage reduces tumor-promoting activity of the macrophage.

161. 161. The antibody of any one of claims 150 to 160, wherein the macrophage is a tumor-associated macrophage.

162. The antibody of any one of claims 153 to 161, wherein the antibody modifies the expression of at least one marker on the macrophage.

163. An antibody described in any one of claims 152 to 162, wherein the CD163 protein is a glycoform of CD163.

164. An antibody described in any one of claims 152 to 163, wherein the CD163 protein is a 150 kDa glycoform of CD163.

165. An antibody described in any one of claims 153 to 164, which does not specifically bind to the 130 kDa glycoform of CD163 expressed by human macrophages.

166. An antibody described in any one of claims 152 to 165, having a constant domain that enables binding to an Fc receptor.

167. The antibody of claim 166, wherein the Fc receptor is expressed on macrophages.

168. Single heavy chain, single light chain, Fab, Fab', F(ab)', F(ab') ２ , Fd, scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, bispecific Fab ２ , trispecific Fab ３ 168. The antibody of any one of claims 152 to 167, having an antibody fragment comprising a single-chain binding polypeptide, a dAb fragment, or a diabody.

169. The antibody of any one of claims 153 to 168, wherein the at least one marker on human macrophages is CD16, CD64, TLR2, or Siglec-15.

170. 170. The antibody or recombinant antibody of any one of claims 153 to 169, wherein the human macrophage is an M2 macrophage or an M2-like macrophage.

171. The antibody of claim 170, wherein the human macrophage is an M2a, M2b, M2c, or M2d macrophage.

172. An antibody described in any one of claims 153 to 171, wherein the CD163 protein is a component of a cell surface complex comprising at least one other protein expressed by the macrophage.

173. 173. The antibody of claim 172, wherein the at least one other protein is a galectin-1 protein, a LILRB2 protein, a casein kinase II protein, or any combination thereof.

174. An antibody described in any one of claims 153 to 173, which is internalized by the human macrophage upon binding to the CD163 protein.

175. An antibody described in any one of claims 153 to 174, wherein binding to the CD163 protein is not cytotoxic to the macrophage.

176. The binding to the CD163 protein is ＋ T cell activation, CD4 ＋ T cell proliferation, or CD4 ＋ An antibody described in any one of claims 152 to 175, which promotes both T cell activation and proliferation.

177. The binding to the CD163 protein is ＋ 177. The antibody of any one of claims 152 to 176, which promotes expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by T cells.

178. The binding to the CD163 protein is ＋ T cell activation, CD8 ＋ T cell proliferation, or CD8 ＋ An antibody described in any one of claims 152 to 177, which promotes both T cell activation and proliferation.

179. The binding to the CD163 protein is ＋ 179. The antibody of any one of claims 152 to 178, which promotes expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by T cells.

180. The antibody of any one of claims 152 to 179, wherein binding to the CD163 protein reduces immunosuppression in the tumor microenvironment.

181. An antibody described in any one of claims 152 to 180, wherein binding to the CD163 protein promotes cell death in the tumor microenvironment.

182. An antibody described in any one of claims 152 to 181, wherein binding to the CD163 protein promotes cytotoxic lymphocyte-mediated cancer cell killing.

183. An antibody described in any one of claims 152 to 182, wherein binding to the CD163 protein promotes NK cell-mediated tumor cell killing.

184. The antibody of any one of claims 152 to 183, wherein binding to the CD163 protein promotes expression of IL-2 by T cells.

185. The binding to the CD163 protein is ＋ T cells, CD196-T cells, CXCR3 ＋ T cells, CCR4 － 185. The antibody of any one of claims 152 to 184, which increases T cells, or any combination thereof.

186. An antibody described in any one of claims 152 to 185, wherein binding to CD163 reduces immunosuppression in the tumor microenvironment caused by macrophages.

187. An antibody that specifically binds to the CD163 protein expressed on human macrophages, wherein the binding has the following effects: (a) a decrease in the expression by macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) internalization of antibodies by macrophages; (c) CD4 ＋ T cells, CD8 ＋ Activation of T cells, NK cells, or any combination thereof; (d) CD4 ＋ T cells, CD8 ＋ Proliferation of T cells, NK cells, or any combination thereof; and (e) promoting tumor cell death in the tumor microenvironment An antibody that provides at least one of the following:

188. The antibody of claim 187, wherein the binding results in two or more of (a) through (e), three or more of (a) through (e), four or more of (a) through (e), or all of (a) through (e).

189. CD163 in the tumor microenvironment ＋ An antibody that specifically binds to immunosuppressive myeloid cells, whereby this binding reduces the inhibition of cytotoxic T cell-mediated killing of tumor cells in the tumor microenvironment.

190. An antibody that specifically binds to CD163 protein expressed on human tumor-associated macrophages and reduces the expression of CD16, CD64, TLR2, Siglec-15, or any combination thereof, by the macrophages, for use in a method for treating cancer.

191. 191. The antibody of any one of claims 153 to 190, wherein binding of the antibody to the macrophage modulates cellular immune function in the tumor microenvironment.

192. 192. The antibody of any one of claims 153 to 191, wherein binding of the antibody to the macrophage promotes anti-tumor immune function.

193. 193. A composition comprising an antibody according to any one of claims 152 to 192 and an excipient.

194. 193. A pharmaceutical composition comprising an antibody according to any one of claims 152 to 192 and a pharmaceutically acceptable carrier.

195. 20. Use of an antibody according to any one of claims 152 to 192 in the manufacture of a medicament for treating cancer in a human subject.

196. 20. Use of an antibody according to any one of claims 152 to 192 in the manufacture of a medicament for reducing immunosuppression by tumor-associated macrophages in a human subject with cancer.

197. 20. Use of an antibody according to any one of claims 152 to 192 in the manufacture of a medicament for promoting T cell-mediated tumor cell killing in a human subject with cancer.

198. 1. A method for promoting immune cell function, comprising: allowing an antibody to specifically bind to CD163 protein expressed on immunosuppressive human myeloid cells; The following parameters (i) CD4 ＋ T cells, CD8 ＋ Activation of T cells, NK cells, or any combination thereof, and (ii) CD4 ＋ T cells, CD8 ＋ Proliferation of T cells, NK cells, or any combination thereof promoting immune cell function as measured by one or both of A method comprising:

199. 199. The method of claim 198, wherein the immunosuppressive human bone marrow cells are macrophages.

200. 199. The method of claim 198, wherein said immunosuppressive human myeloid cells are myeloid-derived suppressor cells.

201. 200. The method of claim 198, wherein the antibody is an antibody described in any one of claims 152 to 192.

202. 20. A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody described in any one of claims 152 to 192, thereby treating the individual's cancer.

203. 20. A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody described in any one of claims 152 to 192, thereby reducing immunosuppression by tumor-associated macrophages in the individual.

204. 20. A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody of any one of claims 152 to 192, thereby increasing T cell-mediated tumor cell killing in the individual.

205. 205. The method of any one of claims 198 to 204, wherein the cancer is lung cancer or sarcoma.

206. The method of claim 205, wherein the lung cancer is lung carcinoma or lung adenocarcinoma.

207. A method for reducing tumor-promoting activity of tumor-associated macrophages in an individual in need thereof, comprising administering to said individual CD4 ＋ T cell activation, CD4 ＋ T cell proliferation, CD8 ＋ T cell activation, CD8 ＋ 195. A method comprising administering to an individual an amount of the pharmaceutical composition described in claim 194 effective to regulate T cell proliferation, T cell proliferation, or any combination thereof.

208. 208. The method of claim 207, further comprising promoting tumor cell death in the tumor microenvironment.

209. 195. A method for promoting lymphocyte-mediated tumor cell killing in an individual in need thereof, comprising administering to said individual an effective amount of the pharmaceutical composition of claim 194.

210. A method for modulating the activity of tumor-associated macrophages in a tumor microenvironment, comprising binding to CD163-expressing human macrophages and exerting the following effects: (a) binding of the antibody reduces the expression on macrophages of at least one marker that is CD16, CD64, TLR2, or Siglec-15; (b) upon binding of the antibody to CD163 protein, the antibody is internalized by human macrophages; (c) the binding of the antibody is not cytotoxic to macrophages; (d) the binding of the antibody is ＋ T cells, CD8 ＋ promotes activation of T cells, NK cells, or any combination thereof; (e) the binding of the antibody is ＋ T cells, CD8 ＋ Promoting the proliferation of T cells, NK cells, or any combination thereof; and (f) binding of the antibody promotes tumor cell killing in the tumor microenvironment. contacting the tumor-associated macrophages with an antibody comprising at least one of:

211. 211. The method of claim 210, wherein the combination results in two or more of (a)-(f), three or more of (a)-(f), four or more of (a)-(f), five or more of (a)-(f), or all of (a)-(f).

212. 212. The method of any one of claims 198 to 211, wherein the method is performed in a tumor microenvironment.

213. 213. The method of any one of claims 198 to 212, wherein the method is performed in vivo.

214. 214. The method of any one of claims 198 to 213, wherein binding of the antibody to the macrophage modulates cellular immune function in the tumor microenvironment.

215. 215. The method of any one of claims 198 to 214, wherein binding of the antibody to the macrophage promotes anti-tumor immune function.

216. The method of any one of claims 198 to 215, wherein the antibody comprises a constant domain, and the constant domain binds to an Fc receptor.

217. 217. The method of claim 216, wherein the Fc receptor is expressed on a macrophage.

218. 218. The method of any one of claims 198 to 217, further comprising the step of internalizing the antibody by the human macrophage upon binding to the CD163 protein.

219. The method of any one of claims 198 to 218, wherein binding to the CD163 protein is not cytotoxic to the human macrophage.

220. CD4 ＋ 220. The method of any one of claims 198 to 219, further comprising promoting expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by T cells.

221. CD8 ＋ 221. The method of any one of claims 198-220, further comprising promoting expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof, by T cells.

222. 222. The method of any one of claims 198 to 221, further comprising reducing immunosuppression in the tumor microenvironment.

223. 223. The method of any one of claims 198 to 222, comprising promoting cytotoxic lymphocyte-mediated cancer cell killing.

224. 224. The method of any one of claims 198 to 223, comprising promoting NK cell-mediated tumor cell killing.

225. 225. The method of any one of claims 198 to 224, comprising promoting expression of IL-2 by T cells.

226. CD4 ＋ T cells, CD196 － T cells, CXCR3 ＋ T cells, CCR4 － 226. The method of any one of claims 198 to 225, comprising increasing T cells, or any combination thereof.

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