Immunomodulatory antibodies and methods of use thereof

Abstract

Antibodies and methods of use thereof are provided herein. Antibodies as disclosed herein bind to CD163+ on cells (such as macrophages). These antibodies can be used in therapeutic methods, such as methods for treating cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 876,580, filed on July 19, 2019, U.S. Provisional Application No. 62 / 876,579, filed on July 19, 2019, and U.S. Provisional Application No. 62 / 878,265, filed on July 24, 2019, the entire contents of each of which are incorporated herein by reference.

[0003] Overview of public content

[0004] Provided herein are antibodies that can be used to treat cancer and other disorders, including antigen binding fragments and other antigen binding polypeptides. In some embodiments, the antibody specifically binds to M2 and M2-like immunosuppressive macrophages but does not specifically bind to M1 and M1-like anti-tumor macrophages. The disclosed antibodies bind to M2 and M2-like tumor-associated macrophages and regulate the physical and functional characteristics of tumor-associated M2 and M2-like macrophages to reduce the immunosuppression of the tumor microenvironment and increase cytotoxic T cell activation and proliferation, which promote tumor cell killing. The antibody molecules of the present disclosure specifically bind to human CD163 expressed on the surface of M2 and M2-like macrophages.

[0005] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO:8.

[0006] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain variable region (VH) having at least 90% identity to the amino acid sequence of SEQ ID NO:8.

[0007] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain variable region (VH) having at least 95% identity to the amino acid sequence of SEQ ID NO:8.

[0008] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain variable region (VH) that is at least 99% identical to the amino acid sequence of SEQ ID NO:8.

[0009] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain variable region (VH) having at least 100% identity to the amino acid sequence 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 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 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 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 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 SEQ ID NO: 7.

[0010] In certain embodiments, disclosed herein are light chain variable regions (VL) having at least 80% identity to the amino acid sequence of SEQ ID NO:7.

[0011] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a light chain variable region (VL) that is at least 90% identical to the amino acid sequence of SEQ ID NO:.

[0012] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a light chain variable region (VL) that is at least 95% identical to the amino acid sequence of SEQ ID NO:.

[0013] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a light chain variable region (VL) that is at least 99% identical to the amino acid sequence of SEQ ID NO:.

[0014] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a light chain variable region (VL) having at least 100% identity to the amino acid sequence SEQ ID NO: . 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 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 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 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 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 SEQ ID NO: 8.

[0015] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain variable region (V L ... H ) and a light chain variable region (V L In some embodiments, the antibody or recombinant antibody comprises a light chain variable region (V L ... L In some embodiments, the antibody or recombinant antibody comprises a light chain variable region (V L L) 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 L L) having at least 95% 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 L L) 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 L ... L In some embodiments, the antibody or recombinant antibody comprises a heavy chain variable region (V H In some embodiments, the antibody or recombinant antibody comprises a heavy chain variable region (V HIn some embodiments, the antibody or recombinant antibody comprises a heavy chain variable region (V H In some embodiments, the antibody or recombinant antibody comprises a heavy chain variable region (V H In some embodiments, the antibody or recombinant antibody comprises a heavy chain variable region (V H ).

[0016] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain sequence comprising a complementary determining region (CDR) H1 having at least 80% identity to the amino acid sequence SEQ ID NO: 4, a CDR H2 having at least 80% identity to the amino acid sequence SEQ ID NO: 5, and a CDR H3 having at least 80% identity to the amino acid sequence SEQ ID NO: 6; and a light chain sequence comprising a CDR L1 having at least 80% identity to the amino acid sequence SEQ ID NO: 1, a CDR L2 having at least 80% identity to the amino acid sequence SEQ ID NO: 2, and a CDR L3 having at least 80% identity to the amino acid sequence 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 SEQ ID NO: 1, a CDR L2 having at least 85% identity to the amino acid sequence SEQ ID NO: 2, and a CDR L3 having at least 85% identity to the amino acid sequence 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 with the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 90% identity with 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 with the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 95% identity with the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 95% identity with 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 with the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 99% identity with the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 99% identity with 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 that is at least 100% identical to the amino acid sequence of SEQ ID NO:1, a CDR L2 that is at least 100% identical to the amino acid sequence of SEQ ID NO:2, and a CDR L3 that is at least 100% identical to the amino acid sequence of SEQ ID NO:3.In some embodiments, the antibody or recombinant antibody comprises a heavy chain sequence comprising a CDR H1 having at least 85% identity to the amino acid sequence of SEQ ID NO: 4, a CDR H2 having at least 85% identity to the amino acid sequence of SEQ ID NO: 5, and a 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 a 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. 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.

[0017] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain sequence comprising at least one of: 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.

[0018] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain sequence comprising at least one of: 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.

[0019] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain sequence comprising at least one of: 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.

[0020] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain sequence comprising at least one of: 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.

[0021] In certain embodiments, disclosed herein are antibodies or recombinant antibodies comprising a heavy chain sequence comprising at least one of: 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.

[0022] In some embodiments, the antibody or recombinant antibody further comprises a light chain sequence comprising at least one of the following: a complementary 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. 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 is at least 99% identical to the amino acid sequence of SEQ ID NO: 1, CDR L2 is at least 99% identical to the amino acid sequence of SEQ ID NO: 2, and CDR L3 is at least 99% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, CDRL1 is at least 100% identical to the amino acid sequence of SEQ ID NO: 1, CDR L2 is at least 100% identical to the amino acid sequence of SEQ ID NO: 2, and CDR L3 is at least 100% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the light chain variable region (VL) is at least 80% identical to the amino acid sequence of SEQ ID NO: 7.

[0023] 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.

[0024] In certain embodiments, disclosed herein are antibodies or recombinant antibodies 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.

[0025] In certain embodiments, disclosed herein are antibodies or recombinant antibodies 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.

[0026] In certain embodiments, disclosed herein are antibodies or recombinant antibodies 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.

[0027] In certain embodiments, disclosed herein are antibodies or recombinant antibodies 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.

[0028] In certain embodiments, disclosed herein is 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.

[0029] In some embodiments, the antibody or recombinant antibody further comprises a heavy chain sequence comprising at least one of the following: a complementary 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. 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 is at least 99% identical to the amino acid sequence of SEQ ID NO: 4, CDR H2 is at least 99% identical to the amino acid sequence of SEQ ID NO: 5, and CDR H3 is at least 99% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, CDR H1 is at least 100% identical to the amino acid sequence of SEQ ID NO: 4, CDR H2 is at least 100% identical to the amino acid sequence of SEQ ID NO: 5, and CDR H3 is at least 100% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy chain variable region (VH) is at least 80% identical to the amino acid sequence of SEQ ID NO: 8.

[0030] In some embodiments, the antibody or recombinant antibody further comprises a heavy chain variable region (VH) that is at least 80% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the light chain variable region (VL) is at least 80% identical to the amino acid sequence of SEQ ID NO:7.

[0031] 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 with the amino acid sequence of SEQ ID NO: 10. In some embodiments, the light chain has at least 80% identity with the amino acid sequence of SEQ ID NO: 9. In some embodiments, wherein the heavy chain has at least 80% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain has at least 80% identity with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the heavy chain has at least 80% identity with 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 with 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, 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 or diabody.

[0032] In some embodiments, the antibody or recombinant antibody specifically binds to CD163 protein expressed on immunosuppressive human bone marrow cells, wherein the binding of the antibody or recombinant antibody to the bone marrow cells promotes immune cell function, as measured by one or both of the following parameters: (i) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; and (ii) CD4 + T cells, CD8 + T cells, NK cells, or any combination thereof. In some embodiments, wherein the CD4 + T cells, CD8 +The activation of T cells, NK cells or any combination thereof is measured as the increased level of IFN-γ, TNF-α or perforin or any combination thereof. In some embodiments, immunosuppressive human bone marrow cells are macrophages or suppressive cells of bone marrow origin. In some embodiments, immune cell function is in the tumor microenvironment. In some embodiments, the tumor microenvironment is in vivo. In some embodiments, antibodies or recombinant antibodies specifically bind to CD163 proteins expressed on human macrophages, wherein the combination of antibodies and macrophages increases the immunostimulatory activity in the tumor microenvironment. In some embodiments, the tumor microenvironment is in vivo. In some embodiments, the combination of antibodies or recombinant antibodies with macrophages reduces the immunosuppressive activity of macrophages. In some embodiments, the combination of antibodies or recombinant antibodies with macrophages reduces the tumor promoting activity of macrophages. In some embodiments, macrophages are tumor-associated macrophages. In some embodiments, antibodies or recombinant antibodies change the expression of at least one marker on macrophages. In some embodiments, CD163 protein is CD163 glycoform (glycoform of CD163). In some embodiments, the CD163 protein is a 150kDa CD163 glycoform. In some embodiments, the antibody does not specifically bind to the 130kDa CD163 glycoform expressed by human macrophages.

[0033] In some embodiments, the antibody or recombinant antibody has a constant domain that can bind to Fc receptors. In some embodiments, Fc receptors are expressed on macrophages. In some embodiments, the antibody or recombinant antibody has an antibody fragment comprising the following: a single heavy chain, a single light chain, 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 double antibody. In some embodiments, at least one marker on human macrophages is CD16, CD64, TLR2 or Siglec-15. In some embodiments, human macrophages are M2 macrophages or M2-like macrophages. In some embodiments, human macrophages are M2a, M2b, M2c or M2d macrophages. In some embodiments, CD163 protein is a component of a cell surface complex comprising at least one other protein expressed by macrophages. In some embodiments, 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, after binding to the CD163 protein, the antibody or recombinant antibody is 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 promotes CD4+ T cell activation, CD4 + T cell proliferation, or CD4 + Both T cell activation and proliferation. In some embodiments, binding to the CD163 protein promotes CD4 + T cells express CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof. In some embodiments, binding to the CD163 protein promotes CD8 + T cell activation, CD8 + T cell proliferation, or CD8 + Both T cell activation and proliferation. In some embodiments, binding to the CD163 protein promotes CD8 + T cells express ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof. In some embodiments, the combination with CD163 protein reduces immunosuppression in the tumor microenvironment. In some embodiments, the combination with CD163 protein promotes tumor cell killing in the tumor microenvironment. In some embodiments, the combination with CD163 protein promotes cytotoxic lymphocyte-mediated cancer cell killing. In some embodiments, the combination with CD163 protein promotes NK cell-mediated tumor cell killing. In some embodiments, the combination with CD163 protein promotes T cells to express IL-2. In some embodiments, the combination with CD163 protein increases CD4 + T cells, CD196 - T cells, CXCR3 + T cells, CCR4 - T cells, or any combination thereof.

[0034] 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, wherein the binding results in at least one of the following effects: (a) macrophages reduce expression of at least one marker, wherein at least one marker is CD16, CD64, TLR2 or Siglec-15; (b) the antibody is internalized by the macrophage; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells or any combination thereof; (d) CD4 + T cells, CD8 +In some embodiments, the binding results in: two or more of (a) to (e); three or more of (a) to (e); four or more of (a) to (e); or all of (a) to (e). In some embodiments, the antibody or recombinant antibody binds to CD163 in the tumor microenvironment. + Immunosuppressive myeloid cells specifically bind, wherein the binding reduces the inhibition of cytotoxic T cell-mediated tumor cell killing 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 macrophages to CD16, CD64, TLR2, Siglec-15 or a combination thereof, for use in a method for treating cancer. In some embodiments, the combination of an antibody or recombinant antibody with a macrophage regulates the immune function of cells in the tumor microenvironment. In some embodiments, the combination of an antibody or recombinant antibody with a macrophage promotes anti-tumor immune function.

[0035] In some embodiments, the antibody or recombinant antibody specifically binds to a CD163 epitope comprising an amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody or recombinant antibody specifically binds to a CD163 epitope comprising an amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody or recombinant antibody specifically binds to a CD163 epitope comprising an amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or recombinant antibody specifically binds to a CD163 epitope comprising an amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In some embodiments, the antibody or recombinant antibody specifically binds to a CD163 epitope comprising each of the amino acid sequences SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In some embodiments, the antibody or recombinant antibody is at a K of 1 nM to 100 nM. D Specifically binds to CD163. In some embodiments, the antibody or recombinant antibody has a K of 1 nM to 50 nM. D Specifically binds to CD163. In some embodiments, the antibody or recombinant antibody has a K of 1 nM to 10 nM. D Specifically binds to CD163. In some embodiments, CD163 is human CD163. In some embodiments, the antibody or recombinant antibody has a K of 1 nM to 100 nM. D Specifically binds to M2c macrophages. In some embodiments, the antibody or recombinant antibody has a K of 1 nM to 50 nM. D Specifically binds to M2c macrophages. In some embodiments, the antibody or recombinant antibody has a K of 1 nM to 10 nM. DSpecifically binds to M2c macrophages. In some embodiments, the M2c macrophages are human M2c macrophages.

[0036] In certain embodiments, disclosed herein are compositions comprising an antibody or a recombinant antibody and an excipient.

[0037] In certain embodiments, disclosed herein is a pharmaceutical composition comprising the antibody or recombinant antibody of any one of the preceding embodiments and a pharmaceutically acceptable carrier.

[0038] In certain embodiments, disclosed herein is the use of the antibody or recombinant antibody of any of the preceding embodiments for the preparation of a medicament for treating cancer in a human subject.

[0039] In certain embodiments, disclosed herein is the use of the antibody or recombinant antibody of any of the preceding embodiments for the preparation of a medicament for reducing immunosuppression of tumor-associated macrophages in a human subject suffering from cancer.

[0040] In certain embodiments, disclosed herein is the use of an antibody or recombinant antibody according to any one of the preceding embodiments for the preparation of a medicament for promoting T cell-mediated killing of tumor cells in a human subject suffering from cancer.

[0041] In certain embodiments, disclosed herein is a method for promoting immune cell function, the method comprising: administering an antibody or recombinant antibody according to any one of the preceding embodiments to an individual in need thereof, and promoting immune cell function, such as measured by one or both of 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. In some embodiments, CD4 + T cells, CD8 + The activation of T cells, NK cells or any combination thereof is measured as an increased 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 suppressor cells of myeloid origin. In some embodiments, the antibody or recombinant antibody is an antibody or recombinant antibody of any of the preceding embodiments.

[0042] In certain embodiments, disclosed herein are methods of treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of the antibody or recombinant antibody of any one of the preceding embodiments, thereby treating the cancer in the individual.

[0043] In certain embodiments, disclosed herein are methods of treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of the antibody or recombinant antibody of any one of the preceding embodiments, thereby reducing immunosuppression of tumor-associated macrophages in the individual.

[0044] In certain embodiments, disclosed herein is a method of treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of the antibody or recombinant antibody of any one of the preceding embodiments, thereby increasing T cell-mediated tumor cell killing in the individual.

[0045] Disclosed herein, in certain embodiments, are methods of reducing the tumor-promoting activity of tumor-associated macrophages in an individual in need thereof, the method comprising administering to the individual a drug that modulates CD4 + T cell activation, CD4 + T cell proliferation, CD8 + T cell activation, CD8 + The pharmaceutical composition of any of the preceding embodiments in an amount effective for T cell proliferation or any combination thereof.

[0046] In certain embodiments, disclosed herein is a method of 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 antibody of any of the preceding embodiments or a pharmaceutical composition of any of the preceding embodiments.

[0047] In some embodiments, the method also includes promoting tumor cell killing in the tumor microenvironment. In some embodiments, cancer is lung cancer or sarcoma. In some embodiments, lung cancer is lung epithelial cancer (lung carcinoma) or lung adenocarcinoma. In some embodiments, the method also includes administering another anticancer therapeutic agent or anticancer therapy to the individual. In some embodiments, another anticancer therapy is surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy and cytokine therapy and a combination thereof. In some embodiments, another anticancer therapy is immunotherapy. In some embodiments, immunotherapy is a composition comprising a checkpoint inhibitor.

[0048] In certain embodiments, disclosed herein is a method for modulating the activity of tumor-associated macrophages in a tumor microenvironment, the method comprising contacting the tumor-associated macrophages with an antibody or a recombinant antibody that binds to human macrophages expressing CD163 and comprises at least one of the following effects: (a) binding of the antibody reduces the expression of at least one marker on the macrophages, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15; (b) after the antibody binds to the CD163 protein, the antibody is internalized by the human macrophages; (c) the binding of the antibody is not cytotoxic to the macrophages; (d) the binding of the antibody or the recombinant antibody inhibits the expression of at least one marker on the macrophages through CD4 + T cells, CD8 + T cells, NK cells or any combination thereof produce IFN-γ, TNF-α, perforin or any group thereof; (e) the binding of the antibody or recombinant antibody promotes CD4 + T cells, CD8 + Activation of T cells, NK cells or any combination thereof; (f) binding of the antibody or recombinant antibody promotes CD4 + T cells, CD8 + Proliferation of T cells, NK cells, or any combination thereof; and (g) the combination of antibodies or recombinant antibodies promotes tumor cell killing in the tumor microenvironment. In some embodiments, the combination results in: two or more of (a) to (g); three or more of (a) to (g); four or more of (a) to (g); five or more of (a) to (g); six or more of (a) to (g); or all of (a) to (g).

[0049] In some embodiments, the method occurs in a tumor microenvironment. In some embodiments, the method occurs in vivo. In some embodiments, the binding of an antibody or recombinant antibody to a macrophage regulates the immune function of cells in the tumor microenvironment. In some embodiments, the binding of an antibody or recombinant antibody to a macrophage 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 causing the antibody or recombinant antibody to be internalized by a human macrophage after binding to the CD163 protein. In some embodiments, the binding to the CD163 protein is not cytotoxic to human macrophages. In some embodiments, the method further comprises promoting CD4 + T cells express CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof. In some embodiments, the method further comprises promoting CD8 +In some embodiments, the method comprises reducing immunosuppression in the tumor microenvironment. In some embodiments, the method comprises promoting cytotoxic lymphocyte-mediated killing of cancer cells. In some embodiments, the method comprises promoting NK cell-mediated killing of tumor cells. In some embodiments, the method comprises promoting T cell expression of IL-2. In some embodiments, the method comprises increasing CD4 + T cells, CD196 - T cells, CXCR3 + T cells, CCR4 - T cells, or any combination thereof.

[0050] In certain embodiments, disclosed herein are antibodies that specifically bind to CD163 protein expressed on immunosuppressive human myeloid cells, wherein binding of the antibody to the myeloid cells promotes immune cell function, as measured by one or both of the following parameters: (i) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; and (ii) CD4 + T cells, CD8 + In some embodiments, the immunosuppressive human myeloid cells are macrophages or myeloid-derived suppressor cells. In some embodiments, the CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof is measured as increased production of IFN-γ, TNF-α, or perforin, or any combination thereof.

[0051] In certain embodiments, disclosed herein are antibodies that specifically bind to the CD163 protein expressed on human macrophages, wherein binding of the antibody to the macrophages promotes immune cell function, as measured by one or both of the following parameters: (i) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof; and (ii) CD4 + T cells, CD8 + In some embodiments, the immune cell function is in the tumor microenvironment. In some embodiments, the tumor microenvironment is in vivo. In some embodiments, CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof is measured as increased production of IFN-γ, TNF-α, or perforin, or any combination thereof.

[0052] In certain embodiments, disclosed herein is an antibody specifically bound to a CD163 protein expressed on human macrophages, wherein the combination of the antibody and the macrophage increases the immunostimulatory activity in the tumor microenvironment. In some embodiments, the tumor microenvironment is in vivo. In some embodiments, the combination of the antibody and the macrophage reduces the immunosuppressive activity of the macrophage. In some embodiments, the combination of the antibody and the macrophage reduces the tumor promoting activity of the macrophage. In some embodiments, the macrophage is a tumor-associated macrophage. In some embodiments, the antibody changes the expression of at least one marker on the macrophage. In some embodiments, the CD163 protein is a CD163 glycoform. In some embodiments, the CD163 protein is a 150kDa CD163 glycoform. In some embodiments, the antibody does not specifically bind to the 130kDa CD163 glycoform expressed by human macrophages. In some embodiments, the antibody has a constant domain capable of binding to an Fc receptor. In some embodiments, the Fc receptor is expressed on macrophages. In some embodiments, the antibody fragment comprises a single heavy chain, a single light chain, 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 double antibody. In some embodiments, at least one marker on human macrophages is CD16, CD64, TLR2 or Siglec-15. In some embodiments, human macrophages are M2 macrophages or M2-like macrophages. In some embodiments, human macrophages are M2a, M2b, M2c or M2d macrophages. In some embodiments, CD163 protein is a component of a cell surface complex comprising at least one other protein expressed by macrophages. In some embodiments, at least one other protein is galectin-1 protein, LILRB2 protein, casein kinase II protein or any combination thereof. In some embodiments, after 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 promotes CD4 + T cell activation, CD4 + T cell proliferation, or CD4 + Both T cell activation and proliferation. In some embodiments, binding to the CD163 protein promotes CD4 + T cells express CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof. In some embodiments, binding to the CD163 protein promotes CD8 + T cell activation, CD8+ T cell proliferation, or CD8 + Both T cell activation and proliferation. In some embodiments, binding to the CD163 protein promotes CD8 + T cells express ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof. In some embodiments, the combination with CD163 protein reduces immunosuppression in the tumor microenvironment. In some embodiments, the combination with CD163 protein promotes tumor cell killing in the tumor microenvironment. In some embodiments, the combination with CD163 protein promotes cytotoxic lymphocyte-mediated cancer cell killing. In some embodiments, the combination with CD163 protein promotes NK cell-mediated tumor cell killing. In some embodiments, the combination with CD163 protein promotes T cells to express IL-2. In some embodiments, the combination with CD163 protein increases CD4 + T cells, CD196 - T cells, CXCR3 + T cells, CCR4 - T cells or any combination thereof. In some embodiments, binding to CD163 reduces immunosuppression in the tumor microenvironment caused by macrophages.

[0053] In certain embodiments, disclosed herein are antibodies that specifically bind to a CD163 protein expressed on human macrophages, wherein the binding results in at least one of the following effects: (a) a decrease in the expression of at least one marker by the macrophages, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15; (b) the antibody is internalized by the macrophages; (c) CD4 + T cells, CD8 + Activation of T cells, NK cells or any combination thereof; (d) CD4 + T cells, CD8 + In some embodiments, the combination results in: two or more of (a) to (e); three or more of (a) to (e); four or more of (a) to (e); or all of (a) to (e). In some embodiments, CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof is measured as increased production of IFN-γ, TNF-α, or perforin, or any combination thereof.

[0054] In certain embodiments, disclosed herein are CD163 in the tumor microenvironment. +Antibodies that specifically bind to immunosuppressive myeloid cells, wherein binding reduces inhibition of cytotoxic T cell-mediated tumor cell killing in the tumor microenvironment.

[0055] In certain embodiments, disclosed herein is 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 a combination thereof by macrophages for use in a method for treating cancer. In some embodiments, the combination of antibodies and macrophages regulates the immune function of cells in the tumor microenvironment. In some embodiments, the combination of antibodies and macrophages promotes anti-tumor immune function.

[0056] In some embodiments, the composition comprises an antibody according to any embodiment as disclosed herein and an excipient.

[0057] In some embodiments, a pharmaceutical composition comprises an antibody according to any embodiment as disclosed herein and a pharmaceutically acceptable carrier.

[0058] In some embodiments, the use of an antibody as disclosed herein is for the preparation of a medicament for treating cancer in a human subject.

[0059] In some embodiments, the use of an antibody as disclosed herein is for the preparation of a medicament for reducing immunosuppression of tumor-associated macrophages in a human subject suffering from cancer.

[0060] In some embodiments, the use of an antibody as disclosed herein is for the preparation of a medicament for promoting T cell-mediated killing of tumor cells in a human subject suffering from cancer.

[0061] In certain embodiments, disclosed herein is a method for promoting immune cell function, the method comprising: allowing an antibody to specifically bind to a CD163 protein expressed on immunosuppressive human bone marrow cells; and promoting immune cell function, as measured by one or both of 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. In some embodiments, the immunosuppressive human myeloid cells are macrophages. In some embodiments, the immunosuppressive human myeloid cells are suppressor cells of myeloid origin. In some embodiments, the antibody is any embodiment as described herein.

[0062] In certain embodiments, disclosed herein are methods of treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of an antibody of any one of the embodiments disclosed herein, thereby treating the cancer in the individual.

[0063] In certain embodiments, disclosed herein are methods of treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of an antibody of any one of the embodiments disclosed herein, thereby reducing immunosuppression of tumor-associated macrophages in the individual.

[0064] In certain embodiments, disclosed herein is 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 of any one of the embodiments disclosed herein, thereby increasing T cell-mediated tumor cell killing in the individual. In some embodiments, the cancer is lung cancer or sarcoma. In some embodiments, the lung cancer is lung epithelial cancer or lung adenocarcinoma.

[0065] Disclosed herein, in certain embodiments, are methods of reducing the tumor-promoting activity of tumor-associated macrophages in an individual in need thereof, the method comprising administering to the individual a drug that modulates CD4 + T cell activation, CD4 + T cell proliferation, CD8 + T cell activation, CD8 + T cell proliferation or any combination thereof in an amount effective for the pharmaceutical composition disclosed herein. In some embodiments, the method further comprises promoting tumor cell killing in the tumor microenvironment.

[0066] In certain embodiments, disclosed herein are methods of promoting lymphocyte-mediated killing of tumor cells in an individual in need thereof, the method comprising administering to the individual an effective amount of a pharmaceutical composition of any one of the embodiments disclosed herein.

[0067] In certain embodiments, disclosed herein is a method for modulating the activity of tumor-associated macrophages in a tumor microenvironment, the method comprising contacting the tumor-associated macrophages with an antibody that binds to human macrophages expressing CD163 and comprises at least one of the following effects: (a) binding of the antibody reduces the expression of at least one marker on the macrophages, wherein the at least one marker is CD16, CD64, TLR2 or Siglec-15; (b) after the antibody binds to the CD163 protein, the antibody is internalized by the human macrophages; (c) the binding of the antibody is not cytotoxic to the macrophages; (d) the binding of the antibody promotes CD4 + T cells, CD8 + Activation of T cells, NK cells or any combination thereof; (e) binding of antibodies promotes CD4 + T cells, CD8+ In some embodiments, the binding results in: two or more of (a) to (f); three or more of (a) to (f); four or more of (a) to (f); five or more of (a) to (f); or all of (a) to (f). In some embodiments, CD4 + T cells, CD8 + The activation of T cells, NK cells or any combination thereof is measured as the increased production of IFN-γ, TNF-α, perforin or any combination thereof. In some embodiments, the method occurs in a tumor microenvironment. In some embodiments, the method occurs in vivo. In some embodiments, the combination of antibodies and macrophages regulates the immune function of cells in the tumor microenvironment. In some embodiments, the combination of antibodies and macrophages promotes anti-tumor immune function. In some embodiments, the antibody comprises a constant domain and the constant domain is combined with Fc receptors. In some embodiments, Fc receptors are expressed on macrophages. In some embodiments, the method also includes being internalized by human macrophages after the antibody is combined with CD163 proteins.

[0068] In some embodiments, binding to the CD163 protein is not cytotoxic to human macrophages.

[0069] In some embodiments, the method further comprises promoting CD4 + T cells express CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof. In some embodiments, the method further comprises promoting CD8 + T cells express ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof. In some embodiments, the method comprises reducing immunosuppression in the tumor microenvironment. In some embodiments, the method comprises promoting cytotoxic lymphocyte-mediated killing of cancer cells.

[0070] In some embodiments, the method comprises promoting NK cell-mediated killing of tumor cells. In some embodiments, the method comprises promoting the expression of IL-2 by T cells. In some embodiments, the method comprises increasing CD4 + T cells, CD196 - T cells, CXCR3 + T cells, CCR4 - T cells, or any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description and the accompanying drawings, which set forth illustrative embodiments in which the principles of the present disclosure are utilized, wherein:

[0073] Figure 1 Binding of the AB101 antibody to human MDSC populations is shown.

[0074] Figure 2 AB101 and CD163 are shown 高 The combination of cells.

[0075] Figure 3 Binding of AB101 to human M2c, M1 and M0 is shown compared to isotype controls.

[0076] Figure 4 Binding of AB101 to human peripheral blood T cells, B cells, NKT cells, neutrophils, monocytes, and dendritic cells is shown, with isotype controls shown in grey and AB101 binding shown in black.

[0077] Figure 5 AB101 was shown not to bind to a panel of human primary cells including small airway epithelial cells (SAEC), renal proximal tubule epithelial cells (RPTEC), pulmonary microvascular endothelial cells (HMVEC), umbilical vein endothelial cells (HUVEC), aortic smooth muscle cells (AOSMC), and keratinocytes.

[0078] Fig. 6A Shown are the top 20 targets of AB102 based on mass spectrometry analysis of post-immunoprecipitation samples.

[0079] Figure 6B Shown are the top cell surface targets of AB102 based on mass spectrometry analysis of post-immunoprecipitation samples.

[0080] Figure 6C Shown are the top cell surface targets of AB102 compared to ISO based on mass spectrometry analysis of AB102 and ISO (isotype negative control) samples after immunoprecipitation.

[0081] Figure 7 A significantly higher molecular weight glycoform of CD163 co-immunoprecipitated by AB102 is shown.

[0082] Figure 8 Binding of AB101, AB102 and a control CD163 antibody to huCD163 is shown, while the isotype control showed no appreciable binding.

[0083] Fig. 9 It was shown that neither AB101 nor the control anti-huCD163 antibody bound to recombinant murine CD163, in contrast to the commercially available anti-muCD163 antibody which did bind to murine CD163.

[0084] Fig.10 It is shown that pretreatment of M2c macrophages with polyclonal anti-CD163 antibody blocked the binding of AB101 antibody, compared to treatment with goat control polyclonal antibody which did not block the binding of AB101.

[0085] Fig.11 It is shown that pretreatment of M2c macrophages with polyclonal anti-CD163 antibodies blocked binding of the control monoclonal anti-huCD163 antibody, compared to treatment with goat control polyclonal antibodies which did not block binding of the control monoclonal anti-huCD163 antibody.

[0086] Fig.12 Shown is treatment of polarized M2c macrophages with siRNA against CD163 that substantially reduced binding of the AB102 antibody compared to M2c macrophages treated with scrambled siRNA (siScramb) or without siRNA, and is representative of three replicates.

[0087] Fig.13 It was shown that siRNA knockdown of CD163 reduced the binding of AB102 antibody, and there was a slight decrease in the binding of AB102 antibody after knockdown with siRNA against FCGR2A+FCGR3A (1 of 3 donors), FCGR2C, or FCGR3A, but there was no evidence of reduced AB102 binding after knockdown with siCD206, siCD163L1, siPPIA, siLGALS1, siLGALS3, siLILRB2, and siUPAR.

[0088] Fig.14 It is shown that treatment of cultured M2 macrophages with LPS resulted in loss of binding of both the AB101 antibody and a control anti-CD163 antibody.

[0089] Fig.15 Shown is increased IL-2 production following treatment of bone marrow cells with the AB101 antibody.

[0090] Fig.16 AB101 antibody treatment during polarization promotes CD4 + T cell proliferation.

[0091] Fig.17 AB101 antibody treatment during polarization was shown to promote CD8 + T cell proliferation.

[0092] Fig.18It is shown that treatment with AB101 antibody resulted in enhanced IL-2 production when compared to isotype antibody treatment during polarization (labeled "Before" on the graph), after polarization (labeled "After" on the graph), or during and after polarization combined (labeled "Before and After" on the graph).

[0093] Fig.19 It is shown that treatment of M2 macrophages with AB101 antibody reduces the expression of CD16, CD64, calreticulin, and Siglec-15.

[0094] Fig. 20 It is shown that treatment of M2c cells with AB101 increases the Th1 / Th2 ratio compared to the isotype control.

[0095] Fig.21 It is shown that treatment of M2c cells with AB101 increased the expression of CD69 on CD4 T cells compared to isotype control.

[0096] Fig. 22 It is shown that treatment of M2c cells with AB101 increased the expression of ICOS on CD4 T cells compared to isotype control.

[0097] Fig.23 It is shown that treatment of M2c cells with AB101 increased the expression of OX40 on CD4 T cells compared to isotype control.

[0098] Fig.24 It is shown that increased CTLs in the presence of BiTEs resulted in increased killing of Raji tumor cells compared to isotype controls.

[0099] Fig.25 It was shown that the internalization of the AB102 antibody was about as good as that of a commercial anti-CD163 antibody (R&D Systems MAB1607-100), and that the internalization of the AB101 antibody was about 2-fold greater than that of the commercial CD163 antibody.

[0100] Fig.26 Tumor volumes plotted for A549 tumors over 30 days are shown. Arrows indicate injected antibody treatments. Each point represents the average measurement from 7 mice. Error bars represent standard error of the mean (SEM). Statistical significance was calculated using the Mann-Whitney test.

[0101] Fig. 27 Tumor volumes plotted for H1975 tumors over 30 days are shown. Arrows indicate injected antibody treatments. Each point represents the average measurement from 7 mice. Error bars represent standard error of the mean (SEM). Statistical significance was calculated using the Mann-Whitney test.

[0102] Fig.28 Shown is the experimental design of the M2c / T cell co-culture assay used to evaluate the effects of AB101 treatment on T cell proliferation and IL-2 production.

[0103] Fig.29 It was shown that treatment with AB101 during M2c macrophage polarization restored T cell proliferation in an M2c / T cell co-culture assay.

[0104] Fig.30 It was shown that in the M2c / T cell co-culture assay, IL-2 secretion by OKT3 activated T cells was enhanced by treatment with AB101 during M2c macrophage polarization.

[0105] Fig.31 It is shown that in the M2c / T cell co-culture assay, treatment with AB101 in the pre-, pre / post-, and post-schedules resulted in CD8 + Increased T cell proliferation.

[0106] Fig.32 Treatment with AB101 in the pre-, pre / post-, and post-protocols in the M2c / T cell co-culture assay resulted in an increase in CD8 + Increased T cell proliferation.

[0107] Fig.33 AB101 treatment was shown to upregulate CD8 in M2c / T cell co-cultures from more than one subject. + T cell proliferation is enhanced.

[0108] Fig.34 AB101 treatment was shown to upregulate CD4 + T cell proliferation is enhanced.

[0109] Fig.35 It was shown that AB101 treatment enhanced IL-2 production by T cells from more than one human during M2c / T cell co-culture.

[0110] Fig.36 It was shown that AB101 was more effective than AB104 and AB102 isotypes in enhancing T cell proliferation in the M2c / T cell co-culture assay.

[0111] Fig.37 AB101 but not AB104 rescues CD8 in both pre / post and post protocols + T cell proliferation is free from M2c-mediated immunosuppression.

[0112] Fig.38 AB101 was shown to enhance CD8 +T cell cytokine responses were restored.

[0113] Fig.39 AB101 rescues CD4 + T cell IFN-γ, TNF-α, and perforin responses protect against M2c macrophage-mediated immunosuppression.

[0114] Fig.40 AB101 treatment showed that it enhanced CD8 + T cell cytotoxic activity.

[0115] Fig.41 AB101 treatment enhancement shown Assisted CD8 + T cell cytotoxic activity.

[0116] Fig.42 AB101 treatment was shown to enhance non-HLA restricted CD8 + T cell cytotoxic activity.

[0117] Fig.43 AB101 treatment alleviates M2c cell-mediated immunosuppression and induces activated CD4 + Unique expression patterns of T cells.

[0118] Fig.44 AB101 treatment was shown to alleviate M2c macrophage immunosuppression and enhance CD4 + T cells and CD8 + Activation of T cells.

[0119] Fig.45 Shows activated CD4 + CXCR3 expression on T cells.

[0120] Fig.46 It is shown that during M2c macrophage polarization, AB101 treatment reduces the expression of CD16, CD64, Siglex-15, and TLR2 by M2c macrophages.

[0121] Fig.47 Shown is the profile of changes in AB101 binding to huCD163 based on pepsin digestion.

[0122] Fig.48 Shown is the profile of changes in AB101 binding to huCD163 based on Nepenthesin II digestion.

[0123] Fig.49 Schematic diagram showing the binding of AB101 to human CD163 ECD based on HDX-MS studies.

[0124] Fig.50 AB101 is shown to bind to a truncated CD163 ECD consisting of SRCR domains 1-5.

[0125] Fig.51 The comparison of human CD163 and cynomolgus monkey CD163 is shown. Signal sequence. The sequence under the black bar represents 9 SRCR domains, and the gray line above the sequence represents the consensus sequence. The protected area and the exposed area are shown based on the AB101 binding epitope determined by the protection observed in HDX-MS. The solid bar below the sequence represents the area protected by Nepenthes protease II. The outline box below the sequence represents the area protected by pepsin. The oblique line box below the sequence represents the area exposed by pepsin. The vertical striped box below the sequence represents the area exposed by Nepenthes protease II. Lysine (K) at position 323 of human CD163 and glutamic acid (E) of cynomolgus monkey CD163 are represented by boxes.

[0126] Fig.52 AB101 was shown to bind to human and cynomolgus monkey E323K mutants, but not to wild-type cynomolgus monkey CD163 ECD.

[0127] Fig.53 SPR detection of AB101 binding to human CD163 is shown. AB101 is serially diluted to different concentrations with (A) EDTA or (B) calcium-containing running buffer. GHI / 61 is then injected into flow cell 2 at a flow rate of 30 μl / min, a concentration of 6.25 / 12.5 / 25 / 50 / 100 / 200 μg / ml, a contact time of 300 s, and a dissociation time of 600 s.

[0128] Fig.54 SPR detection of GHI / 61 binding to human CD163 is shown. Anti-CD163 clone GHI / 61 is serially diluted to different concentrations with (A) EDTA or (B) calcium-containing running buffer. GHI / 61 is then injected into flow cell 2 at a flow rate of 30 μl / min, a concentration of 3.125 / 6.25 / 12.5 / 25 / 50 / 100 μg / ml, a contact time of 300 s, and a dissociation time of 600 s.

[0129] Fig.55 SPR detection of CD163 combined with AB101 is shown. Human CD163 protein is serially diluted into different concentrations with calcium-containing running buffer. Then CD163 protein is injected into flow cell 2 with a flow rate of 30 μl / min, a concentration 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.

[0130] Fig.56 The binding of AB101 to soluble CD163 in AlphaLisa assay is shown. AB101 (circle) or isotype control (triangle) was incubated with 750nM CD163-His at the indicated concentration for 1 h. 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 (GraphPad Prism) was performed using 1-site and 2-site saturation binding models. (R 2 =0.92). (A) Linear and (B) logarithmic x-axis scales.

[0131] Fig.57 AB101 binding to M2c macrophages is shown. M2c macrophages were blocked with stringent FACS blocking buffer containing 0.5 mg / ml human IgG1 and then stained for 30 min with indicated concentrations of AB101 (circles) or isotype controls (triangles). The binding of AlexaFluor 647-labeled AB101 and isotype controls to M2c macrophages was quantified by fluorescence intensity and reported as gMFI. Symbols represent the mean ± standard error of four study subjects. Curve fitting (GraphPad Prism) was performed using a 2-site saturation binding model. (R 2 = 0.99). (A) Linear and (B) logarithmic x-axis scales.

[0132] Details of public content

[0133] This article discloses + In some embodiments, CD163 + The cells are immunosuppressive myeloid cells. In some embodiments, CD163 + The cells are bone marrow cells expressing human CD163. In some embodiments, CD163 + The cell is a tumor cell. In some embodiments, CD163 + 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 高 ). 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.

[0134] Monocytes and macrophages exposed to certain inflammatory cytokines or microorganism-associated molecular patterns differentiate into proinflammatory (M1 or M1-like) or anti-inflammatory M2 or M2-like macrophages. M1 and M2 are used to define macrophages activated in vitro as proinflammatory (when classically activated with IFN-γ and lipopolysaccharide) or anti-inflammatory (when activated with IL-4 or IL-10), respectively, and macrophages with M1 or M2 phenotypes in vivo or in vitro are defined as M1-like or M2-like macrophages. In some embodiments, M2 macrophages are produced by their exposure to certain cytokines. In some embodiments, M2 macrophages are differentiated by IL-4, IL-10, IL-13, or a combination thereof.

[0135] Subtypes of M2 macrophages include M2a, M2b, M2c, and M2d subtypes. M2a macrophages are induced by IL-4 and IL-13, leading to upregulated expression of CD163, arginase-1, mannose receptor MRC1 (CD206), antigen presentation by the MHC II system, and production of IL-10 and TGF-β, resulting in tissue regeneration and inhibition of proinflammatory molecules to prevent inflammatory responses. M2b macrophages produce IL-1, IL-6, IL-10, TNF-α in response to immune complexes. M2c macrophages are induced by IL-10, transforming growth factor β (TGF-β), and glucocorticoid exposure, and produce IL-10 and TGF-β, leading to inhibition of inflammatory responses. The M2d subtype is activated in response to IL-6 and adenosine.

[0136] M2 macrophages have functions and phenotypes corresponding to M2 macrophages and their subtypes. M2-like macrophages are any macrophages that have functional or phenotypic characteristics of a subset of M2 macrophages in vivo or ex vivo.

[0137] In some embodiments, the antibodies of the present disclosure have high affinity and specific binding to immunosuppressive myeloid cells (particularly tumor-associated macrophages, such as M2 and M2-like macrophages). In some embodiments, the antibodies specifically bind to M2 and M2-like TAMs from human primary lung tumors. In some embodiments, the antibodies disclosed herein have no detectable binding to M1 or M1-like macrophages. M1 activated macrophages express transcription factors such as interferon regulatory factor (IRF5), nuclear factor of κ light polypeptide gene enhancer (NF-κB), activating protein (AP-1) and STAT1. M1 macrophages secrete proinflammatory 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 any macrophages that have a subset of M1 macrophage functions or phenotypic characteristics in vivo or in vitro.

[0138] In some embodiments, the antibodies of the present disclosure do not bind to primary human cells. In some embodiments, the antibodies of the present disclosure do not bind to hematopoietic stem cells, leukocytes, T cells, B cells, NK cells, and granulocytes.

[0139] Tumor-associated macrophages (TAMs) are a heterogeneous class of macrophages that are abundantly present in the microenvironment of solid tumors. Most evidence suggests that TAMs have a tumor-promoting phenotype, manifested by involvement in tumor cell proliferation, tumor angiogenesis, motility and invasion, metastasis, anticancer drug tolerance, and tumor immune evasion.

[0140] Direct tumor cell killing by cytotoxic T cells in tumor infiltrating lymphocytes (TILs) plays an important role in the anti-tumor function of the immune system. However, TAMs in the tumor microenvironment (TME) inhibit T cell-mediated anti-tumor 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 inhibit T cell function through surface presentation of programmed death ligand 1 (PD-L1) in hepatocellular carcinoma and B7 homologs in ovarian cancer, which activate programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) on T cells, respectively. The inhibitory signals of PD-1 and CTLA4 are immune checkpoints, and the binding of these inhibitory receptors to their ligands inhibits T cell receptor signaling and T cell cytotoxic function, and promotes T cell apoptosis. HIF-1α induces TAMs to inhibit T cell function. CD163 has been identified as an immunosuppressive molecule expressed only on TAMs and may be a potential therapeutic target for cancer immunotherapy.

[0141] According to the functional characteristics of TAM, including TAM and T helper cells (CD4 + ) type Th1 and Th2, TAMs generally fall into two categories, M1-like anti-tumor macrophages and M2-like immunosuppressive macrophages. M1 macrophages are a "classic" model and can be produced by IFN-γ and innate immune activators such as pathogen-associated molecular patterns (PAMPs) (e.g., lipopolysaccharide (LPS)) or damage-associated molecular patterns (DAMPs) and inflammatory cytokines (e.g., tumor necrosis factor-α (TNF-α)). In addition, T cell-dependent macrophage activation via the CD40-CD40 ligand pathway induces M1 differentiation. M1 macrophages have pro-inflammatory, bactericidal and cytotoxic functions. These macrophages promote antigen-dependent induction of Th1 cells and the differentiation of Th1 and CD8 +Activation of T cells. The promotion of the cytotoxic activity of T cells by M1-like anti-tumor macrophages is crucial for tumor cell elimination. In some embodiments, the M1-like anti-tumor macrophages are characterized by surface marker expression measured by flow cytometry and have CD80 + CD86 + CD163 低 / - or CD206 低 / - Phenotype. M1 macrophages secrete IL-12 and low levels of IL-10 and / or TGF-β.

[0142] In contrast, M2-like immunosuppressive macrophages are an "alternative" or "non-classical" activation model 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 the tumor. M2-like immunosuppressive macrophages are the main macrophage type involved in the pro-tumor functions of TAMs, which include promoting tumor growth, metastasis, and immune evasion. M2-like macrophages express surface markers CD15, CD23, CD64, CD68, CD163 高 、CD204 高 、CD206 高 and / or other M2 macrophage markers as determined by flow cytometry. M2 macrophages secrete high levels of IL-10 and TGF-β1 and low levels of IL-12.

[0143] In many tumor types, the level of TAM infiltration has significant prognostic value. TAMs have been associated with poor prognosis in a variety of tumors. For example, breast cancer patients with more M2-like tumor-associated macrophages have been found to have higher grade tumors, greater microvessel density, and lower overall survival. Patients with more M1-like anti-tumor TAMs show the opposite effect.

[0144] Thus, there remains a need to identify compounds and methods for improving immunotherapy for treating cancer.

[0145] 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 and protects tissues from oxidative damage mediated by free hemoglobin. Four subtypes of CD163 protein have been reported, with molecular weights of 125,451Da, 125,982Da, 121,609Da and 124,958Da, respectively. Subtype 1 is the most common subtype of CD163, with a molecular weight of 125,451Da, and is composed of 1115 amino acid residue polypeptides comprising an extracellular domain, a transmembrane segment and a cytoplasmic tail. The extracellular domain comprises nine cysteine-rich repeat domains. Isoform 1 of the CD163 protein has four N-linked glycosylation sites, and in M2 macrophages the CD163 protein shows two distinct bands at ∼150 kDa and ∼130 kDa in SDS-PAGE under reducing conditions.

[0146] CD163 mRNA expression is generally restricted to myeloid cells, but is also expressed by certain human cancers. CD163 expression on TAMs has been associated with an immunosuppressive M2-like phenotype, which has been shown to be associated with poor clinical outcomes in cancer. CD163 is required for pro-tumor activation of macrophages in human and murine sarcomas. CD163 has also been reported to be a macrophage scavenger receptor and promotes immunosuppression. In some embodiments, the interaction of the hemoglobin-haptoglobin complex with CD163 induces the secretion of the immunosuppressive cytokine IL-10 and the expression of heme-oxygenase-1 (HO-1). HO-1 produces the anti-inflammatory metabolite Fe 2+ , CO and biliverdin.

[0147] Soluble CD163 occurs via ectodomain shedding in humans and has been reported to have anti-inflammatory properties, such as downregulating T cell responses, including lymphocyte proliferation stimulated by phytohemagglutinin (PHA) or 12-O-tetradecanoylphorbol-13-acetate (TPA).

[0148] Antibodies targeting CD163 have been shown to modulate the innate immune response of macrophages expressing CD163. For example, the RM3 / 1 antibody against CD163 is a mouse monoclonal IgG1 (κ light chain) generated against human monocytes. The RM3 / 1 antibody binds to the cysteine-rich domain 9 of human CD163, reduces LPS-induced TNFα, and enhances IL-10 secretion by macrophages.

[0149] Certain terms

[0150] Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those generally understood by those skilled in the art of the claimed subject matter. In general, the nomenclature used in conjunction with the following and the following techniques described herein are those well known and commonly used in the art: immunology, oncology, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization. It should be understood that the foregoing general description and the following detailed description are only exemplary and illustrative, and do not limit any claimed subject matter. The section titles used herein are only for organizational purposes and should not be construed as limiting the described subject matter.

[0151] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes more than one antibody, and in some embodiments reference to "an antibody" includes a plurality of antibodies, and so forth.

[0152] As used herein, all numerical values ​​or numerical ranges include integers within or including such ranges and fractions of values ​​or integers within or including ranges, unless the context clearly indicates otherwise. Thus, for example, 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., and so on. In another example, reference to a range of 1-5,000 fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5 fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5 fold, etc., and so forth.

[0153] As used herein, "about" a number means including the number and a range from 10% below the number to 10% above the number. "About" a range means 10% below the lower limit of the range spanning to 10% above the upper limit of the range.

[0154] "Percent identity" and "% identity" refer to the extent to which two sequences (nucleotides or amino acids) have identical residues at identical positions in an alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" refers to the % identity of an amino acid sequence to SEQ ID NO: Y, and is specified as X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y. Generally, such calculations are performed using computer programs. Exemplary programs for comparing and aligning sequence pairs 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).

[0155] As used herein, "antibody" refers to a protein that binds to an antigen.Antibodies generally contain variable domains and constant domains in each heavy chain and light chain. Accordingly, most antibodies have a heavy chain variable domain (VH) and a light chain variable domain (VL), which together form the part of the antibody that binds to the antigen. There are three complementary determining regions (CDRs) in each variable domain, which form a loop and contact the surface of the antigen in the heavy chain variable domain (VH) and the light chain variable domain (VL). "Antibodies" include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), natural antibodies, humanized antibodies, human antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, grafted antibodies, antibody fragments (e.g., a part of a full-length antibody, typically an antigen-binding fragment or its variable region, e.g., Fab fragments, Fab' fragments, F(ab')2 fragments and Fv fragments) and antibodies with antigen-binding activity produced in vitro. The term also includes single chain antibodies, e.g., single chain Fv (sFv or scFv) antibodies, in which the variable heavy chain and variable light chain are linked together (directly or through a peptide linker) to form a contiguous polypeptide.

[0156] As used herein, "complementarity determining region (CDR)" refers to the variable chain portion of an antibody that binds to a specific antigen. More than one method can be used to define CDR. Current technology utilizes different numbering schemes with different definitions for CDR length and position. For example, the Kabat numbering scheme is based on sequence alignment and uses the "variability parameter" of a given amino acid position (the number of different amino acids at a given position divided by the frequency of the most frequently occurring amino acid at that position) to predict CDR. On the other hand, the Chothia numbering scheme is a structure-based numbering scheme in which loop structures are defined as CDRs by comparing antibody crystal structures. The Martin numbering scheme focuses on the structural alignment of different framework regions of unconventional lengths. The IMGT numbering scheme is a standardized numbering system based on sequence alignments from a complete reference gene database (including the entire immunoglobulin superfamily). The Honneger numbering scheme (AHo's) is based on the structural alignment of the 3D structure of the variable region and uses the structurally conserved Cα position to infer the framework and CDR length. Those skilled in the art will note that the definition of CDR will vary depending on the method used. Any method for defining CDRs is contemplated for the sequences disclosed herein.

[0157] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like. In some embodiments, the mammal is a human.

[0158] As used herein, the terms "treatment", "treating", and the like, in some cases, refer to the administration of an agent or the performance of a procedure for the purpose of obtaining a certain effect. In some embodiments, the effect is preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of achieving a partial or complete cure of a disease and / or symptoms of a disease. As used herein, "treatment" includes treating a disease or disorder (e.g., cancer) in mammals, particularly humans, and includes: (a) preventing the disease or symptoms of a disease from occurring in a subject who is susceptible to the disease but has not yet been diagnosed with the disease (e.g., including a disease associated with or caused by a primary disease); (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing regression of the disease. In some embodiments, treatment refers to any sign of success in treating or ameliorating or preventing cancer, including any objective or subjective parameter, such as a reduction, alleviation, attenuation of symptoms, or making the disease condition more tolerable to the subject; slowing the rate of degeneration or decline; or making the endpoint of degeneration less debilitating. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of a physician's examination. Accordingly, the term "treatment" includes the administration of a compound or agent of the present disclosure to prevent or delay, alleviate or prevent or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject. A subject's disease or disorder is "treated" if, after receiving a therapeutic amount of an antibody of the present disclosure, the patient shows an observable and / or measurable change in a parameter or symptom of the disease or disorder, such as, in the case of treating cancer, an increase in tumor cell killing activity assessed ex vivo, a decrease in the level of immunosuppressive secretory 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 indicator associated with a parameter or symptom of the disease or disorder. In some embodiments, the parameters include conversion of an immune-cold tumor to an immune-hot tumor, for example by increasing the number of cytotoxic lymphocytes and T cell activation markers (CD69, ICOS, OX40, etc.) in a tumor biopsy, or decreasing the expression of CD16, CD64, TLR2, Siglec-15 by TAMs in a tumor biopsy.

[0159] In some embodiments, "inducing a response" refers to alleviating or reducing the signs or symptoms of a disease in a subject, and specifically includes, but is not limited to, prolonging survival.

[0160] The term "avidity" refers to the resistance of a complex of two or more agents to dissociate upon dilution.

[0161] 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 and vary with the antibody isotype.

[0162] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody.

[0163] As used herein, "human effector cells" refer to leukocytes that express one or more FcRs and perform effector functions. For example, the cells express at least FcγRIII and perform ADCC effector functions. 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.

[0164] "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 bind to their cognate antigen. To assess complement activation, for example, a CDC assay is performed.

[0165] An "internalizing" antibody is one that is taken up by (i.e., enters) a cell after 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, internalization of an antibody (e.g., such as disclosed herein) alters the biology of the cell, causing it to alter its function.

[0166] An "antigen binding domain", "antigen binding region" or "antigen binding site" is the portion of an antibody that interacts with an antigen and contributes to the antibody's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least a portion of at least one CDR domain of the antibody.

[0167] The antigen binding region of an antibody is called a "paratope", which binds to an antigenic determinant, the "epitope" of an antigen, that is, the portion of an antigen molecule that can be bound by an antibody. In some embodiments, an antigenic substance has one or more portions that can be recognized by an antibody, i.e., more than one epitope, and thus a single antigenic substance is specifically bound by different antibodies, each antibody being specific for a different epitope. In some embodiments, an epitope comprises a non-contiguous portion of an antigen. For example, in a polypeptide, amino acid residues that are discontinuous in the primary sequence of the polypeptide but are close enough to each other in the tertiary and quaternary structures of the polypeptide to be bound by an antigen binding protein constitute an epitope.

[0168] An "antibody fragment" comprises a portion of an intact antibody. In some embodiments, an antibody fragment comprises the antigen binding region or variable region of an intact antibody.

[0169] The terms "antigen-binding portion of an antibody," "antigen-binding fragment," "antigen-binding domain," and "antibody fragment" are used interchangeably herein to 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 included in these terms include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) an Fv fragment containing the VL and VH domains of a single arm of an antibody; (v) a dAb fragment containing a VH domain (Ward et al., Nature 341(6242):544-6(1989)); and (vi) isolated CDRs. Also included are "one-half" antibodies comprising a single heavy chain and a single light chain. Other forms of single-chain antibodies such as diabodies are also included herein.

[0170] As used herein, "functional antibody fragment" in this context refers to an antibody fragment that not only binds to the antigen of the antibody but also has functional attributes that characterize the complete antibody. For example, if the function of the antibody depends on the function of having an Fc domain that can achieve effector functions (such as ADCC), the functional fragment will have such a function. It is assumed that when the antibody of the present disclosure comprises an Fc portion that binds to a macrophage Fc receptor (such as CD16 (FcγRIIIa) or CD64 (FcγRI) in some embodiments), the antibody of the present disclosure is effective in regulating the functional state of tumor-associated macrophages or redirecting or inhibiting M2 state macrophages.

[0171] The term "functional fragment or analog" of an antibody is a compound that has a qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is an antibody functional fragment or analog that binds to IgE immunoglobulin to prevent or substantially reduce the ability of such a molecule to bind to the high affinity receptor FcγRI.

[0172] An "antigen binding protein" is a protein comprising an antigen binding portion comprising an antibody, optionally also comprising a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that facilitates binding of the antigen binding protein to the antigen.

[0173] A “complete” antibody is one that contains both the antigen-binding site and the C L and at least the heavy chain constant domain C H 1. C H 2 and C H3. In some embodiments, the constant domain is a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof.

[0174] The term "recombinant antibody" as used herein refers to an antibody comprising an antigen binding domain (such as, for example, CDRs, VH regions or a complete light chain) of a first antibody and domains from one or more other antibodies or proteins. Chimeric antibodies, hybrid antibodies and humanized antibodies are examples of recombinant antibodies.

[0175] A "CDR-grafted antibody" is an antibody comprising one or more CDRs derived from an antibody or isotype from one species and a framework from another antibody or isotype from the same or a different species.

[0176] The term "human antibody" includes all antibodies having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable and constant domains of an antibody are derived from human immunoglobulin sequences (referred to as "fully human antibodies").

[0177] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as K. D In one embodiment, the antibody or antigen-binding fragment thereof exhibits CD163 as shown by the following K D (equilibrium dissociation constant) measured binding affinity: at 10 -6 M or less, or range as low as 10 -16 M or less, (e.g., about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 M, 10 -16 M or less). In certain embodiments, the antibodies described herein are expressed in an amount less than or equal to 10 -4 M, less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, less than or equal to 10 - 7 M, or less than or equal to 10 -8 M of K D Specific binding to huCD163 polypeptide.

[0178] The term "preferential binding" or "specific binding" means that the antibody or fragment thereof binds to the epitope with a greater affinity than it binds to an unrelated amino acid sequence and, if cross-reacting with other polypeptides containing the epitope, is non-toxic at the levels of administration at which they are formulated for human use. In some embodiments, such affinity is at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the antibody or fragment thereof for an unrelated amino acid sequence.

[0179] The term "specificity" refers to situations where an antibody will not preferentially bind to molecules other than the antigen containing the epitope recognized by the antibody. The term also applies, for example, to situations where an antigen binding domain is specific for a particular epitope carried by many antigens, in which case the antibody or antigen binding fragment thereof carrying the antigen binding domain will be able to bind to a different antigen carrying that epitope.

[0180] As used herein, an antibody is considered to be 4 M -1 , or greater than or equal to about 10 5 M -1 , greater than or equal to about 10 6 M -1 , greater than or equal to about 10 7 M -1 , or greater than or equal to 10 9 M -1 The affinity constant K a ) reacts with an antigen, then the antibody is said to have "immunological specificity" or "specificity" to the antigen or to "specifically bind" to the antigen.

[0181] As used herein, the term "monospecific" refers to an antibody composition containing antibodies that exhibit preferential affinity for a particular epitope. In some embodiments, a monospecific antibody preparation consists of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 99.9% of antibodies that have specific binding activity to a particular antigen.

[0182] The term "polypeptide" is used with its conventional meaning, i.e., with an amino acid sequence. Polypeptide is not limited to a product of a specific length. Peptide, oligopeptide and protein are included in the definition of polypeptide, and these terms are used interchangeably herein, unless otherwise specifically indicated. The term also does not refer to or exclude post-expression modifications of polypeptide, such as glycosylation, acetylation, phosphorylation, etc., and other modifications known in the art, naturally occurring and non-naturally occurring. In some embodiments, polypeptide is a complete protein or a subsequence thereof. In the context of the antibody of the present disclosure, a particular polypeptide of interest is an amino acid subsequence of a CD163 protein that comprises CDR and can bind to human M2 macrophages or expressed by such cells.

[0183] As used herein, "substantially pure" and "substantially free" refer to solutions or suspensions that contain less than, for example, about 20% or less of foreign matter, about 10% or less of foreign matter, about 5% or less of foreign matter, about 4% or less of foreign matter, about 3% or less of foreign matter, about 2% or less of foreign matter, or about 1% or less of foreign matter.

[0184] The term "isolated" refers to proteins (e.g., antibodies), nucleic acids, or other substances that are substantially free of other cellular material and / or chemicals. In some embodiments, antibodies or antigen-binding fragments thereof and nucleic acids of the present disclosure are isolated. In some embodiments, antibodies or antigen-binding fragments thereof and nucleic acids of the present disclosure are substantially pure.

[0185] When applied to polypeptides, "isolated" generally means a polypeptide that is separated from other proteins and nucleic acids that naturally occur with it. Preferably, the polypeptide is also separated from substances used to purify it, such as antibodies or gel matrices (polyacrylamide). In some cases, the term means a polypeptide or a portion thereof that, in view of its source or manipulation, is: (i) present in a host cell as an expression product of part of an expression vector; or (ii) connected to proteins or other chemical moieties other than those to which it is connected in nature; or (iii) not present in nature, for example, a protein that is chemically manipulated by attaching or adding at least one hydrophobic moiety to the protein so that the protein is in a form that does not exist in nature. "Isolated" also means a protein that is: (i) chemically synthesized; or (ii) expressed in a host cell and purified from related proteins and contaminating proteins.

[0186] The term "effective amount" as used herein refers to an amount of an antibody or antigen-binding portion thereof as described herein, which is sufficient to induce a response, e.g., when administered to a subject, sufficient to achieve treatment, prognosis or diagnosis of a disease associated with macrophage activity or TAM activity, as described herein. The therapeutically effective amount of the antibodies provided herein, when used alone or in combination, will depend on the relative activity of the antibodies and compositions (e.g., in terms of inhibiting tumor cell growth) and will vary depending on the subject and disease condition being treated, the subject's weight and age, the severity of the disease condition, the mode of administration, etc., and in some cases, the therapeutically effective amount of the antibody is readily determined by one of ordinary skill in the art.

[0187] The term "therapeutically effective amount" generally refers to the amount of an antibody or drug that effectively "treats" a disease or disorder in a subject or mammal. In some embodiments, the compositions described herein are administered to a subject in an amount that effectively produces some desired therapeutic effect by inhibiting a disease or disorder as 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 portion of the desired therapeutic or preventive effect in an organ or tissue. The amount of antibodies required to bring about preventive and / or therapeutic treatment of a disease or disorder is not fixed per se. In some embodiments, the amount of antibodies administered varies with the type of disease, the extensiveness of the disease, and the size of the mammal suffering from the disease or disorder. When used in conjunction with a method of treatment involving administering a therapeutic agent after the subject develops symptoms of a disease or disorder, the term "therapeutically effective" means that after treatment, one or more signs or symptoms of the disease or disorder are improved or eliminated.

[0188] The expression "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar adverse reactions (such as stomach upset, dizziness, and the like) when administered to humans.

[0189] The term "contacting" is defined herein as bringing a composition as provided herein into physical proximity with a cell, organ, tissue or fluid as described herein.

[0190] Immunomodulatory antibodies

[0191] In certain embodiments, disclosed herein are proteins that are related to human CD163 + In some embodiments, CD163 + The cell is an immunosuppressive myeloid cell. In some embodiments, the immunosuppressive myeloid cell is a human macrophage. In some embodiments, binding of an antibody disclosed herein alters the expression of at least one marker on a human macrophage.

[0192] In some embodiments, antibodies disclosed herein bind to human CD163 (huCD163) proteins expressed on human M2 or M2-like immunosuppressive macrophages. In some embodiments, antibodies specifically bind to CD163 proteins, and the CD163 proteins are huCD163 glycoforms of about 140kDa. In some embodiments, antibodies specifically bind to the extracellular domain 3 of huCD163. In some embodiments, antibodies specifically bind to the extracellular domain 4 of huCD163. In some embodiments, antibodies specifically bind to the extracellular domain 3 and extracellular domain 4 of huCD163. In some embodiments, antibodies specifically bind to huCD163, resulting in conformational changes of huCD163. In some embodiments, the conformational changes of humCD163 expose the extracellular domains 2, 5 and 9 of humCD163. In some embodiments, antibodies do not specifically bind to huCD163 glycoforms of lower molecular weight (about 115kDa).

[0193] In some embodiments, the antibodies disclosed herein are fused to human CD163 + In some embodiments, the antibodies disclosed herein bind to M2 or M2-like 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 macrophages to non-immunosuppressive or less immunosuppressive and more anti-tumor states. In some embodiments, the antibodies disclosed herein bind to M2 or M2-like immunosuppressive macrophages and cause a decrease in the expression of certain cell markers that characterize M2 or M2-like macrophages, indicating functional differentiation of macrophages to an altered differentiation state. In some embodiments, the antibodies disclosed herein reduce CD163 + Expression of one or more of CD16, CD64, TLR2, and Siglec-15 by immunosuppressive myeloid cells.

[0194] In some embodiments, the antibodies disclosed herein are + Binding of immunosuppressive myeloid cells leads to CD163 + Functional changes of immunosuppressive myeloid cells. In some embodiments, the antibodies disclosed herein are in combination with CD163 + Binding of immunosuppressive myeloid cells results in changes in marker expression in M2 or M2-like immunosuppressive macrophages. In some embodiments, the antibody binds to CD163 + Changes in the function of immunosuppressive myeloid cells lead to altered interactions with other cells, such as effector T cells, which subsequently alter their influence on and interaction with target tumor cells.

[0195] In some embodiments, the antibodies of the present disclosure reduce the immunosuppression caused by tumor-associated macrophages in the tumor microenvironment. In some embodiments, the reduction of immunosuppression of tumor-associated macrophages in the tumor microenvironment corresponds to an increase in immunostimulation, for example, promoting T cell activation, T cell proliferation, NK cell activation, NK cell proliferation, or the production of any combination thereof. In some embodiments, T cell activation and / or NK cell activation lead to an increase in IFN-γ, TNF-α, perforin, or a combination thereof of T cells and / or NK cells. In some embodiments, the antibodies of the present disclosure increase immunostimulation, for example, promoting T cell activation, T cell proliferation, NK cell activation, NK cell proliferation, or the production of any combination thereof. In some embodiments, T cell activation and / or NK cell activation lead to an increase in IFN-γ, TNF-α, perforin, or a combination thereof of T cells and / or NK cells. In some embodiments, the CD163 protein specific binding of the antibody of the present disclosure and human macrophage expression, wherein human macrophage has the first immunosuppressive activity before binding antibody and has the second immunosuppressive activity after binding antibody, and wherein the second immunosuppressive activity is lower than the first immunosuppressive activity.In various embodiments, the first and second immunosuppressive activities are each non-zero.

[0196] In some embodiments, the antibodies of the present disclosure promote T cell activation and proliferation. In some embodiments, the antibodies bias T cell populations toward anti-tumor T cell phenotypes. In some embodiments, the antibodies reduce or block the inhibition of T cell activation by bone marrow cells. In some embodiments, the antibodies reduce the ability of TAMs to inhibit T cell activation, resulting in greater T cell stimulation and IL-2 production. In some embodiments, the antibodies block the ability of TAMs to inhibit T cell activation, resulting in greater T cell stimulation and IL-2 production.

[0197] In some embodiments, the antibodies disclosed herein reduce myeloid suppression of T cell proliferation. In some embodiments, the antibodies reduce TAM suppression of CD4 + T cells and CD8 + T cell activation and proliferation. In some embodiments, the antibody reduces TAM inhibition of Th1 cell proliferation. Proliferating T cells in CD4 + T cells showed enhanced expression of activation markers.

[0198] In some embodiments, the antibodies of the present disclosure alter M2 polarized macrophages such that the macrophages exhibit an M1-like phenotype that alleviates the immunosuppressive effects of M2 macrophages. In some embodiments, the antibodies described herein affect the differentiation of monocyte-derived macrophages toward a less immunosuppressive and more anti-tumor differentiation state.

[0199] In some embodiments, provided herein is an antibody that specifically binds to human CD163 protein (huCD163) expressed on human macrophages and reduces the expression of at least one of macrophages to CD16, CD64, TLR2 or Siglec-15. In some embodiments, human macrophages are tumor-associated immunosuppressive macrophages. In some embodiments, human macrophages are M2-like immunosuppressive macrophages.

[0200] In some embodiments, antibodies disclosed herein bind to the CD163 protein expressed by macrophages, and the CD163 protein is a component of a complex comprising at least one other protein expressed by macrophages. In some embodiments, the complex is a cell surface complex. In some embodiments, the complex comprises at least one other protein selected from galectin-1 protein, LILRB2 protein and casein kinase II protein.

[0201] In some embodiments, the antibodies disclosed herein bind to CD163 protein on macrophages and are internalized by the macrophages.

[0202] In some embodiments, the antibodies disclosed herein are not cytotoxic to macrophages to which they bind.

[0203] In some embodiments, the antibodies disclosed herein promote CD4 + T cell activity or proliferation. In some embodiments, the antibody promotes CD4 + T cell expression of CD69, ICOS, OX40, PD1, LAG3, or CTLA4.

[0204] In some embodiments, the antibodies disclosed herein promote CD8 + T cell activity or proliferation. In some embodiments, the antibody promotes CD8 + T cell expression of ICOS, OX40, PD1, LAG3, or CTLA4.

[0205] In some embodiments, the antibodies disclosed herein promote CD8 + In some embodiments, the antibody promotes cytotoxic lymphocyte-mediated killing of cancer cells. In some embodiments, the antibody promotes NK cell-mediated killing of tumor cells.

[0206] In some embodiments, the antibodies disclosed herein promote the expression of IL-2 by T cells. In some embodiments, the binding of the antibodies of the present disclosure to the CD163 protein increases the expression of CD4 + T cells, CD196-T cells, CXCR3+ T cells, CCR4 - T cells or any combination thereof. In some embodiments, the antibody increases CD4 + CD196 - CXCR3 + CCR4 - T cells.

[0207] In some embodiments, the antibodies disclosed herein have a constant domain that binds to an Fc receptor expressed on macrophages. In some embodiments, the antibodies specifically bind to huCD163 and have a constant domain that binds to an Fc receptor. In some embodiments, the antibodies have a constant domain that binds to CD163. + The constant domain that Fc receptors (such as CD16 (FcγRIIIa) or CD64 (FcγRI)) expressed on immunosuppressive myeloid cells bind to. In some embodiments, huCD163 and Fc receptors are expressed on the same cell. In some embodiments, huCD163 and Fc receptors are expressed on different cells. In some embodiments, the antibody variable domains specifically bind to huCD163 and the antibody constant domains simultaneously bind to Fc receptors.

[0208] In certain embodiments, disclosed herein are antibodies that specifically bind to CD163 proteins expressed on human M2 and M2-like macrophages, wherein the binding results in at least one of the following effects:

[0209] (a) human macrophages have reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2 or Siglec-15;

[0210] (b) Antibodies are internalized by human macrophages;

[0211] (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof;

[0212] (d) CD4 + T cells, CD8 + Proliferation of T cells, NK cells, or any combination thereof; and

[0213] (e) Promote tumor cell killing in the tumor microenvironment.

[0214] In certain embodiments, disclosed herein are antibodies that specifically bind to CD163 proteins expressed on human M2 and M2-like macrophages, wherein the binding results in at least two of the following effects:

[0215] (a) human macrophages have reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2 or Siglec-15;

[0216] (b) Antibodies are internalized by human macrophages;

[0217] (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof;

[0218] (d) CD4 + T cells, CD8 + Proliferation of T cells, NK cells, or any combination thereof; and

[0219] (e) Promote tumor cell killing in the tumor microenvironment.

[0220] In certain embodiments, disclosed herein are antibodies that specifically bind to CD163 proteins expressed on human M2 and M2-like macrophages, wherein the binding results in at least three of the following effects:

[0221] (a) human macrophages have reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2 or Siglec-15;

[0222] (b) Antibodies are internalized by human macrophages;

[0223] (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof;

[0224] (d) CD4 + T cells, CD8 + Proliferation of T cells, NK cells, or any combination thereof; and

[0225] (e) Promote tumor cell killing in the tumor microenvironment.

[0226] In certain embodiments, disclosed herein are antibodies that specifically bind to CD163 proteins expressed on human M2 and M2-like macrophages, wherein the binding results in at least four of the following effects:

[0227] (a) human macrophages have reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2 or Siglec-15;

[0228] (b) Antibodies are internalized by human macrophages;

[0229] (c)CD4 +T cells, CD8 + Activation of T cells, NK cells, or any combination thereof;

[0230] (d) CD4 + T cells, CD8 + Proliferation of T cells, NK cells, or any combination thereof; and

[0231] (e) Promote tumor cell killing in the tumor microenvironment.

[0232] In certain embodiments, disclosed herein are antibodies that specifically bind to CD163 proteins expressed on human M2 and M2-like macrophages, wherein the binding results in at least five of the following effects:

[0233] (a) human macrophages have reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2 or Siglec-15;

[0234] (b) Antibodies are internalized by human macrophages;

[0235] (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof;

[0236] (d) CD4 + T cells, CD8 + Proliferation of T cells, NK cells, or any combination thereof; and

[0237] (e) Promote tumor cell killing in the tumor microenvironment.

[0238] In some embodiments, the antibodies disclosed herein bind to human CD163 in a tumor-associated macrophage (TAM) population. + Selective binding of immunosuppressive myeloid cells, where the antibody specifically binds to the CD163 protein expressed on M2 macrophages and reduces the immunosuppressive activity of the TAM population.

[0239] In some embodiments, the antibodies disclosed herein bind to human CD163 in the tumor microenvironment. + Immunosuppressive myeloid cells selectively bind, wherein the antibody specifically binds to the CD163 protein expressed on M2 macrophages and reduces the inhibition mediated by M2 macrophages. In some embodiments, the antibodies disclosed herein are human, humanized or chimeric. In some embodiments, the antibodies disclosed herein are antigen-binding fragments combined as described.

[0240] In some embodiments, the antibodies of the present disclosure are complete immunoglobulin molecules, such as, for example, human antibodies, and those portions or single heavy chains and single light chains containing antigen binding sites (i.e., paratopes) in humanized Ig molecules, including those portions known in the art, such as Fab, Fab', F(ab'), F(ab')2, Fd, scFv, variable heavy domains, variable light domains, variable NAR domains, bispecific scFv, bispecific Fab2, trispecific Fab3, single-chain binding polypeptides, dAb fragments, diabodies, and others also referred to as antigen binding fragments. When constructing immunoglobulin molecules or fragments thereof, in some embodiments, the variable region or a portion thereof is fused, linked, or otherwise linked to one or more constant regions or a portion thereof to produce any antibody or fragment thereof described herein. Therefore, in some embodiments, the antigen binding fragment of any of the antibodies described above is Fab, Fab', Fd, F(ab')2, Fv, scFv, single-chain binding polypeptides (e.g., scFv with an Fc portion), or any other functional fragment thereof as described herein.

[0241] In some embodiments, the antibodies of the present disclosure belong to any immunoglobulin class and thus, in some embodiments, have a γ, μ, α, δ, or ε heavy chain. In some embodiments, the γ chain is γ1, γ2, γ3, or γ4. In some embodiments, the α chain is α1 or α2.

[0242] In some embodiments, the antibodies of the present disclosure are IgG immunoglobulins. In some embodiments, the antibodies of the present disclosure belong to any IgG subclass. In some embodiments, the antibody is IgG1.

[0243] In some embodiments, the antibodies of the present disclosure comprise a variable light chain that is κ or λ. In some embodiments, the λ chain belongs to any subtype including, for example, λ1, λ2, λ3, and λ4. In some embodiments, the light chain is κ.

[0244] 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.

[0245] 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.

[0246] 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 comprising SEQ ID NO: 11, AB102 comprises the variable region of AB101, and wherein the heavy chain constant region is a human IgG1 in an Fc null form. AB102 is further described in the Examples below.

[0247] In some embodiments, the heavy chain constant region is a human IgG1 modified to enhance ADCC function. An exemplary antibody with enhanced ADCC function of the present disclosure is AB103. AB103 comprises a light chain comprising SEQ ID NO: 9 and a heavy chain comprising SEQ ID NO: 12, AB103 comprises the variable region of AB101, and wherein the heavy chain constant region is a human IgG1 in an enhanced ADCC form.

[0248] 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 comprising SEQ ID NO: 13, AB104 comprises the variable region of AB101, and wherein the heavy chain constant region is human IgG4.

[0249] In some embodiments, the antibodies of the present disclosure comprise a human variable framework region and a mouse constant region. In some embodiments, the antibodies of the present disclosure comprise a human heavy chain variable framework region and a mouse heavy chain constant region. In some embodiments, the antibodies of the present disclosure comprise a human light chain variable framework region, a mouse light chain constant region, a human heavy chain variable framework region, and a mouse heavy chain constant region.

[0250] In some embodiments, the heavy chain constant region is murine IgG2A. An example of an antibody having murine IgG2A is AB211. AB211 comprises a light chain comprising SEQ ID NO: 14 and a heavy chain comprising SEQ ID NO: 15, AB211 comprises the human variable regions of AB101, and wherein the heavy chain constant region is murine IgG1 in an Fc-null form and the light chain constant region is murine κ. AB211 is further described in the Examples below.

[0251] In some embodiments, the heavy chain constant region is murine IgG2A. An example of an antibody having a murine IgG2A heavy chain is AB212. AB212 comprises a light chain comprising SEQ ID NO: 14 and a heavy chain comprising SEQ ID NO: 16, AB212 comprises the human variable regions of AB101, and wherein the heavy chain constant region is murine IgG2a and the light chain constant region is murine κ. AB212 is further described in the Examples below.

[0252] In some embodiments, the binding portion of the CD163 protein expressed on the antibody or Fab and M2 macrophages (for example, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or any number therein) or completely regulates the biological function of such M2 macrophages. The activity of the antibody or Fab, for example, is determined using in vitro assays and / or in vivo using art-recognized assays, such as those described herein or other art-known.

[0253] In some embodiments, if desired, the antibodies of the present disclosure are further modified to alter specific properties of the antibodies while retaining desired functionality. For example, in one embodiment, the antibodies of the present disclosure are modified to alter the pharmacokinetic properties of the antibodies, including, but not limited to, in vivo stability, solubility, bioavailability, or half-life.

[0254] In some embodiments, the antibodies described herein have a dissociation constant (Kd) of about 1 pM to about 10 pM, about 10 pM to about 20 pM, about 1 pM to about 29 pM, about 30 pM to about 40 pM, about 10 pM to about 100 pM, or about 20 pM to about 500 pM.

[0255] In some embodiments, the antibodies described herein have a dissociation constant (Kd) of 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 7.5 pM, less than about 5 pM, less than about 2.5 pM, or less than about 1 pM.

[0256] In some embodiments, the antibodies described herein have about 10 -9 To about 10 -14 , about 10 -10 To about 10 -14 , about 10 -11 To about 10 -14 , about 10-12 To about 10 -14 , about 10 -13 To about 10 -14 , about 10 -10 To about 10 -11 , about 10 -11 To about 10 -12 , about 10 -12 To about 10 -13 or 10 -13 To about 10 -14 M's affinity.

[0257] In some embodiments, the antibodies described herein have more than one binding site. In some embodiments, the binding sites are identical to each other. In some embodiments, the binding sites are different from each other. Naturally occurring human immunoglobulins typically have two identical binding sites, while engineered antibodies, for example, have two or more different binding sites.

[0258] In some embodiments, the antibodies of the present disclosure are bispecific or multispecific. Bispecific antibodies are antibodies that have binding specificity to at least two different epitopes. In some embodiments, exemplary bispecific antibodies bind to two different epitopes of a single antigen. In some embodiments, other such antibodies 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, the binding of a bispecific antibody to one or more epitopes is simultaneous with the binding of the constant domain of the bispecific antibody to an Fc receptor.

[0259] In some embodiments, the antibodies of the present disclosure have two or more valencies, which are also referred to as multivalent. In some embodiments, the antibodies of the present disclosure are trispecific. In some embodiments, multivalent antibodies are internalized (and / or decomposed) faster than bivalent antibodies by cells expressing antigens bound to the antibodies. In some embodiments, the antibodies of the present disclosure are multivalent antibodies (e.g., tetravalent antibodies) with three or more antigen binding sites. In some embodiments, the multivalent antibodies of the present disclosure are produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. In some embodiments, the multivalent antibodies comprise a dimerization domain and three or more antigen binding sites. In some embodiments, the dimerization domain comprises an Fc region or a hinge region (or is composed of an Fc region or a hinge region). In this case, the antibody will comprise three or more antigen binding sites at the amino terminus of the Fc region and the Fc region. In some embodiments, the multivalent antibodies herein comprise about three to about eight (but preferably four) antigen binding sites. The 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, a polypeptide chain containing VD1-(X1) n -VD2-(X2) n -Fc, wherein VD1 is the first variable region, VD2 is the second variable region, Fc is a polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. In some embodiments, each polypeptide chain independently comprises: V H -C H 1-Flexible joint-V H -C H 1-Fc region chain; or V H -C H 1-V H -C H 1-Fc region chain. In some embodiments, the multivalent antibodies herein further comprise at least two (and preferably four) light chain variable region polypeptides. In some embodiments, the multivalent antibodies herein comprise 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 further comprise C L Structural domain.

[0260] In some embodiments, the antibodies of the present disclosure are constructed to fold into a multivalent form, which in some embodiments improves binding affinity, specificity and / or increases half-life in the blood. Multivalent forms of antibodies are prepared, for example, by techniques known in the art.

[0261] In some embodiments, the antibodies of the present disclosure are SMIPs or binding domain immunoglobulin fusion proteins specific for a target protein. These constructs are single-chain polypeptides comprising an antigen binding domain fused to the immunoglobulin domains necessary to achieve antibody effector functions.

[0262] In some embodiments, the antibodies of the present disclosure comprise a single-chain binding polypeptide having a heavy chain variable region and / or a light chain variable region that binds an epitope disclosed herein, and optionally an immunoglobulin Fc region. Such a molecule is a single chain variable fragment (scFv), optionally with effector function or increased half-life through the presence of an immunoglobulin Fc region.

[0263] Anti-CD163 antibody

[0264] In certain embodiments, provided herein is an antibody specifically bound to CD163 protein. In some embodiments, CD163 binding antibodies include at least one heavy chain and at least one light chain. In some embodiments, CD163 binding antibodies include 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 include three complementary determining regions (CDRs). The amino acid sequence of VH and VL and CDR determines the antigen binding specificity and antigen binding strength of the antibody. VH and VL domains are summarized in Table 1. Light chain and heavy chain are summarized in Table 2. The amino acid sequence of CDR is summarized in Table 3.

[0265] 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 complete immunoglobulins, scFv, Fab, F(ab')2 or disulfide bonded Fvs. In some embodiments, the antibodies disclosed herein are IgG or IgM. In some embodiments, the antibodies disclosed herein are humanized. In some embodiments, the antibodies disclosed herein are chimeric.

[0266] Anti-CD163 antibody variable domain

[0267] Table 1. Anti-CD163 variable domain sequences.

[0268]

[0269] 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 in 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 in SEQ ID NO: 7. In some embodiments, the VL has an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 7.

[0270] 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 in 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 in SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 8.

[0271] In certain embodiments, disclosed herein are antibodies comprising a light chain variable domain (VL) and a heavy chain variable domain (VH), wherein the light chain variable domain (VL) has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:7, and the heavy chain variable domain (VH) has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the VL has 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 in SEQ ID NO:7, and the VH has 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 in SEQ ID NO:8. In some embodiments, VL has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:7, and VH has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:8.

[0272] Anti-CD163 antibody light and heavy chains

[0273] Table 2. Anti-CD163 light chain and heavy chain sequences

[0274]

[0275]

[0276]

[0277] 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 in 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 in SEQ ID NO: 9. In some embodiments, the light chain has an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 9.

[0278] In certain embodiments, disclosed herein are antibodies comprising a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the heavy chain has 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 in SEQ ID NO: 10. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 10.

[0279] In certain embodiments, disclosed herein are antibodies comprising a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the heavy chain has 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 in SEQ ID NO: 11. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 11.

[0280] In certain embodiments, disclosed herein are antibodies comprising a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the heavy chain has 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 in SEQ ID NO: 12. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0281] In certain embodiments, disclosed herein are antibodies comprising a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the heavy chain has 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 in SEQ ID NO: 13. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 13.

[0282] In certain embodiments, disclosed herein are antibodies comprising a light chain and a heavy chain, wherein the light chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the light chain has 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 in SEQ ID NO:9, and the heavy chain has 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 in 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 in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:10.

[0283] In certain embodiments, disclosed herein are antibodies comprising a light chain and a heavy chain, wherein the light chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the light chain has 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 in SEQ ID NO:9, and the heavy chain has 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 in 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 in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:11.

[0284] In certain embodiments, disclosed herein are antibodies comprising a light chain and a heavy chain, wherein the light chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the light chain has 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 in SEQ ID NO:9, and the heavy chain has 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 in 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 in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:12.

[0285] In certain embodiments, disclosed herein are antibodies comprising a light chain and a heavy chain, wherein the light chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:13. In some embodiments, the light chain has 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 in SEQ ID NO:9, and the heavy chain has 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 in 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 in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:13.

[0286] In certain embodiments, disclosed herein are antibodies comprising a light chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the light chain has 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 in SEQ ID NO: 14. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 14.

[0287] In certain embodiments, disclosed herein are antibodies comprising a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the heavy chain has 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 in SEQ ID NO: 15. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 15.

[0288] In certain embodiments, disclosed herein are antibodies comprising a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the heavy chain has 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 in SEQ ID NO: 16. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 16.

[0289] In certain embodiments, disclosed herein are antibodies comprising a light chain and a heavy chain, wherein the light chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:14, and the heavy chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the light chain has 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 in SEQ ID NO:14, and the heavy chain has 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 in 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 in SEQ ID NO:14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:15.

[0290] In certain embodiments, disclosed herein are antibodies comprising a light chain and a heavy chain, wherein the light chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:14, and the heavy chain has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the light chain has 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 in SEQ ID NO:14, and the heavy chain has 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 in 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 in SEQ ID NO:14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:16.

[0291] Exemplary anti-CD163 complementarity determining regions

[0292] Table 3. Anti-CD163 CDR sequences

[0293]

[0294] In certain embodiments, disclosed herein are antibodies comprising at least one of: a light chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence listed in SEQ ID NO:1, a light chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence listed in SEQ ID NO:2, and a light chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence listed in SEQ ID NO:3. In some embodiments, the antibody that binds to CD163 comprises at least one of the following: a light chain CDR1 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 listed in SEQ ID NO: 1, a light chain CDR2 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 listed in SEQ ID NO: 2, and a light 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 listed in SEQ ID NO: NO:3 lists 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 amino acid sequence of the light chain CDR3. In some embodiments, the antibody combined with CD163 comprises at least one of the following: there is a light chain CDR1 with an amino acid sequence 100% identical to the amino acid sequence listed in SEQ ID NO:1, there is a light chain CDR2 with an amino acid sequence 100% identical to the amino acid sequence listed in SEQ ID NO:2, and there is a light chain CDR3 with an amino acid sequence 100% identical to the amino acid sequence listed in SEQ ID NO:3.

[0295] In certain embodiments, disclosed herein are antibodies comprising at least one of: a heavy chain CDR having an amino acid sequence that is at least about 70% identical to the amino acid sequence listed in SEQ ID NO:4, a heavy chain CDR2 having an amino acid sequence that is at least about 70% identical to the amino acid sequence listed in SEQ ID NO:5, and a heavy chain CDR3 having an amino acid sequence that is at least about 70% identical to the amino acid sequence listed in SEQ ID NO:6. In some embodiments, the antibody that binds to CD163 comprises at least one of the following: a heavy chain CDR1 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 listed in SEQ ID NO:4, a heavy chain CDR2 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 listed in SEQ ID NO:5, IDNO:6 amino acid sequence listed 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 amino acid sequence of heavy chain CDR3. In some embodiments, the antibody combined with CD163 comprises at least one of the following: there is a heavy chain CDR1 with an amino acid sequence 100% identical to the amino acid sequence listed in SEQ ID NO:4, there is a heavy chain CDR2 with an amino acid sequence 100% identical to the amino acid sequence listed in SEQ ID NO:5, there is a heavy chain CDR3 with an amino acid sequence 100% identical to the amino acid sequence listed in SEQ ID NO:6.

[0296] In certain embodiments, disclosed herein are antibodies comprising at least one of the following: a light chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in 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 in 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 in 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 in 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 in 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 in SEQ ID NO:6.In some embodiments, the antibody that binds to CD163 comprises at least one of the following: a light chain CDR1 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 listed in SEQ ID NO: 1, a light chain CDR2 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 listed in SEQ ID NO: 2, a light 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 listed in SEQ ID NO: NO:3 has a light chain CDR3 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 in SEQ ID NO:3, a heavy chain CDR1 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 in SEQ ID NO:4, and a heavy chain CDR2 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 in SEQ ID NO:4. NO:5 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 in SEQ ID NO:5, and a heavy chain CDR2 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 in SEQ ID NO:6.In some embodiments, the antibody that binds to CD163 comprises at least one of the following: a light chain CDR1 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO: 2, a light chain CDR3 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO: 3, a heavy chain CDR1 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO: 5, and a heavy chain CDR3 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO: 6.

[0297] Binding affinity and immunoreactivity

[0298] The binding affinity and / or avidity of an antibody or its Fab is improved by modifying the framework regions. Any suitable method for modifying the framework regions is known in the art and is contemplated herein. The selection of one or more related framework amino acid positions to be changed depends on a number of criteria. A criterion for selecting the related framework amino acids to be changed is, for example, the relative difference of the amino acid framework residues between the donor molecule and the acceptor molecule. Using this method to select the related framework positions to be changed has the advantage of avoiding any subjective bias in the residue determination or any bias in the contribution of the residue to the CDR binding affinity.

[0299] In some embodiments, the binding interaction is manifested as intermolecular contact with one or more amino acid residues of one or more CDRs. Antigen binding involves, for example, a CDR or CDR pair, or in some cases, a V H Chain and V L Interaction of up to all six CDRs of the chain.

[0300] The binding affinity and avidity of antibodies or antigen-binding fragments can be measured by surface plasmon resonance (SPR) measurements, AlphaLisa assays or equilibrium dissociation constants (K D ) by flow cytometry.

[0301] Disclosed herein are K values ​​ranging from 0.1 nM to 1000 nM. DAntibodies that specifically bind to human CD163. In some embodiments, the antibody is at about 0.1nM to about 500nM, about 0.1nM to about 100nM, about 0.1nM to about 50nM, about 0.1nM to about 20nM, about 0.1nM to about 10nM, about 0.1nM to about 5nM, about 0.1nM to about 2nM, about 0.1nM to about 1nM, about 0.1nM to about 0.5nM, about 0.5nM to about 1000nM, about 0.5nM to about 500nM, about 0.5nM to about 100nM, about 0.5 nM to about 50 nM, about 0.5 nM to about 20 nM, about 0.5 nM to about 10 nM, about 0.5 nM to about 5 nM, about 0.5 nM to about 2 nM, about 0.5 nM to about 1 nM, about 1 nM to about 1000 nM, about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 1 nM to about 2 nM, about 2 nM to about 1 000nM, about 2nM to about 500nM, about 2nM to about 100nM, about 2nM to about 50nM, about 2nM to about 20nM, about 2nM to about 10nM, about 2nM to about 5nM, about 5nM to about 1000nM, about 5nM to about 500nM, about 5nM to about 100nM, about 5nM to about 50nM, about 5nM to about 20nM, about 5nM to about 10nM, about 10nM to about 1000nM, about 10nM to about 500nM, about 1 K of about 0 nM to about 100 nM, about 10 nM to about 50 nM, about 10 nM to about 20 nM, about 20 nM to about 1000 nM, about 20 nM to about 500 nM, about 20 nM to about 100 nM, about 20 nM to about 50 nM, about 50 nM to about 1000 nM, about 50 nM to about 500 nM, about 50 nM to about 100 nM, about 100 nM to about 500 nM, about 100 nM to about 1000 nM, about 500 nM to about 1000 nM D Specifically binds to human CD163. In some embodiments, the antibody has a K of 1.8 nM, 12 nM, 45 nM or 89 nM. D Binds specifically to human CD163.

[0302] The antibodies disclosed herein bind to the bone marrow scavenger receptor CD163, which is highly expressed on tumor-associated macrophages (TAMs) and has been detected on different tumor cells from different sources. The expression of CD163 in tumor tissue is associated with poor prognosis. The binding affinity between the antibodies disclosed herein and IL-10 polarized M2c macrophages was measured by flow cytometry.

[0303] Disclosed herein are K values ​​ranging from 0.1 nM to 1000 nM. D Antibodies that specifically bind to M2c macrophages. In some embodiments, the antibodies are present at about 0.1 nM to about 500 nM, about 0.1 nM to about 100 nM, about 0.1 nM to about 50 nM, about 0.1 nM to about 20 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, about 0.1 nM to about 0.5 nM, about 0.5 nM to about 1000 nM, about 0.5 nM to about 500 nM, about 0.5 nM to about 100 nM, about 0.5 nM to about 50 nM, about 0.5 nM to about 20 nM, about 0.5 nM to about 10 nM, about 0.5 nM to about 5 nM, about 0.5 nM to about 2 nM, about 0.5 nM to about 1 nM, about 1 nM to about 1000 nM, about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 1 nM to about 2 nM, about 2 nM to about 1 000nM, about 2nM to about 500nM, about 2nM to about 100nM, about 2nM to about 50nM, about 2nM to about 20nM, about 2nM to about 10nM, about 2nM to about 5nM, about 5nM to about 1000nM, about 5nM to about 500nM, about 5nM to about 100nM, about 5nM to about 50nM, about 5nM to about 20nM, about 5nM to about 10nM, about 10nM to about 1000nM, about 10nM to about 500nM, about 1 K of about 0 nM to about 100 nM, about 10 nM to about 50 nM, about 10 nM to about 20 nM, about 20 nM to about 1000 nM, about 20 nM to about 500 nM, about 20 nM to about 100 nM, about 20 nM to about 50 nM, about 50 nM to about 1000 nM, about 50 nM to about 500 nM, about 50 nM to about 100 nM, about 100 nM to about 500 nM, about 100 nM to about 1000 nM, about 500 nM to about 1000 nM D Specifically binds to M2c macrophages. In some embodiments, the antibody has a K of 7.7 nM D Binds specifically to M2c macrophages.

[0304] Binding epitope

[0305] Antibody epitopes can be linear peptide sequences (i.e., "continuous") or can be composed of discontinuous amino acid sequences (i.e., "conformational" or "discontinuous"). In some embodiments, antibodies recognize one or more amino acid sequences; therefore, an epitope defines more than one different amino acid sequence. The 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 a CDR.

[0306] Human CD163 protein is a protein encoded by the CD163 gene in humans. The amino acid sequence of human CD163 is:

[0307]

[0308] Disclosed herein are antibodies that specifically bind to epitopes in human CD163. In some embodiments, antibodies disclosed herein bind to epitopes comprising non-continuous amino acid sequences. In some embodiments, antibodies bind to epitopes of human CD163 comprising the amino acid sequence IGRVNASKGFGHIWLDSVSCQGHEPAI (SEQ ID NO: 18). In some embodiments, antibodies bind to epitopes of human CD163 comprising the amino acid sequence VVCRQLGCGSA (SEQ ID NO: 19). In some embodiments, antibodies bind to epitopes of human CD163 comprising the amino acid sequence WDCKNWQWGGLTCD (SEQ ID NO: 20). In some embodiments, antibodies bind to epitopes of human CD163 comprising the amino acid sequence SEQ ID NO: 18-20.

[0309] Also disclosed herein are other antibodies specifically bound to the epitopes disclosed herein.These other antibodies or their antigen-binding fragments specifically bound to the epitopes disclosed herein can be identified using techniques known in the art.For example, computational methods are used to design epitope-specific antibodies.Nimrod et al., Computational Design of Epitope-Specific Functional Antibodies, Cell Reports 25, 2121-2131, Nov. 20, 2018, (incorporated herein by reference).Another method can be used to identify antibodies bound to specific epitopes from a library of antibodies bound to antigens, such as the following: first, atypical amino acids (ncAA) p-benzoyl-L-phenylalanine (pBpa) and p-azido-L-phenylalanine (pAzF) are incorporated into the target epitope, and then the antibody cross-linked with the epitope incorporated with ncAA after ultraviolet irradiation is selected.Because cross-linking will only occur when the distance between the antibody and the epitope is close enough, this method can effectively select antibodies specifically bound to the target epitope. Chen et al. Epitope-directed antibody selection by site-specific photocrosslinking, Science Advances, Vol. 6, no. 14, eaaz7825, 01Apr2020 (incorporated herein by reference).

[0310] Antibody modification

[0311] In some cases, antibodies or their antigen-binding fragments are modified using techniques known in the art for various purposes, such as, for example, by adding polyethylene glycol (PEG). In some embodiments, PEG modification (PEGylation) results in one or more of the following: improved circulation time, improved solubility, improved proteolytic resistance, reduced antigenicity and immunogenicity, improved bioavailability, reduced toxicity, improved stability, and easier formulation.

[0312] In some cases, when the antigen binding fragment does not contain an Fc portion, the Fc portion is added (e.g., recombinantly) to the fragment, for example, to increase the half-life of the antigen binding fragment in the blood circulation when administered to a subject. The selection of an appropriate Fc region and methods for incorporating such a fragment are known in the art. For example, by using conventional techniques known in the art, the Fc region of an IgG is incorporated into a polypeptide of interest so as to increase its circulation half-life without losing its biological activity. In some embodiments, the Fc portion of the antibody is further modified to increase the half-life of the antigen binding fragment in the blood circulation when administered to a subject. The modification is determined, for example, using conventional means in the art.

[0313] In addition, in some embodiments, antibodies and antigen-binding fragments thereof are produced or expressed so that they do not contain fucose on their composite N-glycoside-linked sugar chains. It is known that removing fucose from composite N-glycoside-linked sugar chains increases the effector functions of antibodies and antigen-binding fragments, including but not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Similarly, in some cases, an antibody or antigen-binding fragment thereof that binds an epitope is attached at its C-terminus to all or part of an immunoglobulin heavy chain derived from any antibody isotype (e.g., IgG, IgA, IgE, IgD, and IgM) and any isotype subclass (particularly IgG1, IgG2, IgG3, and IgG4).

[0314] In addition, in some embodiments, the antibodies or Fabs described herein are also modified so that they can pass through the blood-brain barrier. Such modifications of the antibodies or Fabs described herein allow for the treatment of brain diseases such as glioblastoma multiforme (GBM). Exemplary modifications that allow proteins such as antibodies or Fabs to pass through the blood-brain barrier are described in U.S. Patent Publication 2007 / 0082380.

[0315] Glycosylation of immunoglobulins has been shown to have a significant impact on their effector functions, structural stability, and secretion from antibody-producing cells. The carbohydrate groups responsible for these properties are generally attached to the constant (C) region of the antibody. For example, IgG has a C HGlycosylation at asparagine 297 in the 2 domain is essential for the full ability of IgG to activate the classical pathway of complement-dependent cell lysis (Tao and Morrison, J Immunol 143:2595 (1989)). H Glycosylation at asparagine 402 in the 3 domain is essential for the proper assembly and cytolytic activity of the antibody (Muraoka and Shulman, J Immunol 142:695 (1989)). H 1 and C H Removal of glycosylation sites such as positions 162 and 419 in the 3 domain results in intracellular degradation and at least 90% inhibition of secretion (Taylor and Wall, Mol Cell Biol 8:4197 (1988)). In addition, in some embodiments, antibodies and antigen-binding fragments thereof are produced or expressed such that they do not contain fucose on their complex N-glycoside-linked sugar chains. It is known that removal of fucose from complex N-glycoside-linked sugar chains increases the effector functions of antibodies and antigen-binding fragments, including, but not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In some embodiments, these "defucosylated" antibodies and antigen-binding fragments are produced by various systems utilizing molecular cloning techniques known in the art, including, but not limited to, transgenic animals, transgenic plants, or cell lines that have been genetically engineered to no longer contain the enzymes and biochemical pathways necessary to incorporate fucose into complex N-glycoside-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.

[0316] Glycosylation in the variable (V) region of immunoglobulins has also been observed. Sox and Hood 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 believed to be caused by the occasional appearance of the N-linked glycosylation signal Asn-Xaa-Ser / Thr in the V region sequence and has not been considered in the art to play a role in immunoglobulin function.

[0317] In some cases, glycosylation at variable domain framework residues alters the binding interaction of the antibody with the antigen. The present disclosure includes criteria for selecting a limited number of amino acids in the framework or CDRs of a humanized immunoglobulin chain for mutation (e.g., by substitution, deletion or addition of residues) to increase antibody affinity.

[0318] In some embodiments, cysteine ​​residues are removed or introduced into the Fc region of an antibody or an Fc-containing polypeptide, thereby eliminating or increasing interchain disulfide bond formation in this region. In some embodiments, homodimeric specific binding agents or antibodies produced using such methods exhibit improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).

[0319] It has been shown that sequences within the CDRs cause the antibody to bind to MHC class II and in some cases trigger unwanted helper T cell responses. In some embodiments, conservative substitutions allow the antibody to retain binding activity but reduce its ability to trigger unwanted T cell responses. In one embodiment, one or more of the 20 amino acids at the N-terminus of the heavy or light chain are removed.

[0320] In some embodiments, antibody molecules with altered carbohydrate structures are generated, resulting in altered effector activities, including antibody molecules with absent or reduced fucosylation that exhibit improved ADCC activity. Various methods are known in the art to accomplish this. For example, ADCC effector activity is mediated by binding of antibody molecules to the FcγRIII receptor, which has been shown to be dependent on C H The non-fucosylated antibody binds to the FcγRIII receptor with increased affinity and triggers FcγRIII-mediated effector functions more effectively than the naturally fucosylated antibody. Some host cell lines, such as Lec13 or rat hybridoma YB2 / 0 cell lines, naturally produce antibodies with lower fucosylation levels. Increased levels of bisected carbohydrates (e.g., by recombinantly producing antibodies in cells overexpressing the GnTIII enzyme) have also been determined to increase ADCC activity. In some embodiments, the absence of only one of the two fucose residues is sufficient to increase ADCC activity.

[0321] Covalent modification of antibodies is also included herein. In some embodiments, if applicable, covalent modification of antibodies is carried out by chemical synthesis or by enzymatic cleavage or chemical cleavage of antibodies. In some embodiments, other types of covalent modifications are introduced by reacting the targeted amino acid residues with an organic derivatizing agent that can react with selected side chains or N-terminal or C-terminal residues.

[0322] Cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines) such as chloroacetic acid or chloroacetamide to generate carboxymethyl or carboxamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercurybenzoate, 2-chloromercury-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0323] In some embodiments, histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0, as this agent is relatively specific for histidyl side chains. In some embodiments, p-bromobenzoyl bromide is also useful; in some embodiments, the reaction is carried out in 0.1 M sodium dimethylarsenate at pH 6.0.

[0324] In some embodiments, lysyl and amino terminal residues are reacted with succinic anhydride or other carboxylic anhydrides. Derivatization with these agents has the effect of reversing the charge of lysyl residues. Other suitable reagents for making residues containing α-amino groups derivatize include imidoesters, such as picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione and transaminase-catalyzed reaction with glyoxylic acid.

[0325] 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. Due to the high pKa of the guanidine functional group, the derivatization of arginine residues requires the reaction to be carried out under alkaline conditions. In addition, in some embodiments, these reagents react with lysine groups and arginine epsilon-amino groups.

[0326] In some embodiments, due to the particular interest in introducing spectral labels to tyrosyl residues, tyrosyl residues are specifically modified by reaction with aromatic diazonium compounds or tetranitromethane. In some embodiments, most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. 125 I or 131 I iodinated tyrosyl residues to prepare labeled proteins for radioimmunoassay.

[0327] The carboxyl side groups (aspartyl or glutamyl) are specifically modified by reaction with carbodiimides (RN=C=N-R'), wherein R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0328] In some embodiments, glutaminyl residues and asparaginyl residues are deamidated to the corresponding glutamyl residues and aspartyl residues, respectively. These residues are deamidated under neutral or alkaline conditions.

[0329] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups 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.

[0330] Another type of covalent modification includes chemical or enzymatic coupling of glycosides to specific binding agents or antibodies. These procedures do not require the production of polypeptides or antibodies in host cells with glycosylation capabilities for N-linked or O-linked glycosylation. In some embodiments, depending on the coupling mode used, 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) amide groups of glutamine.

[0331] In some embodiments, the removal of any carbohydrate moieties present on a polypeptide or antibody is accomplished chemically or enzymatically. Chemical deglycosylation includes exposing the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all carbohydrates except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine) while keeping the antibody intact. In some embodiments, enzymatic cleavage of carbohydrate moieties on an antibody is achieved by using various endo- and exo-glycosidases.

[0332] Another type of covalent modification includes linking the antibody to one of various nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran. Such methods are known in the art.

[0333] The affinity for binding to a predetermined polypeptide antigen is generally adjusted by introducing one or more mutations into the V region framework (usually in a region adjacent to one or more CDRs and / or in one or more framework regions). Typically, such mutations include conservative amino acid substitutions that introduce a glycosylation site sequence that destroys or generates a glycosylation site sequence but does not substantially affect the hydrophilic / hydrophobic structural properties of the polypeptide. Typically, mutations that introduce proline residues are avoided.

[0334] Effector function

[0335] 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. Typically, Fc-mediated functions involve the binding of the Fc portion of an antibody to specific receptor molecules, "Fc receptors," or "FcRs," which are expressed by cells whose cellular function is to be affected.

[0336] IgG is considered to be the most functional immunoglobulin because it performs all functions of immunoglobulin molecules in some embodiments. IgG is the main Ig in serum, and is also the only class of Ig that crosses the placenta. IgG also fixes complement, although the IgG4 subclass does not fix complement. Macrophages, monocytes, polymorphonuclear leukocytes (PMN) and some lymphocytes have receptors for the Fc region of IgG. Not all subclasses combine equally well: IgG2 and IgG4 do not bind to Fc receptors. The result of binding to the Fc receptors on PMN, monocytes and macrophages is that, in some cases, cells can now better internalize antigens. IgG is an opsonin that enhances phagocytosis. The combination of IgG with the Fc receptors on other types of cells causes the activation of other functions.

[0337] In certain embodiments, the FcR is a native sequence human FcR. In addition, a preferred FcR is an FcR that binds to an IgG antibody (a gamma ("γ") receptor), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.

[0338] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are required for such killing. Among the main cells that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. In order to assess the ADCC activity of a molecule of interest, an in vitro ADCC assay is performed in some embodiments. Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0339] Alternatively or additionally, in some embodiments, ADCC activity of the molecule of interest is assessed in vivo, eg, in an animal model.

[0340] In some embodiments, the antibodies of the present disclosure bind to surface membrane proteins of M2-like macrophages and are internalized by M2-like macrophages. This internalization process is believed to be involved in the observed changes in the functional immunosuppressive characteristics of these cells, i.e., the cells differentiate from the M2 state to a subtly activated state without killing them or inhibiting their proliferation. In some embodiments, after internalization, the antibodies reduce the expression of immunosuppressive soluble factors while stimulating or promoting T cells (including CD4 + Increased expression of soluble factors that regulate the activity or proliferation of T helper cells and cytotoxic lymphocytes.

[0341] For some therapeutic applications, the internalization process is adopted to kill the target cell expressing CD163 protein or reduce the activity or propagation purpose of the target cell expressing CD163 protein.The quantity of the antibody molecule of internalization will be enough or enough to kill cells or inhibit its growth, especially cancer cells.Depending on the effectiveness of antibody or antibody conjugates, in some cases, a single antibody molecule is taken into the target cell that is enough to kill antibody binding in the cell.For example, some toxin has efficient lethality, makes the internalization of a molecule of the toxin conjugated with antibody just enough to kill target cells.

[0342] In some embodiments, the antibody or antigen binding fragment provided herein is conjugated or connected to a therapeutic moiety, imaging or detectable moiety or affinity tag. Methods for conjugating or connecting polypeptides are well known in the art. The association (binding) between the compound and the label includes any manner known in the art, including but not limited to covalent and non-covalent interactions, chemical conjugation, and recombinant techniques. In some embodiments, the antibody or its antigen binding fragment is conjugated to an affinity tag (e.g., a purification tag) or is recombinantly engineered to have an affinity tag (e.g., a purification tag). Affinity tags, such as, for example, polyhistidine (e.g., His6) tags are conventional in the art.

[0343] In some embodiments, antibody or Fab also comprise detectable part.Detection, for example, in vitro, in vivo or in vitro completion.Use antibody or its Fab for, for example, by the detection of huCD163 protein expressed by macrophage and / or determine (quantitative, qualitative etc.) in vitro assay including but not limited to, for example, ELISA, RIA and protein blot.In some embodiments, the in vitro detection, diagnosis or monitoring of the antigen of antibody is by obtaining sample (for example, blood sample) from subject and testing the sample in for example standard ELISA assay and occur.

[0344] Also disclosed herein, in certain embodiments, are compositions comprising an antibody as disclosed herein and a carrier.

[0345] Pharmaceutical composition

[0346] Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising an antibody disclosed herein and a pharmaceutically acceptable excipient.

[0347] Such compositions can be used in in vitro or in vivo assays, or, in the case of pharmaceutical compositions, for in vivo or ex vivo administration to a subject to treat the subject with the disclosed antibodies.

[0348] In some embodiments, the excipient is a carrier, buffer, stabilizer or other suitable substance known to those skilled in the art. Such a substance should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance will depend on the route of administration.

[0349] In some embodiments, pharmaceutical formulations comprising antibodies or antigen-binding fragments identified by the methods described herein are prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing a protein having a desired degree of purity with an optional physiologically acceptable carrier, excipient, or stabilizer (see, e.g., Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)). Acceptable carriers or stabilizers are those that are nontoxic to recipients at the doses and concentrations employed, and include: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than 1% to 1% hydroxybenzoic acid) and / or 1% to 2% hydroxybenzoic acid. about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinyl pyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as or polyethylene glycol (PEG). In certain embodiments, the pharmaceutical composition comprises the antibody at a concentration between 5 mg / mL-200 mg / mL; preferably between 10 mg / mL-100 mg / mL.

[0350] An acceptable carrier is physiologically acceptable to the subject to be administered, and retains the therapeutic properties of the compound to be administered therewith / administered therein. Acceptable carriers and their formulations are generally described, for example, in the following: Remington's Pharmaceutical Sciences, supra. An exemplary carrier is normal saline. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, which participates in carrying or transporting the subject compound from an organ or a portion of the site of administration of the body to another organ or a portion of the body or in an in vitro assay system. Each carrier is "acceptable" in the sense that it is compatible with the other ingredients of the preparation and is non-damaging to the subject to be administered thereto. An acceptable carrier should also not change the specific activity of the subject compound.

[0351] In another embodiment, the pharmaceutical composition disclosed herein also includes an acceptable additive to improve the stability of the compound in the composition and / or control the release rate of the composition. Acceptable additives do not change the specific activity of the subject compound. Exemplary acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, seaweed, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof. In some embodiments, acceptable additives are combined with acceptable carriers and / or excipients such as dextrose. Alternatively, exemplary acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 to increase the stability of the peptide and reduce the gelation of the solution. In some embodiments, the surfactant is 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 in storage.

[0352] In one embodiment, the pharmaceutical compositions disclosed herein contain an isotonic buffer such as phosphate, acetate or TRIS buffer, in combination with a tonicity agent such as a polyol, sorbitol, sucrose or sodium chloride, which increases tonicity and stabilizes. In one embodiment, the tonicity agent is present in the composition in an amount of about 5%.

[0353] In another embodiment, the pharmaceutical compositions disclosed herein comprise 0.01% to 0.02% wt / vol of a surfactant to prevent aggregation and for stabilization.

[0354] In another embodiment, the pH range of the pharmaceutical compositions disclosed herein is 4.5-6.5 or 4.5-5.5.

[0355] In some embodiments, the pharmaceutical compositions disclosed herein also contain more than one active compound necessary for the indication being treated, such as those with complementary activities that do not adversely affect each other. For example, the treatment method also provides an immunosuppressant. Such molecules are suitably combined in an amount effective for the intended purpose.

[0356] In some embodiments, the active ingredient is embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in: Remington's Pharmaceutical Sciences, supra.

[0357] Suspension and crystalline forms of the antibodies are also contemplated herein; methods of preparing suspensions and crystalline forms are known to those skilled in the art.

[0358] 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 is easily completed by filtering through a sterile filtration membrane. In some embodiments, the resulting solution is packaged for use or filtered and lyophilized under aseptic conditions, and the lyophilized preparation is combined with a sterile solution before administration.

[0359] In some embodiments, freeze-drying is employed to stabilize the polypeptide for long-term storage, such as when the polypeptide is relatively unstable in a liquid composition.

[0360] In some embodiments, some excipients, such as, for example, polyols (including mannitol, sorbitol and glycerol); sugars (including glucose and sucrose); and amino acids (including alanine, glycine and glutamic acid), act as stabilizers for freeze-dried products. In some embodiments, polyols and sugars are also used to protect polypeptides from damage induced by freezing and drying and to enhance stability during storage in a dry state. In some embodiments, sugars are effective both during the freeze-drying process and during storage. Other classes of molecules, including monosaccharides and disaccharides and polymers such as PVP, have also been reported as stabilizers for freeze-dried products.

[0361] For injection, in some embodiments, the pharmaceutical compositions disclosed herein are powders suitable for reconstitution with the appropriate solutions described above. Examples of these include, but are not limited to, freeze-dried powders, rotary dried powders or spray dried powders, amorphous powders, granules, precipitates or microparticles. For injection, the composition optionally contains stabilizers, pH regulators, surfactants, bioavailability regulators and combinations of these.

[0362] In some embodiments, a sustained release formulation is prepared.Sustained release formulations are suitable examples including semipermeable matrices of solid hydrophobic polymers containing antibodies, wherein the matrix is ​​in the form of molded articles, for example, films or microcapsules.Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (methacrylic acid -2- hydroxyethyl ester) or poly (vinyl alcohol)), polylactides (see, e.g., U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot.TM. (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable the release of molecules to exceed 100 days, while some hydrogels enable the release of proteins to be shorter. In some embodiments, although the encapsulated antibodies are retained in vivo for a long time, they denature or aggregate due to exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. In some cases, the rational strategy designed for stabilization depends on the mechanism involved. For example, if the aggregation mechanism is found to be the formation of intermolecular S--S bonds through sulfo-disulfide exchange, stabilization is achieved in some cases by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0363] In some embodiments, the pharmaceutical compositions disclosed herein are designed to be short-acting, fast-release, long-acting, or sustained-release as described herein. In one embodiment, the pharmaceutical compositions disclosed herein are formulated for controlled release or for slow release.

[0364] Pharmaceutical compositions, for example, are administered by injection, including but not limited to, subcutaneous, intravitreal, intradermal, intravenous, intraarterial, intraperitoneal, intracerebrospinal or intramuscular injection. Excipients and carriers for preparing compositions for each type of injection are contemplated herein. The following description is exemplary only and is not intended to limit the scope of the composition. Compositions for injection include but are not limited to aqueous solutions (in the case of water soluble) or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL.TM. (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In some embodiments, the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Maintain fluidity, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In some embodiments, isotonic agents are included in the composition, for example, sugars, polyols such as mannitol, sorbitol, and sodium chloride. In some embodiments, the resulting solution is packaged for use as is or lyophilized; in some embodiments, the lyophilized product is combined with a sterile solution before administration. For intravenous injection or injection at the site of disease, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity, and stability. Those skilled in the art are well able to use, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection to prepare suitable solutions. In some embodiments, preservatives, stabilizers, buffers, antioxidants, and / or other additives are included as needed. In some embodiments, sterile injectable solutions are prepared by: the required amount of active ingredient is mixed with one or a combination of the ingredients listed above as needed in an appropriate solvent, and then filtered and sterilized. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0365] In some embodiments, the composition is routinely administered intravenously, for example, such as by injecting a unit dose. For injection, in some embodiments, the active ingredient is substantially free of pyrogens and has a parenteral acceptable aqueous solution form of suitable pH, isotonicity and stability. In some embodiments, people use, for example, isotonic vehicles (such as sodium chloride injection, Ringer's injection, lactated Ringer's injection) to prepare suitable solutions. In some embodiments, preservatives, stabilizers, buffers, antioxidants and / or other additives are included as required. In addition, in some embodiments, the composition is administered via aerosolization. (Lahn et al., Int Arch Allergy Immunol 134:49-55 (2004)).

[0366] For parenteral administration, the antibody is formulated into a unit dose injectable form (solution, suspension, emulsion) together with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as non-volatile oils and ethyl oleate are also used. Liposomes are used as carriers. The vehicle contains a small amount of additives, such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives. The antibody is typically formulated in such a vehicle at a concentration of about 1 mg / mL to 10 mg / mL.

[0367] In one embodiment, the pharmaceutical compositions disclosed herein are lyophilized, for example, to increase shelf life in storage. When considering the use of the composition for the medicaments or any methods provided herein, in some embodiments, it is envisioned that the composition is substantially free of pyrogens so that the composition does not cause an inflammatory response or an unsafe allergic reaction when administered to a human subject. Testing the composition for pyrogens and preparing compositions that are substantially free of pyrogens are well known to those of ordinary skill in the art and are accomplished in some embodiments using commercially available kits.

[0368] In some embodiments, an acceptable carrier contains a compound that stabilizes, increases or delays absorption or clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose or dextran; low molecular weight proteins; compositions that reduce clearance or hydrolysis of 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 or increase or decrease the absorption of pharmaceutical compositions including liposome carriers. To prevent digestion, in some embodiments, the compound is complexed with a composition that is resistant to acid hydrolysis and enzymatic hydrolysis, or in some embodiments, the compound is complexed in an appropriate resistant carrier such as a liposome. Methods for protecting compounds from digestion are known in the art.

[0369] In some embodiments, the composition is used in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be applied depends on the ability of the subject to be treated, the subject's immune system to utilize the active ingredient and the degree of the desired binding ability. The precise amount of the active ingredient needed to be applied depends on the judgment of the professional and is unique to each individual. The appropriate scheme for initial application and booster injection is also variable, but it is typical to initially apply and then apply repeatedly at intervals of one or more hours by subsequent injections or other applications. Alternatively, it is envisioned that the continuous intravenous infusion that is enough to maintain concentration in the blood is infused.

[0370] In some embodiments, the disclosure provides the use of compositions described herein for the preparation of medicines for treating conditions, diseases or disorders described herein. In some embodiments, the medicine is formulated based on the physical characteristics of the subject in need of treatment, and is formulated as a single or more than one formulation based on the stage of the condition, disease or disorder. In some embodiments, the medicine is packaged in suitable packaging with appropriate labels for distribution to hospitals and clinics, wherein the label is used to indicate the treatment of subjects with diseases described herein. In some embodiments, the medicine is packaged as a single or more than one unit. In some embodiments, the dosage of the composition and the instructions for administration are included with the packaging as described below. The disclosure also relates to medicines comprising antibodies or antigen-binding fragments thereof described herein and pharmaceutically acceptable carriers.

[0371] 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 condition to be treated. In one embodiment, the second therapeutic treatment is an anticancer therapy or anticancer therapeutic. When two or more compositions are administered, the compositions, for example, are administered in combination (sequentially or simultaneously). In some embodiments, the composition is administered in a single dose or more than one dose.

[0372] In some embodiments, the composition is formulated to be pyrogen-free when formulated for administration to a human subject. Testing compositions for pyrogens and preparing pyrogen-free pharmaceutical compositions are well known to those of ordinary skill in the art.

[0373] In some embodiments, the antibody or antigen-binding fragment thereof is formulated for administration to a subject by any suitable route, including but not limited to injection. Injection includes, for example, subcutaneous, peritoneal, intravenous, intramuscular, or spinal injection into the cerebrospinal fluid (CSF). In some embodiments, administration is performed at one, two, three, four, five, six, seven or more injection sites. In one embodiment, administration is performed via six injection sites.

[0374] For in vivo application, contact occurs, for example, via administering compositions (such as described herein) to a subject in any suitable manner. In some embodiments, antibodies described herein are administered systemically or topically in any suitable manner, including administration via parenteral, subcutaneous, intraperitoneal, intracerebrospinal, intrapulmonary and intranasal, and if desired local treatment, intralesional administration. Parenteral approaches include, for example, intravenous, intraarterial, intraperitoneal, epidural, intramuscular and intrathecal administration. In some embodiments, such administration is such as bolus, continuous infusion or pulse infusion. In some embodiments, depending in part on whether administration is short-lived or long-term, compositions are administered by injection. Other modes of administration methods are also contemplated, including topical administration, particularly percutaneous, transmucosal, rectal, oral or topical administration, for example, by a catheter placed near the desired site.

[0375] Treatment and use

[0376] In certain embodiments, disclosed herein is a method for treating a cancer of an individual in need thereof, the method comprising administering an antibody disclosed herein to an individual. In some embodiments, the disclosure provides the use of an antibody as described herein for preparing 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 macrophages.

[0377] In certain embodiments, disclosed herein is a method for regulating the immune activity in a subject in need thereof, the method comprising administering an antibody described herein to the subject. In some embodiments, the antibody specifically binds to the CD163 protein expressed on human tumor-associated macrophages and reduces the expression of at least one of CD16, CD64, TLR2 or Siglec-15 by macrophages.

[0378] In certain embodiments, disclosed herein is a method for treating a subject with pathological or inappropriately elevated M2 macrophage levels (e.g., relative to an inappropriately elevated level that can be used to promote immune-mediated tumor cell killing in a subject), the method comprising administering an antibody described herein to the subject. In some embodiments, the antibody specifically binds to the CD163 protein expressed on human tumor-associated macrophages and reduces the expression of at least one of macrophages to CD16, CD64, TLR2 or Siglec-15.

[0379] 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 in 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 in 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 in SEQ ID NO:3. In some embodiments, the antibody comprises at least one of: a light chain CDR1 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 in SEQ ID NO: 1, a light chain CDR2 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 in SEQ ID NO: 2, and a light 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 in SEQ ID NO: NO:3 set forth 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 light chain CDR3 of the amino acid sequence. In some embodiments, the antibody comprises at least one of the following: a light chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 2, and a light chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 3.

[0380] In some embodiments, the antibody comprises at least one of the following: a heavy chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence listed in SEQ ID NO:4, a heavy chain CDR2 having an amino acid sequence at least about 70% identical to the amino acid sequence listed in SEQ ID NO:5, and a heavy chain CDR3 having an amino acid sequence at least about 70% identical to the amino acid sequence listed in SEQ ID NO:6. In some embodiments, the antibody comprises at least one of: a heavy chain CDR1 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 in SEQ ID NO:4, a heavy chain CDR2 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 in SEQ ID NO:5, a 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 in SEQ ID NO: NO:6 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 listed in SEQ ID NO:6. In some embodiments, the antibody comprises at least one of the following: a heavy chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence listed in SEQ ID NO:4, a heavy chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence listed in SEQ ID NO:5, and a heavy chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence listed in SEQ ID NO:6.

[0381] In some embodiments, the antibody comprises at least one of the following: a light chain CDR1 having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in 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 in 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 in 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 in 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 in 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 in SEQ ID NO:6.In some embodiments, the antibody comprises at least one of: a light chain CDR1 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 in SEQ ID NO: 1, a light chain CDR2 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 in SEQ ID NO: 2, a light 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 in SEQ ID NO: NO:3 has a light chain CDR3 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 in SEQ ID NO:3, a heavy chain CDR1 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 in SEQ ID NO:4, and a heavy chain CDR2 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 in SEQ ID NO:4. ID NO:5 is set forth in the present invention. The present invention further comprises a heavy chain CDR2 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 in SEQ ID NO:5, and a 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 in SEQ ID NO:6.In some embodiments, the antibody comprises at least one of the following: a light chain CDR1 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO:1, a light chain CDR2 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO:2, a light chain CDR3 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO:3, a heavy chain CDR1 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO:4, a heavy chain CDR2 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO:5, and a heavy chain CDR3 having an amino acid sequence that is 100% identical to the amino acid sequence listed in SEQ ID NO:6.

[0382] 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 in 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 in SEQ ID NO: 7. In some embodiments, the VL has an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 7.

[0383] 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 in 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 in SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 8.

[0384] In some embodiments, the antibody comprises a light chain variable domain (VL) and a heavy chain variable domain (VH), wherein the light chain variable domain (VL) has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:7, and the heavy chain variable domain (VH) has an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the VL has 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 in SEQ ID NO:7, and the VH has 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 in SEQ ID NO:8. In some embodiments, VL has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:7, and VH has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:8.

[0385] In some embodiments, the antibody comprises a light chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the light chain has 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 in SEQ ID NO: 9. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 9.

[0386] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the heavy chain has 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 in SEQ ID NO: 10. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 10.

[0387] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the heavy chain has 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 in SEQ ID NO: 11. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 11.

[0388] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the heavy chain has 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 in SEQ ID NO: 12. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0389] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the heavy chain has 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 in SEQ ID NO: 13. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 13.

[0390] 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 in 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 in SEQ ID NO:10. In some embodiments, the light chain has 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 in SEQ ID NO:9, and the heavy chain has 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 in 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 in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:10.

[0391] In some embodiments, the antibody comprises a light chain and a heavy chain, the light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the light chain has 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 in SEQ ID NO:9, and the heavy chain has 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 in 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 in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:11.

[0392] In some embodiments, the antibody comprises a light chain and a heavy chain, the light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the light chain has 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 in SEQ ID NO:9, and the heavy chain has 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 in 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 in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:12.

[0393] In some embodiments, the antibody comprises a light chain and a heavy chain, the light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:13. In some embodiments, the light chain has 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 in SEQ ID NO:9, and the heavy chain has 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 in 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 in SEQ ID NO:9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:13.

[0394] In some embodiments, the antibody comprises a light chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the light chain has 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 in SEQ ID NO: 14. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 14.

[0395] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the heavy chain has 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 in SEQ ID NO: 15. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 15.

[0396] In some embodiments, the antibody comprises a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the heavy chain has 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 in SEQ ID NO: 16. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 16.

[0397] In some embodiments, the antibody comprises a light chain and a heavy chain, the light chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:14, and the heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the light chain has 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 in SEQ ID NO:14, and the heavy chain has 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 in 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 in SEQ ID NO:14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:15.

[0398] 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 in 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 in SEQ ID NO:16. In some embodiments, the light chain has 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 in SEQ ID NO:14, and the heavy chain has 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 in 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 in SEQ ID NO:14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO:16.

[0399] In some embodiments, the disclosure provides use of an antibody as described herein for the preparation of a medicament for reducing immunosuppression of tumor-associated macrophages in a human subject having cancer.

[0400] In some embodiments, the disclosure provides for the use of an antibody as described herein for the preparation of a medicament for promoting T cell-mediated killing of tumor cells in a human subject having cancer.

[0401] In some embodiments, the disclosure provides methods of treating a human subject having cancer, the methods comprising administering to the subject a therapeutically effective amount of an antibody as described herein, thereby reducing immunosuppression of tumor-associated macrophages in the subject.

[0402] In some embodiments, the disclosure provides a method of treating a human subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody as described herein, thereby increasing T cell-mediated tumor cell killing in the subject.

[0403] Disclosed herein, in certain embodiments, are methods of functionally redirecting tumor-associated macrophages to reduce immunosuppression in a patient suffering from cancer, the method comprising administering to the patient an amount of a pharmaceutical composition comprising an antibody described herein that improves CD4 in the tumor microenvironment. + or CD8 + T cell activity or proliferation is effective.

[0404] Disclosed herein, in certain embodiments, are methods of promoting lymphocyte-mediated killing of tumor cells in a human subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody as described herein.

[0405] In certain embodiments, disclosed herein are methods of modulating the activity of tumor-associated macrophages in a tumor microenvironment, the method comprising contacting the tumor-associated macrophages with an antibody disclosed herein, wherein the method results in at least one of the following effects:

[0406] (a) human macrophages have reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2 or Siglec-15;

[0407] (b) Antibodies are internalized by human macrophages;

[0408] (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof;

[0409] (d) CD4 + T cells, CD8 + Proliferation of T cells, NK cells, or any combination thereof; and

[0410] (e) Promote tumor cell killing in the tumor microenvironment.

[0411] In certain embodiments, disclosed herein are methods of modulating the activity of tumor-associated macrophages in a tumor microenvironment, the method comprising contacting the tumor-associated macrophages with an antibody disclosed herein, wherein the method results in at least two of the following effects:

[0412] (a) human macrophages have reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2 or Siglec-15;

[0413] (b) Antibodies are internalized by human macrophages;

[0414] (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof;

[0415] (d) CD4 + T cells, CD8 + Proliferation of T cells, NK cells, or any combination thereof; and

[0416] (e) Promote tumor cell killing in the tumor microenvironment.

[0417] In certain embodiments, disclosed herein are methods of modulating the activity of tumor-associated macrophages in a tumor microenvironment, the method comprising contacting the tumor-associated macrophages with an antibody disclosed herein, wherein the method results in at least three of the following effects:

[0418] (a) human macrophages have reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2 or Siglec-15;

[0419] (b) Antibodies are internalized by human macrophages;

[0420] (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof;

[0421] (d) CD4 + T cells, CD8 + Proliferation of T cells, NK cells, or any combination thereof; and

[0422] (e) Promote tumor cell killing in the tumor microenvironment.

[0423] In certain embodiments, disclosed herein are methods of modulating the activity of tumor-associated macrophages in a tumor microenvironment, the method comprising contacting the tumor-associated macrophages with an antibody disclosed herein, wherein the method results in at least four of the following effects:

[0424] (a) human macrophages have reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2 or Siglec-15;

[0425] (b) Antibodies are internalized by human macrophages;

[0426] (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof;

[0427] (d) CD4 + T cells, CD8 + Proliferation of T cells, NK cells, or any combination thereof; and

[0428] (e) Promote tumor cell killing in the tumor microenvironment.

[0429] In certain embodiments, disclosed herein are methods of modulating the activity of tumor-associated macrophages in a tumor microenvironment, the method comprising contacting the tumor-associated macrophages with an antibody disclosed herein, wherein the method results in at least five of the following effects:

[0430] (a) human macrophages have reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2 or Siglec-15;

[0431] (b) Antibodies are internalized by human macrophages;

[0432] (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof;

[0433] (d) CD4 + T cells, CD8 + Proliferation of T cells, NK cells, or any combination thereof; and

[0434] (e) Promote tumor cell killing in the tumor microenvironment.

[0435] In some embodiments, the methods disclosed herein reduce the suppression of CD8 T cell activation and proliferation by myeloid cells.

[0436] M2 to M1 macrophage polarization refers to the process by which an M2 or M2-like macrophage of the present disclosure is altered so that the resulting macrophage has certain functional or phenotypic attributes associated with an M1 or M1-like macrophage. In some embodiments, an M2 or M2-like macrophage is polarized when it no longer expresses CD163.

[0437] In some embodiments, the antibody reduces myeloid cell inhibition of CD19-CD3 BiTE-mediated Raji cell killing of CD8 T cells.

[0438] In some embodiments, the antibody reduces myeloid cell inhibition of CAR T cell-mediated killing of cancer cells.

[0439] In some embodiments, the antibody reduces myeloid cell inhibition of NK cell-mediated killing of cancer cells by ADCC.

[0440] In some cases, any method disclosed herein further comprises administering to the subject an additional anti-cancer therapy. Anti-cancer therapy includes, but is not limited to, surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone 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.

[0441] 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.

[0442] In some embodiments, the disclosure provides an in vitro or ex vivo method for identifying macrophages expressing huCD163 in a cell sample suspected of containing macrophages expressing huCD163, the method comprising contacting the cell sample with an antibody described herein and measuring binding to cells in the sample, a positive signal indicating the presence of macrophages expressing huCD163 in the sample.

[0443] In some embodiments, the present disclosure provides a method for identifying human M2c macrophages in a cell sample, the method comprising contacting a cell sample containing blood cells suspected of containing human M2c macrophages with an antibody as described herein and measuring binding to cells in the sample, a positive signal indicating the presence of human M2c 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 method for detecting M2 cells in a sample comprises using an antibody or fragment that is not internalized by the cell upon binding, but remains bound to the outside of the cell to facilitate detection.

[0444] In some embodiments, the disclosure provides a method for identifying cancer cells expressing huCD163 in a cell sample, the method comprising contacting a cell sample containing cells suspected of containing cancer cells expressing huCD163 with an antibody described herein and measuring the binding to the cells in the sample, a positive signal indicating the presence of cancer cells expressing huCD163 in the sample. In some embodiments, the cell sample includes cells obtained from a human subject. In some embodiments, the cell sample includes cultured cells.

[0445] In some embodiments, the disclosure provides methods for regulating 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.

[0446] In some embodiments, the disclosure provides methods for reducing the inhibition of myeloid cell activation to T cells. In some embodiments, the method induces the increased IL-2 production of T cells. In some embodiments, the disclosure provides methods for increasing the proliferation of Th1 cells. In some embodiments, the disclosure provides methods for increasing the proportion of Th1 cells in proliferating T cells. In each of these aspects, the method includes administering an antibody as described herein in vivo, or contacting the antibody with a composite immune cell system in vitro or ex vivo, wherein macrophages and effector T cells and optional tumor target cells are allowed to interact in a manner that recapitulates or simulates in vivo interactions.

[0447] In some embodiments, the present disclosure provides methods for reducing the inhibition of T cell proliferation by bone marrow cells. In some embodiments, the present disclosure provides methods for increasing CD4 + Cell proliferation or CD8 + T cell proliferation or both.

[0448] In some embodiments, the present disclosure provides methods for increasing the expression of at least one of CD69, ICOS, OX40, PD-1, LAG3, and CTLA4 by T cells in a patient in need thereof. In some embodiments, the method increases the expression of at least one of CD69, ICOS, OX40, PD-1, LAG3, and CTLA4 by CD4+ T cells. In some embodiments, the method increases the expression of at least one of CD8 + Expression of at least one of ICOS, OX40, PD-1, LAG3 and CTLA4 by T cells.

[0449] In some embodiments, the present disclosure provides methods for increasing cancer cell killing. In some embodiments, cytotoxic lymphocyte (CTL) killing of cancer cells is increased. In some embodiments, T cell-mediated killing of MHC mismatched cancer cells is increased.

[0450] In some embodiments, the disclosure provides methods for contacting bone marrow cells with an antibody described herein to reduce macrophage-mediated suppression of CD8 + T cell activation or CD8 + Methods for inhibiting T cell proliferation. In some embodiments, the antibody is contacted with a bone marrow cell comprising an M0 macrophage. In some embodiments, the antibody is contacted with a bone marrow cell comprising an M2 macrophage. In some embodiments, the antibody is contacted with a bone marrow cell comprising an M0 and M2 macrophage.

[0451] In some embodiments, the 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 as described herein. In some embodiments, the lung cancer is an epithelial cancer or adenocarcinoma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0452] When the subject experiences partial or complete remission or reduction of the signs or symptoms of the disease, the effective response of the present disclosure is achieved, and in the case of cancer treatment, particularly includes, but is not limited to, cure, remission, extended survival or other objective responses. In some embodiments, depending on the prognostic factors including the number of recurrences, the stage of the disease and other factors, the expected progression-free survival time is measured in months to years. Extended survival includes but is not limited to at least 1 month (mo.), about at least 2 months, about at least 3 months, about at least 4 months, about at least 6 months, about at least 1 year, about at least 2 years, about at least 3 years, etc. The overall survival is also measured, for example, in months to years. Alternatively, in some embodiments, the effective response is that the subject's symptoms remain static. Further instructions for treatment indications are described in more detail below.

[0453] In some embodiments, the administration of the therapeutic agent is performed in a preventive manner, before the symptoms of an undesirable disease or disorder are manifested, so that the disease or disorder is prevented or, alternatively, its progression is delayed. Thus, when used in conjunction with a preventive approach, the term "therapeutically effective" means that, following treatment, a smaller number of subjects (on average) develop an undesirable disease or disorder or the severity of symptoms progresses.

[0454] In some embodiments, the factors of variation include the amount of active ingredient in the composition, the composition formulation, and the mode of administration, to provide an amount of active ingredient that is effective to achieve the desired therapeutic response for each subject without being overly toxic to the subject. The selected dosage level will depend on various factors, including the activity of the specific compound employed, the route of administration, the time of administration, the rate of excretion or metabolism of the specific compound employed, the duration of the treatment, other drugs, compounds and / or substances used in combination with the specific composition employed, the age, sex, weight, condition, general health, diet, and previous medical history of the subject being treated, and similar factors well known in the medical arts.

[0455] In some embodiments, the antibody and Fab described herein are administered to the subject in different doses and in different time frames. Non-limiting dosage includes about 0.01mg / kg, about 0.05mg / kg, about 0.1mg / kg, about 0.5mg / kg, about 1mg / kg, about 5mg / kg, about 10mg / kg, about 20mg / kg, about 30mg / kg, about 40mg / kg, about 50mg / kg, about 60mg / kg, about 70mg / kg, about 80mg / kg, about 90mg / kg, about 100mg / kg, about 125mg / kg, about 150mg / kg, about 175mg / kg, about 200mg / kg or any integer therebetween. In addition, in some embodiments, the dosage of antibody or Fab is administered twice a week, once a week, once every two weeks, once every three weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks, once every 12 weeks, or any combination of weeks thereof. Also contemplated are dosing cycles such as, for example, once or twice weekly administration of the antibody or antigen-binding fragment thereof for 4 weeks, followed by two weeks of no treatment.Alternative dosing cycles are also contemplated within the present disclosure including, for example, different combinations of the doses and weekly cycles described herein.

[0456] In some embodiments, the therapeutically effective amount of the composition varies and depends on the severity of the disease and the weight and general state of the subject being treated, but generally ranges from about 1.0 μg / kg to about 100 mg / kg body weight, or about 10 μg / kg to about 30 mg / kg, or about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 10 mg / kg per application. Depending on the response to the disorder or condition and the subject's tolerance to treatment, administration may be once a day, once a few days, once a week, twice a month, once a month, or more or less frequently as needed. In some embodiments, it is necessary to maintain the dose over a longer period of time, such as 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, or 12 weeks or longer, until the desired inhibition of the disorder symptoms occurs, and the dose is adjusted as needed. The progress of this therapy is easily monitored by conventional techniques and assays.

[0457] In some embodiments, an antibody of the present disclosure is administered intravenously in a physiological solution at a dose range of between 0.01 mg / kg to 100 mg / kg at a frequency range of once daily to once weekly to once monthly (e.g., once daily, once every two days, once every three days, or 2, 3, 4, 5, or 6 times per week), preferably at a dose range of 0.1 mg / kg to 45 mg / kg, 0.1 mg / kg to 15 mg / kg, or 0.1 mg / kg to 10 mg / kg, at a frequency of 2 or 3 times per week, or up to 45 mg / kg once per month.

[0458] When the subject experiences partial or complete relief or alleviation of the signs or symptoms of the disease and particularly includes but is not limited to the extension of the survival period, response is achieved.Depending on the prognostic factors including the number of relapses, the stage of the disease and other factors, the expected progression-free survival time is, for example, measured in months to years.Extended survival includes but is not limited to at least 1 month (mo), about at least 2 months (mo), about at least 3 months, about at least 4 months, about at least 6 months, about at least 1 year, about at least 2 years, about at least 3 years or longer.In some embodiments, the total survival period is also measured in months to years.In some embodiments, the subject's symptoms remain static or reduce.

[0459] In some cases, a physician or veterinarian having ordinary skills in the art can readily determine and prescribe the effective amount (ED) of the composition required. 50 ). For example, a physician or veterinarian may start the dosage of the compound employed in the composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Alternatively, in some embodiments the dosage remains constant.

[0460] 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 simultaneous administration or administration of one or more compositions within 30 minutes of each other. In some embodiments, administration occurs at the same or different sites.

[0461] In some embodiments, the toxicity and therapeutic efficacy of such components are determined in cell cultures or experimental animals by standard pharmaceutical procedures, e.g., for determining the LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). In some embodiments, the dose ratio between toxic and therapeutic effects is the therapeutic index and it is expressed as LD 50 / ED 50 In some embodiments, while using compounds that exhibit toxic side effects, care should be taken to design delivery systems that target such compounds to the affected tissue site to minimize potential damage to healthy cells, thereby reducing side effects.

[0462] In some embodiments, data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in humans. The dosage of such compounds preferably falls within the range that includes the ED 50 In some embodiments, the dosage varies within this range depending on the dosage form employed and the route of administration utilized. In some embodiments, for any compound used in the methods of the present disclosure, the therapeutically effective dose is initially estimated from cell culture assays. In some embodiments, a dose is formulated in animal models to achieve an IC as determined in cell culture. 50 The circulating plasma concentration range (i.e., the concentration of the test compound that achieves half-maximal inhibition) can be determined. Levels in plasma are measured, for example, by high performance liquid chromatography. Such information is used, in some cases, to more accurately determine useful doses in humans.

[0463] In some embodiments, the present disclosure provides a method for treating a patient suffering from cancer, the method comprising administering to the patient a therapeutically effective amount of an antibody as 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.

[0464] Diagnostic Products and Methods

[0465] In some embodiments, disclosed herein are methods of detecting huCD163 protein or M2 macrophages in a sample or subject to assess the patient's treatment status or diagnose a disease or disorder associated or related to the activity of M2 macrophages or TAMs, such as those described herein.

[0466] In the in vivo detection, diagnosis or monitoring of the soluble huCD163 protein of cell or tissue, the expression of huCD163 protein or the presence of M2 macrophage or activity, the subject is administered an antibody or Fab as described herein, wherein the antibody or Fab is combined with a detectable portion. In some embodiments, the detectable portion is visualized using the following methods recognized in the art: such as, but not limited to, magnetic resonance imaging (MRI), fluorescence, radiography, the light source provided by endoscope, laparoscope or intravascular catheter (i.e., via the detection of photoactive agent), light scanning, positron emission tomography (PET) scanning, whole body nuclear magnetic resonance (NMR), radioscintigraphy, single photon emission computed tomography (SPECT), targeted near infrared region (NIR) scanning, X-rays, ultrasound. It is also known in the art to detect the labeling of compounds using such methods. In some embodiments, the visualization of the detectable portion allows detection, diagnosis and / or monitoring of the condition or disease related to the activity of another cell of M2 macrophage activity or expression of 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.

[0467] For in vitro detection methods, samples to be obtained from a subject include, but are not limited to, blood, tissue biopsy samples, and fluids therefrom.

[0468] Therefore, the present disclosure provides antibodies and antigen-binding fragments thereof, which can be used to detect or diagnose the level of M2 macrophages or TAM macrophages associated with a disease or disorder, potentially indicating the need for therapeutic treatment. In other embodiments, the antibody also includes a second agent. In some embodiments, such an agent is a molecule or part such as, for example, a reporter molecule or a detectable marker. The detectable marker / part for such a detection method is known in the art and is described in more detail below. For example, a reporter molecule is any part detected using an assay. Non-limiting examples of reporter molecules conjugated to polypeptides include enzymes, radioactive labels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin. In some embodiments, detectable markers include compounds and / or elements detected due to their specific functional properties and / or chemical characteristics, and their use allows the polypeptides to which they are attached to be detected, and / or further quantified if necessary. Many suitable detectable (imaging) agents are known in the art, as are methods for attaching to polypeptides.

[0469] In some embodiments, the polypeptides are conjugated to a variety of fluorescent dyes, quenchers, and haptens such as fluorescein, R-phycoerythrin, and biotin. In some embodiments, conjugation occurs during polypeptide synthesis or after polypeptide synthesis and purification.

[0470] Alternatively, in some embodiments, the antibody, antigen binding fragment or binding protein is conjugated to a fluorescent moiety. Conjugation of a polypeptide to a fluorescent moiety (e.g., R-phycoerythrin, fluorescein isothiocyanate (FITC), etc.) is accomplished, for example, using art-recognized techniques. Many commercially available fluorescent dyes and dye conjugation kits for specific applications (such as fluorescence microscopy, flow cytometry, fluorescence activated cell sorting (FACS), etc.) are commercially available.

[0471] In one non-limiting embodiment, the antibody antigen-binding fragment is associated (conjugated) with a detectable label, such as a radionuclide, a dye, an imaging agent, or a fluorescent agent for immunodetection of binding to the antigen, which is used to visualize the binding of the antibody to M2 macrophages or soluble or bound huCD163 protein in vitro and / or in vivo.

[0472] Non-limiting examples of radioactive labels 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 Such as 99 Tc, 99m Tc, 100 Pd, 101 Rh, 103 Pb, 105 Rh, 109 Pd, 111 Ag, 111 In, 113 In, 119 Sb, 121 Sn, 123 I. 125 I. 127 Cs, 128 8. 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 Bihe 213 Bi. In some embodiments, radiolabels are attached to compounds using conventional chemistry known in the field of antibody imaging. Radiolabeled compounds can be used in in vitro diagnostic techniques and in vivo radioimaging techniques and radioimmunotherapy.

[0473] In some embodiments, the compositions of the antibodies and antigen-binding fragments described herein are also used as non-therapeutic agents (eg, as affinity purification agents).

[0474] Antibody Technology

[0475] As will be appreciated by those skilled in the art, the general description herein of antibodies and methods of making and using the same also applies to individual antibody polypeptide components and antibody fragments.

[0476] The antibodies of the present disclosure are polyclonal antibodies or monoclonal antibodies. However, in preferred embodiments, they are monoclonal. In specific embodiments, the antibodies of the present disclosure are human antibodies. Methods for producing polyclonal antibodies and monoclonal antibodies are known in the art.

[0477] In some embodiments, antibodies, antigen-binding fragments, and other proteins generated using such methods that bind to huCD163 expressed by M2 macrophages are tested for one or more of binding affinity, avidity, and modulatory capacity.

[0478] In some embodiments, conventional methods are utilized to identify antibodies or their Fabs that are bound to huCD163 proteins. In some embodiments, one or more of the binding affinity, binding rate, dissociation rate, and avidity of antibodies and Fabs are assessed. The measurement of such parameters, for example, is completed using an assay including but not limited to: enzyme-linked immunosorbent assay (ELISA), ELISpot assay, Scatchard analysis, surface plasmon resonance (e.g., BIACORE) analysis, competitive binding assay, etc. In a non-limiting embodiment, ELISA assays are used to measure the binding capacity of specific antibodies or Fabs that are bound to huCD163 proteins. Surface plasmon resonance technology is described in Liljeblad et al., Glyco J 17:323-9 (2000).

[0479] In some embodiments, antibodies according to the present disclosure are recombinantly produced using vectors and methods available in the art, as described further below. In some embodiments, human antibodies are also produced by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

[0480] In some embodiments, human antibodies are produced in transgenic animals (e.g., mice) that are capable of producing a full human antibody repertoire in the absence of endogenous immunoglobulin production. For example, the antibody heavy chain joining region (JCR) in chimeric and germline mutant mice has been described. H) gene results in complete inhibition of endogenous antibody production. Transferring 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. Pat. Nos. 5,545,806, 5,569,825, 5,591,669; U.S. Pat. No. 5,545,807 and WO 97 / 17852. In some embodiments, such animals are genetically engineered to produce human antibodies comprising polypeptides of the present disclosure.

[0481] For example, antibodies are isolated and purified from culture supernatants or ascites fluid (if the antibodies are produced in animals) using methods known in the art, such as by saturated ammonium sulfate precipitation, euglobulin precipitation method, hexanoic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52), or affinity chromatography using an anti-Ig column or protein A, G or L column.

[0482] As described above, the present disclosure also provides antibody fragments. In some cases, there are advantages to using antibody fragments rather than complete antibodies. For example, in some embodiments, smaller-sized fragments allow for rapid clearance and result in improved 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.

[0483] Various techniques have been developed to produce antibody fragments. Traditionally, these fragments are 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, in some embodiments, these fragments are now directly produced by recombinant host cells. In some embodiments, Fab, Fv and ScFv antibody fragments are expressed in Escherichia coli and secreted from Escherichia coli, thereby allowing a large number of easy production of these fragments. In some embodiments, Fab'-SH fragments are directly recovered from Escherichia coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another method, in some embodiments, F(ab')2 fragments are directly separated from recombinant host cell cultures. The C-terminal fragment comprising the Fc region taken from IgG is preferably a fragment of the Fab'-SH fragment. H Fab and F(ab')2 fragments with increased in vivo half-life that bind to salvage receptor epitopes of both loops of the 2 domain are described in US Pat. Nos. 5,869,046 and 6,121,022. Other techniques for the production of antibody fragments will be apparent to the skilled artisan.

[0484] 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 complete binding sites that do not contain constant regions. Therefore, they are suitable for reducing nonspecific binding during in vivo utilization. In some embodiments, sFv fusion proteins are constructed to produce fusions of effector proteins at the amino or carboxyl termini of sFv. See Antibody Engineering, ed. Borrebaeck, supra. In some embodiments, antibody fragments are also "linear antibodies", for example, as described in, for example, U.S. Patent No. 5,641,870. In some embodiments, such linear antibody fragments are monospecific or bispecific.

[0485] Methods for making 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 chelated recombinant antibodies (Neri et al., J Mol Biol 246:367-73 (1995)).

[0486] Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature 305:537-9 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is accomplished, for example, by affinity chromatography. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J 10:3655-9 (1991).

[0487] According to different approaches, antibody variable regions with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is to an Ig heavy chain constant domain, comprising at least part of the hinge, C H Zone 2 and C H Preferably, the first heavy chain constant region (C H 1) contains the site required for light chain bonding, present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host cell. When the unequal ratios of the four polypeptide chains used in the construction provide the optimal yield of the desired bispecific antibody, this provides greater flexibility in adjusting the mutual ratio of the four polypeptide fragments in the embodiment. However, when the 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, the coding sequences of two or all four polypeptide chains can be inserted into a single expression vector.

[0488] Bispecific antibodies are composed of, for example, a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure helps to separate the desired bispecific compound from the unwanted immunoglobulin chain combination, because there is only an immunoglobulin light chain in one half of the bispecific molecule, providing an easy separation mode. The method is disclosed in WO 94 / 04690. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods Enzymol 121: 210 (1986).

[0489] In some embodiments, the interface between a pair of antibody molecules is engineered to maximize the percentage of heterodimers recovered from recombinant cell culture according to another approach described in U.S. Pat. No. 5,731,168. Preferred interfaces include C H At least a portion of the 3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is generated on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers rather than other unwanted end products such as homodimers.

[0490] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate is coupled to avidin and the other is coupled to biotin. For example, such antibodies have been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and to treat HIV infection (WO 91 / 00360, WO 92 / 20373 and EP03089). In some embodiments, heteroconjugate antibodies are prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 together with some cross-linking techniques. Another method is designed to prepare tetramers by adding a streptavidin coding sequence at the C-terminus of scFv. Streptavidin is composed of four subunits, so when scFv-streptavidin is folded, the four subunits associate to form a tetramer (Kipriyanov et al., Hum Antibodies Hybridomas 6(3):93-101 (1995)).

[0491] In some embodiments, according to another method for making bispecific antibodies, the interface between a pair of antibody molecules is engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. One interface comprises the CH In one embodiment, the present invention relates to a method for preparing a second antibody molecule, wherein the first antibody molecule is provided with a plurality of small amino acid side chains, each of which is a portion of a plurality of domains. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensation "cavity" of the same or similar size as the large side chain is produced on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers rather than other unwanted end products such as homodimers. Referring to WO 96 / 27011.

[0492] The technology for producing bispecific antibodies from antibody fragments is also described in the literature. For example, bispecific antibodies are prepared using chemical connections. 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 dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent the formation of intermolecular disulfides. The Fab' fragments produced are then converted into thionitrobenzoate (TNB) derivatives. Then one of the Fab'-TNB derivatives is converted back into Fab'-thiol by reduction with mercaptoethylamine, and mixed with another Fab'-TNB derivative of equimolar amount to form a bispecific antibody. In some embodiments, the bispecific antibody produced is used as an agent for selective fixation of an enzyme.

[0493] Recent progress has promoted the direct recovery of Fab'-SH fragments from Escherichia coli, which are chemically coupled, for example, to form bispecific antibodies. Shalaby et al., J Exp Med 175:217-25 (1992) described the production of humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment is secreted separately by Escherichia coli and undergoes in vitro directed chemical coupling to form bispecific antibodies. The bispecific antibodies thus formed can bind to cells and normal human T cells that overexpress ErbB2 receptors, and trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0494] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, leucine zippers have been used to produce bispecific antibodies. Kostelny et al., J Immunol 148(5):1547-53(1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced in the hinge region to form monomers, and then reoxidized to form antibody heterodimers. In some embodiments, the method is used to produce antibody heterodimers.

[0495] Antibody identification and preparation

[0496] In some embodiments, the polynucleotide sequence encoding antibody, its variable region or its antigen binding fragment is determined using conventional sequencing technology, and is subcloned into an expression vector for the recombinant production of an antibody. This is accomplished by obtaining mononuclear cells from the blood of a subject (e.g., a cancer patient); B cell clones are produced from mononuclear cells; B cells are induced to become plasma cells that produce antibodies; and the supernatant produced by screening plasma cells is determined to determine whether it contains antibodies. In some embodiments, a similar method is used to complete the identification of other antibodies with the specificity of an antibody of the present disclosure. For example, after identifying the B cell clones that produce antibodies, reverse transcription polymerase chain reaction (RT-PCR) is performed to clone the DNA encoding antibody variable region or its part. These sequences are then subcloned into an expression vector suitable for recombinant production of human antibodies. In some embodiments, binding specificity is confirmed by determining that antibody or recombinant antibodies bind to M2 cells or express the ability of other cells of the human CD163 polypeptides expressed by M2 cells.

[0497] In the specific embodiment of the method described herein, the B cells separated from peripheral blood or lymph nodes are sorted, for example, based on that they are CD19 positive, and in, for example, 96 holes, 384 holes or 1536 holes configuration, for example, as low as each hole single cell specificity plate.Cells are induced to differentiate into cells, such as plasma cells, producing antibodies, and harvest culture supernatants, and use, for example, FMAT or FACS analysis to test the combination with cells expressing target polypeptides on their surfaces.Then positive wells undergo full-well RT-PCR to amplify the heavy chain variable region and light chain variable region of the IgG molecules expressed by cloned progeny plasma cells.The PCR product encoding heavy chain variable region and light chain variable region or its part thereof is subcloned into a human antibody expression vector for recombinant expression.Then the obtained recombinant antibodies are tested to confirm its original binding specificity, and in some embodiments further tested for the cross-reactivity of other cells or proteins.

[0498] Therefore, in one embodiment, the method for identifying antibody is implemented as follows.First, full length or about full length CD163 cDNA is transfected into cell line for expressing CD163 polypeptide.Secondly, the antibody of the polypeptide expressed in combination with cell in test individual human plasma or serum sample.And finally, characterize the combination of the CD163 polypeptide expressed by the MAb and same cell that is derived from plasma or seropositive individual.Now the fine specificity of MAb is further determined in some embodiments.

[0499] In some embodiments, according to methods available in the art and methods described herein, including using primers specific to the conserved regions of human antibody polypeptides to amplify by polymerase chain reaction, separate polynucleotides encoding antibodies or parts thereof of the present disclosure from cells expressing antibodies. For example, according to WO 92 / 02551; U.S. Patent No. 5,627,052; or Babcook et al., Proc Natl Acad Sci USA 93: Molecular biology techniques described in 7843-48 (1996), light chain variable regions and heavy chain variable regions are cloned from B cells. In certain embodiments, all or part of the polynucleotides encoding the heavy chain variable regions and light chain variable regions of the IgG molecules expressed by cloned progeny plasma cells expressing antibodies are subcloned and sequenced. In some embodiments, the sequence of the encoded polypeptide is easily determined according to the polynucleotide sequence.

[0500] Using procedures known in the art and described herein, isolated polynucleotides encoding polypeptides of the disclosure are subcloned into expression vectors for recombinant production of antibodies and polypeptides of the disclosure.

[0501] In some embodiments, the binding properties of antibody (or its fragment) and CD163 polypeptide or M2 cells are generally determined and assessed using immunodetection methods, including, for example, based on immunofluorescence assays, such as immunohistochemistry (IHC) and / or fluorescence activated cell sorting (FACS). In some embodiments, immunoassays include control and program to determine whether antibody is specifically bound to CD163 polypeptide or M2 macrophages, and do not identify control cells, for example, M1 cells, or are transfected with host cells expressing control proteins or cross-react with them.

[0502] After serum pre-screening to identify the patient producing the antibody for CD163 polypeptide or for M2 macrophage or TAM, the method of the present disclosure generally includes separating or purifying B cells from the biological sample previously obtained from a patient or subject. In some embodiments, the patient or subject is currently or previously diagnosed as suffering from or suspected of suffering from cancer or a particular disease of interest, or the patient or subject is considered to have no cancer or disease. Usually, the patient or subject is a mammal, and in a specific embodiment is the mankind. In some embodiments, the biological sample is any sample comprising B cells, including but not limited to, lymph node or lymph node tissue, pleural effusion, peripheral blood, ascites, tumor tissue or cerebrospinal fluid (CSF). In some embodiments, B cells are separated from different types of biological samples, such as tumor biopsy or other biological samples affected by a particular disease. However, in some embodiments, it should be understood that any biological sample comprising B cells is used for any embodiment of the present disclosure.

[0503] After separation, B cells are induced to produce antibodies, for example, by cultivating B cells under the conditions of supporting B cell proliferation or developing into plasma cells (plasmacyte), plasmablasts or plasma cells (plasma cell). Antibodies are then screened, usually using high throughput techniques, to identify antibodies that are specifically bound to target antigens such as specific tissues, cells or polypeptides. In certain embodiments, specific antigens, for example, the cell surface polypeptides bound by antibodies are unknown, and in other embodiments, the antigens bound by antibody specificity are known.

[0504] In some embodiments, according to the present disclosure, B cells are separated from biological samples by any means known in the art and available, for example, tumor, tissue, peripheral blood or lymph node samples. B cells are usually sorted by FACS based on the presence of B cell-specific markers (e.g., CD19, CD138 and / or surface IgG) on its cell surface. However, in some embodiments, other methods known in the art are adopted, such as, for example, column purification is carried out using CD19 magnetic beads or IgG-specific magnetic beads, and then eluted from the column. However, in some embodiments, magnetic separation of B cells using any marker causes the loss of some B cells. Therefore, in certain embodiments, the cells separated are not sorted, but the mononuclear cells purified by Ficoll from tumor separation are directly plated to the specificity of the appropriate or desired number of each well.

[0505] In order to identify the B cells that produce antibodies, B cells are usually plated 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, less than 10 cells per well, or less than 100 cells per well) in multiwell plates or microtiter plates, for example, in 96 wells, 384 wells or 1536 well configurations. In some embodiments, when B cells are initially plated at a density of more than one cell per well, the method of the present disclosure includes subsequently diluting the cells in the wells identified as producing antigen-specific antibodies until the step of reaching single cell specificity per well is reached, thereby helping to identify the B cells that produce antigen-specific antibodies. In some embodiments, the cell supernatant or a portion thereof and / or the cells are frozen and stored for future testing and subsequent recovery of antibody polynucleotides.

[0506] In certain embodiments, B cells are cultured under conditions that are conducive to B cell production of antibodies. For example, B cells are cultured under conditions that are conducive to B cell proliferation and differentiation to obtain plasmablasts, plasma cells or plasma cells that produce antibodies. In a specific embodiment, B cells are cultured in the presence of B cell mitogens such as lipopolysaccharide (LPS) or CD40 ligands. In a specific embodiment, by culturing B cells with feeder cells and / or other B cell activators such as CD40 ligands, B cells are differentiated into cells that produce antibodies.

[0507] In some embodiments, the ability of the cell culture supernatant or the antibody obtained therefrom to bind to the target antigen is tested using conventional methods available in the art, including those described herein. In a specific embodiment, the presence of antibodies that bind to the target antigen in the culture supernatant is tested using a high-throughput method. For example, B cells are cultured in a multi-porous microtiter dish so that a robotic plate processor is used to sample more than one cell supernatant at the same time and test the presence of antibodies that bind to the target antigen. In a specific embodiment, the antigen is combined with a bead (e.g., paramagnetic beads or latex beads) to facilitate the 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, FMAT is used. TM Antibody binding was determined using the ANALYZER(R) assay and instrumentation (Applied Biosystems, Foster City, Calif.). FMAT is a fluorescence macro-confocal platform for high throughput screening that enables mix-and-read, non-radioactive assays using living cells or beads.

[0508] When comparing the binding of an antibody to a particular target antigen (e.g., a biological sample such as a cancer tissue or cell or an infectious agent) and the binding of the antibody to a control sample (e.g., a biological sample such as normal cells, comparison cells from another species, a different cancer tissue or cell, or a different infectious agent), in some embodiments, an antibody is considered to bind preferentially to a particular target antigen if the antibody binds to the particular target antigen at least two-fold, at least three-fold, at least five-fold, or at least ten-fold more than the amount bound to the control sample.

[0509] In some embodiments, the polynucleotides encoding antibody chains, their variable regions or their fragments are separated from cells using any means available in the art. In one embodiment, oligonucleotide primers are used to separate polynucleotides using polymerase chain reaction (PCR) such as reverse transcription PCR (RT-PCR), and the oligonucleotide primers are specifically bound to heavy chain or light chain encoding polynucleotide sequences or their complementary sequences using conventional procedures available in the art. In one embodiment, positive wells undergo full-well RT-PCR to amplify the heavy chain variable region and light chain variable region of the IgG molecules expressed by cloned progeny plasma cells. In some embodiments, according to conventional procedures in the art, these PCR products are sequenced, and then the products encoding heavy chain variable region and light chain variable region or their parts are subcloned into human antibody expression vectors, and recombinantly expressed (see, e.g., U.S. Patent No. 7,112,439). In some embodiments, nucleic acid molecules encoding M2 macrophage-specific antibodies or their fragments as described herein are propagated and expressed according to any of the various well-known procedures for nucleic acid excision, connection, conversion and transfection. Therefore, in certain embodiments, it is preferred to express antibody fragments in prokaryotic host cells such as Escherichia coli (see, e.g., Pluckthun et al., Methods Enzymol 178: 497-515 (1989)). In certain other embodiments, the expression of antibodies or antigen-binding fragments thereof is preferably 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 cells, corn cells, soybean cells and rice cells. In some embodiments, nucleic acid vectors for expressing exogenous sequences in specific host systems are designed by methods known to those of ordinary skill in the art and based on the present disclosure, and then polynucleotide sequences encoding tumor-specific antibodies (or fragments thereof) are inserted. Regulatory elements will vary according to the specific host.

[0510] In some embodiments, one or more replicable expression vectors containing polynucleotides encoding variable and / or constant regions are prepared and used to transform a suitable cell line, e.g., a non-producing myeloma cell line such as the mouse NSO line, or bacteria such as E. coli, where antibody production will occur. In order to obtain efficient transcription and translation, the polynucleotide sequence in each vector should contain appropriate regulatory sequences, particularly a promoter and leader sequence operably linked to the variable region sequence.

[0511] Specific methods for producing antibodies in this way are generally well known and routinely used. For example, molecular biology procedures described below: Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, New York, 1989; See also Sambrook et al., Third Edition, Cold Spring Harbor Laboratory, New York, (2001). Although not required, in certain embodiments, the polynucleotide region encoding the recombinant antibody is sequenced. DNA sequencing is performed, for example, in any manner known in the art 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 Amersham International plc sequencing manual (and including improvements thereto).

[0512] In certain embodiments, the resulting recombinant antibody or fragment thereof is then tested to confirm its original specificity and, in some embodiments, further tested for cross-reactivity, such as cross-reactivity with related polypeptides. In certain embodiments, antibodies identified or generated according to the methods described herein are tested for internalization ability or other effector functions using conventional methods.

[0513] Packaging, Kits and Prefilled Containers

[0514] Also provided herein are kits comprising one or more of the compounds described above. In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof as described herein in suitable container means.

[0515] In some embodiments, a container device comprising a composition described herein is provided. In some embodiments, the container device is any suitable container for holding, for example, a liquid or a 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 any volume of liquid suitable for injection into a subject, including but not limited to 0.5cc, 1cc, 2cc, 5cc, 10cc or more.

[0516] Provided herein are kits comprising one or more compositions described herein. In some embodiments, provided herein are kits for treating a subject having cancer, the kit comprising an antibody as described herein and an anti-cancer therapy.

[0517] In some embodiments, provided herein are kits for treating cancer, the kits comprising an antibody as described herein and a label attached to or packaged with the container, the label describing the use of the antibody in combination with an anti-cancer therapy.

[0518] In some embodiments, provided herein are kits for treating cancer, the kits comprising an anti-cancer therapy and a label attached to or packaged with the container, the label describing the use of the anti-cancer therapy with an antibody as described herein.

[0519] In some embodiments, the container means of the kit will generally include at least one vial, test tube, flask, bottle, ampoule, syringe, intravenous (IV) bag, and / or other container means, in which at least one polypeptide is placed and / or preferably suitably aliquoted. Provided herein are container means comprising the compositions described herein.

[0520] In some embodiments, the kit comprises a tightly closed device for containing at least one fusion protein, a detectable moiety, a reporter molecule, and / or other reagent containers for commercial sale. In some embodiments, such containers include injection and / or blow molded plastic containers in which the desired vials are stored. In some embodiments, the kit also includes printed materials for use of the materials in the kit.

[0521] In some embodiments, packaging and kits additionally comprise buffers, preservatives and / or stabilizers in pharmaceutical preparations. In some embodiments, each component of the kit is packaged in a separate container and all of the different containers can be in a single package. In some embodiments, the kits of the disclosure are designed to be used for refrigeration or room temperature storage.

[0522] In addition, in some embodiments, the formulation contains a stabilizer to increase the shelf life of the kit and includes, for example, bovine serum albumin (BSA). In some embodiments, where the composition is lyophilized, the kit contains an additional solution formulation to reconstitute the lyophilized formulation. Acceptable reconstitution solutions are well known in the art and include, for example, pharmaceutically acceptable phosphate buffered saline (PBS).

[0523] In some embodiments, packaging and kits also include one or more components for measuring, such as, for example, ELISA assays. Samples to be tested in the present application include, for example, blood, plasma, tissue sections and secretions, urine, lymph and products thereof. In some embodiments, packaging and kits also include one or more components (e.g., syringes, cups, swabs, etc.) for collecting samples.

[0524] In some embodiments, the packages and kits further comprise a label specifying information required by the US FDA or similar regulatory agency, for example, a description of the product, amount and mode of administration, and / or treatment instructions.The packages provided herein can contain any composition as described herein.

[0525] The term "packaging material" refers to a physical structure that holds the components of a test kit. In some embodiments, the packaging material maintains the components sterile and is made of materials (e.g., paper, corrugated fiber, glass, plastic, foil, ampoule, etc.) that are commonly used for such purposes. In some embodiments, the label or package insert comprises appropriate written instructions. Therefore, in some embodiments, the test kit additionally comprises labels or instructions for using the components of the test kit with any method of the present disclosure. In some embodiments, the test kit comprises a compound in a package or dispenser and instructions for applying the compound with the methods described herein.

[0526] In still further embodiments, the kit further comprises container means for an anti-cancer therapy.

[0527] In some embodiments, the instructions include instructions for implementing any of the methods described herein, including methods of treatment. In some embodiments, the instructions additionally include satisfactory clinical endpoints or indications of any adverse symptoms that occur, or additional information required by regulatory agencies, such as the Food and Drug Administration, for human subjects.

[0528] In some embodiments, the instructions are on "printed matter," e.g., on paper or cardboard within or affixed to the kit, or on a label affixed to the kit or packaging material, or attached to vials or tubes containing components of the kit. In some embodiments, the instructions are additionally contained on a computer-readable medium, such as, for example, a CD-ROM, DVD, flash memory device, solid-state memory, magnetic disk and magnetic disk device, magnetic tape, cloud computing system and server, etc. In some cases, the program and instructions are permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the medium.

[0529] Provided herein are container devices comprising compositions described herein. In some embodiments, the container device is any suitable container for holding 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 any volume of liquid suitable for injection into a subject, including but not limited to 0.5cc, 1cc, 2cc, 5cc, 10cc or more.

[0530] Provided herein are kits comprising the compositions described herein. In some embodiments, provided herein are kits for treating cancer, the kits comprising a combination of an antibody as described herein and an anti-cancer therapeutic.

[0531] In some embodiments, provided herein are kits for treating cancer, the kits comprising an antibody as described herein and a label attached to or packaged with the container, the label describing the use of the antibody or antigen-binding fragment thereof with an anti-cancer therapy.

[0532] In some embodiments, provided herein are kits for treating cancer, the kits comprising an anti-cancer therapy and a label attached to or packaged with the container, the label describing the use of the anti-cancer therapy with an antibody as described herein. Example

[0533] The present disclosure will be further illustrated in the following examples, which are provided for illustrative purposes only and are not intended to limit the present disclosure in any way.

[0534] Example 1 - Identification and cloning

[0535] Antibodies that specifically bind to human myeloid-derived suppressor cells (MDSCs) produced by patients in response to checkpoint inhibitor anti-PD-1 therapy were isolated and cloned. The immunomodulatory properties of these monoclonal antibodies were further investigated with the goal of identifying antibodies with therapeutic potential to target and reverse the immunosuppressive effects of MDSCs, thereby enhancing tumor clearance.

[0536] Identify and select cancer patients who have achieved partial or complete response to immune checkpoint inhibitors for at least 6 months, and perform memory B cell library analysis via the I-STAR platform. The platform uses a short-term B cell culture system to study the memory B cell library. Based on CD19 and IgG surface expression, more than 15,000 memory B cells were isolated from 10 million peripheral blood mononuclear cells (PBMCs) from each donor patient. Then, these memory B cells were inoculated into 40 384-well microtiter plates at about 1 cell / well under conditions that promote B cell activation, proliferation, terminal differentiation, and antibody secretion. The plating density of 1 cell / well allows the expansion of single B cell clones, so that real antibody heavy and light chain pairs can be reconstructed from each culture well. Using high-throughput and miniaturized multiplex flow cytometry assays, secreted IgG antibodies in each well bound to MDSC were screened. 49 positive B cell clones were identified. A selected subset of antibodies sorted according to the MDSC binding spectrum and antibody variable region sequence was sequenced, cloned, and expressed as recombinant IgG1 for further in vitro characterization.

[0537] The heavy chain variable region (VH) and light chain variable region (VL) of the immunoglobulin gene from the B cell clone that produces MDSC-specific antibodies are amplified by RT-PCR amplification using a family-specific primer set. The pool of VH or VL region clones from the positive family-specific PCR reaction is cloned upstream of the human IgG1 constant domain sequence in the expression vector, thereby producing functional antibodies with the same binding characteristics as the antibodies produced by the B cell clone. Design DNA plasmids and require GenScript, NJ, USA to perform gene synthesis of the constant region. These plasmids combine all possible heavy chain and light chain family-specific pairs and are used for transient transfection of HEK293 cells. All transfectant supernatants containing secreted recombinant antibodies are screened in a flow-based MDSC binding assay. For wells containing more than one B cell clone per well, more than one VH and VL domain sequences are amplified and expressed as previously described. MDSC screening is then used to identify the heavy chain and light chain combination library that recapitulates the binding activity observed for antibodies found in mixed cultures. The DNA sequences of the VH and light VL variable regions of all binding mAbs were confirmed by multiple sequencing reactions using DNA purified from maxipreps (plasmids from GenScript origin and amplified by Macherey Nagel).

[0538] One B cell clone (with a germline ID of heavy chain VH3.30-3 / IGHG1 and light chain VK1.012) was identified from the MDSC screen and named AB101, which contains a light chain containing SEQ ID NO:9 and a heavy chain containing 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 chemotherapy progressive disease and then had a complete remission after anti-PD-1 treatment and was still receiving treatment at the time of the blood draw. Sequencing confirmed that the B cell clone well had only one heavy chain and one light chain. As observed for secreted IgG antibodies from a single B cell clone, the recapitulated antibody also had significant bimodal binding to MDSC, indicating that the antibody target is highly expressed on a selected subset of MDSC. See Figure 1 Results from recapitulation screening of two MDSC donors with relaxed blocking (10 μg / mL of recombinant Fc blocker (Abcam)) and strict blocking (10 μg / mL of recombinant Fc blocker from Abcam + 1 μg / mL of anti-CD16, anti-CD32, and anti-CD64 antibodies) conditions showed dose-dependent saturable binding of AB101 to human MDSCs, with an IC50 of approximately 10 nM under relaxed blocking conditions. Under strict blocking conditions, overall binding of AB101 was reduced, as indicated by a reduction in MFI.

[0539] CD163 is a marker for cells from the monocyte / macrophage lineage. The expression of CD163 on MDSC differentiated in vitro is reported to be bimodal. It is assumed that the bimodal binding of AB101 may be related to CD163 expression. In order to test this hypothesis, in vitro MDSC (see Example 11) was produced and co-stained with anti-CD163 antibody (BioLegend333611) and AB101 conjugated with AF647 as described in Example 8, and combined by FACS analysis. First, the cells were gated as CD163 high or low, and then the binding with different concentrations of AB101 or human IgG1 isotype controls was checked. The cell subset bound by the AB101 antibody is CD163 高 Cell. See Figure 2 .

[0540] Example 2 - Isolation of autologous monocytes and T cells

[0541] This example illustrates the separation of autologous monocytes and T cells. Human monocytes and T cells were obtained using techniques commonly used in the art. During plateletpheresis, human monocytes and T cells were separated from white blood cells (WBC) captured in an integrated chamber (called a LeukoReduction system (LRS) chamber). The platelets were centrifuged using standard density gradient centrifugation (Ficoll). TM Peripheral blood mononuclear cells (PBMCs) were purified from LRS samples using Paque Premium 1.073, GE Healthcare No. 17-5449-52. The supernatant was discarded and the pellet was resuspended in 20 mL EasySep TM Buffer (StemCell Technologies No.20144) for PBMC counting and further separation of monocytes and T cells.

[0542] Monocytes were isolated using the EasySep Human Monocyte Isolation Kit (Stem Cell No. 19359) according to the manufacturer's instructions.

[0543] Human CD3 + T cell isolation kit (StemCell 19051), EasySep TM Human CD4 + T cell isolation kit (StemCell No.17952), EasySep TM Human CD8 +T cell isolation kit (StemCell No. 17953) was used to isolate total CD3 T cells, CD4 T cells, or CD8 T cells according to the manufacturer's instructions. These negative selection kits use antibodies to label unwanted cell types for removal, allowing the desired target cells to be isolated from the sample.

[0544] Example 3 - Specific binding of AB101 to immunosuppressive myeloid cells

[0545] To evaluate specificity, the combination of the antibody of the present invention (antibody AB101) conjugated with the far-red fluorescent dye AF647 was tested on different cell types (including MDSC, immunosuppressive M2c and pro-inflammatory M1 macrophages produced as described in Example 11 and below). In addition, the antibody binding of the individual immune population of PBMC from healthy donors was evaluated. For each of these studies, at least 3 individual donors were used. PBMCs were separated from blood using a Ficoll gradient using standard procedures. To differentiate immune cell populations, PBMCs were then stained for hematopoietic lineage markers (CD45-BV421 (BD642275), CD3-BV510 (BD 563109), CD11c-PE-Cy7 (BD561356), CD14-FITC (BD 347493), CD20-APC-Cy7 (BD 562643), CD56-PE (BD 347747), and CD66c (BD 551478)). Individual lineage populations were further characterized by the following expression patterns and evaluated for binding to AB101: T cells CD45 + CD3 + , B cell CD45 + CD20 + , monocyte CD45 + CD14 + , NK cell CD45 + SSC 低 CD14 - CD3 - CD56 + , granulocyte CD45 + SSC 高 CD14 - CD66 + and dendritic cell CD45 + CD14 - CD66 - CD11c +Antibodies were also evaluated for binding to primary human non-immune cells (Lonza) including: small airway epithelial cells (SAEC, #CC-2547), renal proximal tubule epithelial cells (RPTEC, #CC-2553), pulmonary microvascular endothelial cells (HMVEC, #CC-2527), umbilical vein endothelial cells (HUVEC, #C2519A), aortic smooth muscle cells (AOSMC, #CC2571), and keratinocytes (#00192627). These cells were cultured in cell type specific media and conditions according to the manufacturer's instructions until 60%-70% confluence and then harvested to test antibody binding by flow cytometry ( Figure 4 and Figure 5 ).

[0546] In vitro mononuclear MDSCs were generated from isolated monocytes by standard methods: on day 0, monocytes were cultured at 1.5 × 10 5 Pieces / cm 2 Plated in RPMI 1640 (Hyclone SH30027.02, serum-free), incubated at 5% CO2 and 37°C for 1 hour, then washed with pre-warmed RPMI, and then MDSC medium (RMPI + 10% FBS (Hyclone SH30070.03) + 50ng / mL GM-CSF (R&D Systems 215-GM-010) + 50ng / mL IL-6 (R&D Systems 206-IL-010), 20mL per T75 bottle) was added to the cells. The cells were then cultured at 5% CO2 and 37°C for 7 days without changing the medium. After 7 days, cells were harvested by washing twice with PBS (Hyclone SH30028.03) + 2mM EDTA, then adding cold macrophage dissociation solution (PromoCell C-41330) to each T75 bottle at 15mL, and then incubating at 2-8°C for 40min. Cells were removed by tapping the bottle with the palm of the hand, collected and diluted 1:1 with PBS + 2mM EDTA. Cells were pelleted in conical tubes by centrifugation at 450×g for 15min, washed once with PBS + 2mM EDTA, counted and precipitated at 1×10 7Cells were resuspended in FACS blocking buffer (PBS + 1% FBS + 0.1 μg / mL Fc blocker (Abcam Ab90285) for relaxed staining conditions, or PBS + 1% FBS + Fc blocker + 0.01 μg / mL CDR blocker (antibodies against FcR CD16, CD32, and CD64; BD Biosciences 556617, 557333, and 555525, respectively) for stringent staining conditions). Cells were incubated in FACS blocking buffer for 20 minutes (min) at room temperature (RT) and then incubated at 4°C for 30 min. Cells were then diluted to 1×10 with FACS buffer + 5% BSA (Sigma A3059). 6 cells / mL, and 40 μL of cells (4×10 4 Cells were aliquoted into wells for staining. Primary antibodies (20 μg / mL, 6 μg / mL, 2 μg / mL, 0.75 μg / mL, 0.25 μg / mL, 0.08 μg / mL and 0.02 μg / mL of AB101 or 6 μg / mL of human IgG1 isotype control) were added to cells and incubated at room temperature for 90 min. Cells were washed 3 times with 250 μL / well of FACS buffer + 5% BSA. APC goat anti-human IgG secondary antibody (Jackson IR 109-136-097) was prepared at 1:250 in FACS buffer + BSA + e780 viability dye (diluted at 1:1000) and added to cells (50 μL per well). After incubation at 4°C for 45 minutes, cells were washed 3 times in 250 μL FACS buffer. The cells were then fixed in 100 μL / well of 4% PFA for 10-15 min at room temperature, washed once with 250 μL FACS buffer, pelleted at 650×g for 5 min, and then resuspended in 100 μL FACS buffer for flow cytometric analysis.

[0547] AB101 antibody binds to human immunosuppressive myeloid cells (M2c macrophages and monocytes MDSC), such as Figure 3 As shown, the MFI of AB101 or isotype staining on M2c, M1, and M0 is plotted. The AB101 antibody does not bind to B cells, T cells, NK cells, and granulocytes, as shown in FIG. Figure 4 As shown, it shows that AB101 stains T cells, B cells and NKT cells, neutrophils, monocytes and dendritic cells (black curve) compared to isotype control (grey curve), and AB101 antibody does not bind to non-immune cells such as SAEC, RPTEC, HMVEC, HUVEC, AOSMC and keratinocytes, as shown Figure 5Thus, AB101 antibody specifically binds to immunosuppressive myeloid cells expressing CD163 without affecting other immune cells or non-immune cells.

[0548] Example 4-AB101 Fc invalid antibody immunoprecipitation CD163 polypeptide

[0549] This example shows the immunoprecipitation of CD163 using an Fc-invalid antibody comprising an AB101 variable domain in an IgG1 sequence that is modified to substantially reduce the binding of the antibody to an Fc receptor (Fc-invalid), the Fc-invalid antibody being named AB102, comprising a light chain comprising SEQ ID NO:9 and a heavy chain comprising SEQ ID NO:11. Immunoprecipitation (IP) was performed based on Klockenbusch and Kast, J Biomed Biotechnol article ID 927585 (2010). Antibodies were added prior to paraformaldehyde (PFA) fixation in a more classical cross-linked IP method, or prior to cross-linking with bis(sulfosuccinimidyl) suberate (BS3), followed by IP.

[0550] For IP involving cross-linking using the PFA method, monocytes were isolated from human blood and then polarized to M2 cells using the protocols of Examples 2 and 11. M2 macrophages were dissociated from the plate after incubation with macrophage dissociation solution (macrophage dissociation solution DXF; PromoCell, No. C-41330) at 37°C for 10 min, during which the cells became rounded up and began to dissociate. The bottle was tapped vigorously to promote cell dissociation. After dissociation, the macrophage dissociation solution was quenched by adding FACS buffer to the cells. The cells were precipitated at 300 × g for 10 min, 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 min.

[0551] The cells were divided into 6 aliquots, with 15 × 10 6Cells (5 mL per aliquot), and biotinylated antibodies are added to each aliquot. Two aliquots each receive 50 μg of mouse IgG1 anti-hCD163 (R&D, No.MAB1607), two aliquots each receive 100 μg of isotype control antibody ISO2 (with Fc invalid framework), and two aliquots each receive 100 μg of test antibody AB102 (including AB101 variable domains in the IgG1 sequence, and the IgG1 sequence is modified to significantly reduce the binding of antibodies to Fc receptors (Fc invalid)). Cells are incubated at 4 ° C for 1 hour (hr), and occasionally by gently mixing by inverting the tube. Cells are precipitated 5 min at 300 × g, and washed 3 times with PBS-EDTA, and then resuspended in 5 mL PBS (without magnesium or calcium; HyClone, No.SH30028.02). Paraformaldehyde (5mL 0.8% PFA) is added to each tube to obtain a final PFA concentration of 0.4%. Cells are incubated in PFA for 5min at room temperature with gentle shaking. Cells are precipitated by centrifugation at 800 × g for 5min, and the supernatant is removed. Cells are resuspended in 10mL of ice-cold PBS containing 1.25M glycine to quench. Cells are precipitated at 800 × g for 5min, and resuspended in ice-cold PBS. Cells are precipitated at 800 × g for 5min, and resuspended in 1.0mL of RIPA buffer containing 1 × protease inhibitors (ThermoFisherScientific, No.89900). Cells are incubated on ice for 2hr, and then passed through a 2mL Dounce homogenizer 15 times.

[0552] The cell lysate was centrifuged in a bucket centrifuge to precipitate the nucleus, and the supernatant was used for IP. Protein lysate (50 μL) was reserved as the input fraction, and 2.0 mL of cold PBS containing 1 × protease inhibitors was added to the remaining supernatant. Dynabeads MyOne Streptavidin (250 μL) (ThermoFisher Scientific, No.65601) was added to each sample, and they were rotated overnight at 4 ° C. The beads were collected with a StemCell magnet the next day and the supernatant was removed. The beads were washed sequentially for 5 minutes at 4 ° C with 5 mL Paro buffer I, 5 mL Paro buffer II, and 5 mL Paro buffer III (Oncotarget.2017; 8 (7): 11105-11113). The beads were then washed 3 times with cold PBS, and finally resuspended in 100 μL PBS and frozen at -80 ° C.

[0553] The beads were analyzed by mass spectrometry. The analysis was generally performed according to the method described by Yan et al., Mol Cell Proteomics 10(3):M110.005611(2011). In this method, 6M guanidine, 150mM Tris buffer (pH8.3) were continuously stirred for 3hr at 60°C for reversal of formaldehyde cross-linking and elution of proteins from streptavidin beads. The supernatant was denatured, reduced and alkylated at 95°C in the same buffer containing 10mM tris (2-carboxyethyl)-phosphine (TCEP) and 50mM chloroacetamide (CAA) for 10min. The samples were then diluted 10× and digested with 1.3μg trypsin at 37°C overnight. The peptides were cleaned by C18 cartridges. One repeat of AB102 IP and one repeat of anti-CD163 IP were eluted from the C18 cartridge for direct MS / MS analysis. On C18 column, one repeat of AB102 IP and one repeat of anti-CD163 were incubated with 0.1M formaldehyde-d2, 0.4M sodium cyanoborohydride in PBS buffer pH 7.5 for 1 hour to mark heavy dimethylation (d4), and two repeats of ISO2 (Fc invalid) IP were incubated with 0.1M formaldehyde, 0.4M sodium cyanoborohydride in PBS buffer pH 7.5 for 1 hour to mark light dimethylation (d0). Then the C18 column was washed with 0.1% trifluoroacetic acid (TFA) and eluted with 80% acetonitrile (ACN). The d0 / d4 dimethylated peptides were resuspended in buffer A (20% ACN, 0.1% TFA) and then mixed. The peptides were fractionated using a microcapillary HPLC strong cation exchange column (SCX) (200mm×20cm; Proteomix SCX 3μm, Sepax Technologies) prepared in the institution. The peptides were loaded onto a microcapillary column equilibrated with buffer A and washed with buffer A. The bound peptides were eluted with 20 μL of buffer A containing 30%, 50% buffer B (800 mM ammonium formate,...

Claims

1. An antibody comprising a heavy chain variable region (V H ) and a light chain variable region (V L ... L ), wherein the V H comprising a complementary determining region (CDR) H1 of the amino acid sequence SEQ ID NO: 4, a CDRH2 of the amino acid sequence SEQ ID NO: 5, and a CDR H3 of the amino acid sequence SEQ ID NO: 6; and said V L The invention relates to an antibody comprising a CDR L1 having an amino acid sequence of SEQ ID NO: 1, a CDR L2 having an amino acid sequence of SEQ ID NO: 2, and a CDR L3 having an amino acid sequence of SEQ ID NO: 3, wherein the antibody specifically binds to human CD163 protein.

2. The antibody according to claim 1, wherein the V L It has at least 95% identity with the amino acid sequence of SEQ ID NO:

7.

3. The antibody according to claim 1, wherein the V L It has at least 99% identity with the amino acid sequence of SEQ ID NO:

7.

4. The antibody according to claim 1, wherein the V L Identical to the amino acid sequence SEQ ID NO:

7.

5. The antibody according to claim 1, wherein the V H It has at least 95% identity with the amino acid sequence of SEQ ID NO:

8.

6. The antibody according to claim 1, wherein the V H It has at least 99% identity with the amino acid sequence of SEQ ID NO:

8.

7. The antibody according to claim 1, wherein the V H Identical to the amino acid sequence of SEQ ID NO:

8.

8. The antibody according to claim 1, wherein the V L has at least 95% identity to the amino acid sequence of SEQ ID NO: 7 and wherein V H It has at least 95% identity with the amino acid sequence of SEQ ID NO:

8.

9. The antibody according to claim 1, wherein the V L The amino acid sequence is at least 99% identical to SEQ ID NO: 7 and the V H It has at least 99% identity with the amino acid sequence of SEQ ID NO:

8.

10. The antibody of claim 1, further comprising a human heavy chain constant region or a human light chain constant region. The antibody of claim 10 , wherein the human heavy chain constant region is IgG1 or IgG4 or a fragment thereof.

12. The antibody of claim 1, wherein the heavy chain is at least 80% identical to the amino acid sequence of SEQ ID NO: 10, and the light chain is at least 80% identical to the amino acid sequence of SEQ ID NO:

9.

13. The antibody of claim 1, comprising human variable framework regions and murine constant regions.

14. The antibody according to any one of claims 1 to 13, wherein the antibody is a recombinant antibody.

15. An antibody comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the antibody specifically binds to human CD163 protein.

16. an antibody, the antibody comprising a heavy chain sequence, the heavy chain sequence comprising a complementary determining region (CDR) H1 of an amino acid sequence SEQ ID NO: 4, a CDR H2 of an amino acid sequence SEQ ID NO: 5, and a CDR H3 of an amino acid sequence SEQ ID NO: 6; and a light chain sequence, the light chain sequence comprising a CDR L1 of an amino acid sequence SEQ ID NO: 1, a CDR L2 of an amino acid sequence SEQ ID NO: 2, and a CDR L3 of an amino acid sequence SEQ ID NO: 3, wherein the antibody specifically binds to human CD163 protein.

17. The antibody of claim 16, further comprising a human heavy chain constant region or a human light chain constant region.

18. The antibody of claim 17, wherein the human heavy chain constant region is IgG1 or IgG4 or a fragment thereof.

19. The antibody of claim 16, wherein the heavy chain is at least 80% identical to the amino acid sequence of SEQ ID NO: 10, and the light chain is at least 80% identical to the amino acid sequence of SEQ ID NO:

9.

20. The antibody of claim 16, comprising human variable framework regions and murine constant regions.

21. The antibody according to any one of claims 15-20, wherein the antibody is a recombinant antibody.

22. A pharmaceutical composition comprising the antibody according to any one of claims 1-21 and a pharmaceutically acceptable excipient.

23. Use of an antibody according to any one of claims 1-21 for the preparation of a medicament for treating cancer in a human subject, wherein the cancer is characterized by the presence of CD163 on tumor-associated macrophages in the human subject.

24. The use according to claim 23, wherein the cancer is lung cancer.

25. The use according to claim 24, wherein the lung cancer is lung epithelial cancer or lung adenocarcinoma.

26. Use of an antibody according to any one of claims 1-21 for the preparation of a medicament for treating lung cancer in a human subject.

27. The use according to claim 26, wherein the lung cancer is lung epithelial cancer or lung adenocarcinoma.

28. The use according to any one of claims 26-27, wherein when the medicament is used, an additional anti-cancer therapy is administered to the human subject.

29. The use according to claim 28, wherein the additional anti-cancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy and combinations thereof.

30. The use according to claim 29, wherein the additional anti-cancer therapy is immunotherapy.

31. The use of claim 30, wherein the immunotherapy is a composition comprising a checkpoint inhibitor.

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