Immunomodulatory antibodies and their methods of use

Abstract

Provided herein are antibodies and methods for using the same. The antibodies disclosed herein bind to CD163 on cells such as macrophages. + These antibodies can be used in methods of treatment, such as methods of treating cancer.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority and benefits under U.S. Provisional Application No. 62 / 876,580, filed July 19, 2019, U.S. Provisional Application No. 62 / 876,579, filed July 19, 2019, and U.S. Provisional Application No. 62 / 878,265, filed July 24, 2019, all of which are incorporated herein by reference. [Overview of the Initiative]

[0002] This specification provides antibodies useful for the treatment of cancer and other disorders, which comprise antigen-binding fragments and other antigen-binding polypeptides. In some embodiments, the antibodies specifically bind to M2 and M2-like immunosuppressive macrophages but not to M1 and M1-like antitumor macrophages. The disclosed antibodies bind to M2 and M2-like tumor-associated macrophages and modulate the physical and functional characteristics of tumor-associated M2 and M2-like macrophages to alleviate immunosuppression in the tumor microenvironment and increase the activation and proliferation of cytotoxic T cells, thereby promoting tumor cell death. The antibody molecules of this disclosure specifically bind to human CD163 expressed on the surface of M2 and M2-like macrophages.

[0003] In certain embodiments of this specification, antibodies or recombinant antibodies comprising a heavy chain variable region (VH) having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 are disclosed.

[0004] In certain embodiments of this specification, antibodies or recombinant antibodies comprising a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 8 are disclosed.

[0005] In certain embodiments of this specification, antibodies or recombinant antibodies comprising a heavy chain variable region (VH) having at least 95% identity with the amino acid sequence of SEQ ID NO: 8 are disclosed.

[0006] In certain embodiments of this specification, antibodies or recombinant antibodies comprising a heavy chain variable region (VH) having at least 99% identity with the amino acid sequence of SEQ ID NO: 8 are disclosed.

[0007] In certain embodiments of this specification, antibodies or recombinant antibodies are disclosed that include a heavy chain variable region (VH) having at least 100% identity with the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody or recombinant antibody further includes a light chain variable region (VL) having at least 80% identity with the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or recombinant antibody further includes a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or recombinant antibody further includes a light chain variable region (VL) having at least 95% identity with the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or recombinant antibody further includes a light chain variable region (VL) having at least 99% identity with the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or recombinant antibody further includes a light chain variable region (VL) having at least 100% identity with the amino acid sequence of SEQ ID NO: 7.

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

[0009] In certain embodiments of this specification, antibodies or recombinant antibodies comprising a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 7 are disclosed.

[0010] In certain embodiments of this specification, antibodies or recombinant antibodies comprising a light chain variable region (VL) having at least 95% identity with the amino acid sequence of SEQ ID NO: 7 are disclosed.

[0011] In certain embodiments of this specification, antibodies or recombinant antibodies comprising a light chain variable region (VL) having at least 99% identity with the amino acid sequence of SEQ ID NO: 7 are disclosed.

[0012] In certain embodiments of this specification, an antibody or recombinant antibody is disclosed that includes a light chain variable region (VL) having at least 100% identity with the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or recombinant antibody further includes a heavy chain variable region (VH) having at least 80% identity with the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody or recombinant antibody further includes a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody or recombinant antibody further includes a heavy chain variable region (VH) having at least 95% identity with the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody or recombinant antibody further includes a heavy chain variable region (VH) having at least 99% identity with the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody or recombinant antibody further includes a heavy chain variable region (VH) having at least 100% identity with the amino acid sequence of SEQ ID NO: 8.

[0013] In certain embodiments of this specification, a heavy chain variable region (V) having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 is used. H ) and a light chain variable region (V) that has at least 80% identity with the amino acid sequence of SEQ ID NO: 7 L An antibody or recombinant antibody comprising ) is disclosed. In some embodiments, the antibody or recombinant antibody comprises a light chain variable region (V) having at least 85% identity with the amino acid sequence of SEQ ID NO: 7. L ) includes. In some embodiments, the antibody or recombinant antibody has a light chain variable region (V) that has at least 90% identity with the amino acid sequence of SEQ ID NO: 7. L ) includes. In some embodiments, the antibody or recombinant antibody has a light chain variable region (V) that has at least 95% identity with the amino acid sequence of SEQ ID NO: 7. L) includes. In some embodiments, the antibody or recombinant antibody has a light chain variable region (V having at least 99% identity to the amino acid sequence of SEQ ID NO: 7 L ) includes. In some embodiments, the antibody or recombinant antibody has a light chain variable region (V having at least 99% identity to the amino acid sequence of SEQ ID NO: 7 L ) includes. In some embodiments, the antibody or recombinant antibody has a light chain variable region (V having at least 85% identity to the amino acid sequence of SEQ ID NO: 8 H ) includes. In some embodiments, the antibody or recombinant antibody has a heavy chain variable region (V having at least 90% identity to the amino acid sequence of SEQ ID NO: 8 H ) includes. In some embodiments, the antibody or recombinant antibody has a light chain variable region (V having at least 95% identity to the amino acid sequence of SEQ ID NO: 8 H ) includes. In some embodiments, the antibody or recombinant antibody has a heavy chain variable region (V having at least 99% identity to the amino acid sequence of SEQ ID NO: 8 H ) includes. In some embodiments, the antibody or recombinant antibody has a light chain variable region (V having at least 100% identity to the amino acid sequence of SEQ ID NO: 8 H ) includes.

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

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

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

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

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

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

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

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

[0022] In certain embodiments of this specification, antibodies or recombinant antibodies are disclosed that include a light chain sequence comprising a complementation-determining region (CDR) L1 having at least 80% identity with the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 80% identity with the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 80% identity with the amino acid sequence of SEQ ID NO: 3.

[0023] In certain embodiments of this specification, an antibody or recombinant antibody is disclosed that includes a light chain sequence comprising a complementation-determining region (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.

[0024] In certain embodiments of this specification, an antibody or recombinant antibody is disclosed that includes a light chain sequence comprising a complementation-determining region (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.

[0025] In certain embodiments of this specification, an antibody or recombinant antibody is disclosed that includes a light chain sequence comprising a complementation-determining region (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.

[0026] In certain embodiments of this specification, antibodies or recombinant antibodies are disclosed that include a light chain sequence comprising a complementation-determining region (CDR) L1 having at least 100% identity with the amino acid sequence of SEQ ID NO: 1, a CDR L2 having at least 100% identity with the amino acid sequence of SEQ ID NO: 2, and a CDR L3 having at least 100% identity with the amino acid sequence of SEQ ID NO: 3.

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

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

[0029] In some embodiments, the antibody or recombinant antibody further comprises a human heavy chain constant region or a human light chain constant region. In some embodiments, the human heavy chain constant region is IgG1 or IgG4, or a fragment thereof. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the light chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody or recombinant antibody comprises a human variable framework region and a mouse constant region. In some embodiments, the antibody or recombinant antibody further comprises a mouse heavy chain constant region or a mouse light chain constant region. In some embodiments, the antibody or mouse heavy chain constant region is IgG2A. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the light chain has at least 80% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody or recombinant antibody is an antibody fragment comprising a single heavy chain, a single light chain, 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 triplicate Fab3, a single-chain linked polypeptide, a dAb fragment, or a diabody.

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

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

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

[0033] In some embodiments, the antibody or recombinant antibody specifically binds to the CD163 epitope containing the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody or recombinant antibody specifically binds to the CD163 epitope containing the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody or recombinant antibody specifically binds to the CD163 epitope containing the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or recombinant antibody specifically binds to the CD163 epitopes containing SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively. In some embodiments, the antibody or recombinant antibody has a K content of 1 nM to 100 nM. D It specifically binds to CD163. In some embodiments, the antibody or recombinant antibody has a K content of 1 nM to 50 nM. D It specifically binds to CD163. In some embodiments, the antibody or recombinant antibody has a concentration of 1 nM to 10 nM of K DIt specifically binds to CD163. In some embodiments, the antibody or CD163 is human CD163. In some embodiments, the antibody or recombinant antibody is 1 nM to 100 nM K D It specifically binds to M2c macrophages. In some embodiments, the antibody or recombinant antibody has a concentration of 1 nM to 50 nM of K. D It specifically binds to M2c macrophages. In some embodiments, the antibody or recombinant antibody has a concentration of 1 nM to 10 nM of K. D It specifically binds to M2c macrophages. In some embodiments, the M2c macrophages are human M2c macrophages.

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

[0035] In certain embodiments of this specification, a pharmaceutical composition is disclosed comprising an antibody or recombinant antibody described in any one of the embodiments described above and a pharmaceutically acceptable carrier.

[0036] In certain embodiments of this specification, the use of an antibody or recombinant antibody described in any one of the above embodiments in the manufacture of a drug for treating cancer in human subjects is disclosed.

[0037] In certain embodiments of this specification, the use of an antibody or recombinant antibody described in any one of the above embodiments is disclosed in the manufacture of a drug that reduces immunosuppression by tumor-associated macrophages in human cancer subjects.

[0038] In certain embodiments of this specification, the use of an antibody or recombinant antibody described in any one of the above embodiments is disclosed in the manufacture of a drug that promotes T cell-mediated tumor cell death in human cancer subjects.

[0039] In certain embodiments of this specification, a method for promoting immune cell function is disclosed, comprising the steps of administering an antibody or recombinant antibody described in any one of the above embodiments to an individual in need, and the following parameter: (i) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, and (ii) CD4 + T cells, CD8 + The process includes a step of promoting immune cell function as measured by one or both of T cells, NK cells, or any combination thereof. In some embodiments, CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof is measured as an increase in the levels of IFN-γ, TNF-α, or perforin, or any combination thereof. In some embodiments, immunosuppressive human myeloid cells are macrophages. In some embodiments, immunosuppressive human myeloid cells are myeloid-derived suppressor cells. In some embodiments, the antibody or recombinant antibody is the antibody or recombinant antibody described in any one of the embodiments described above.

[0040] In certain embodiments of this specification, a method for treating cancer in an individual as needed is disclosed, which comprises administering to the individual a therapeutically effective amount of an antibody or recombinant antibody described in any one of the embodiments described above, thereby treating the individual's cancer.

[0041] In certain embodiments of this specification, a method for treating cancer in an individual as needed is disclosed, which comprises administering to the individual a therapeutically effective dose of an antibody or recombinant antibody described in any of the embodiments described above, thereby reducing immunosuppression by tumor-associated macrophages in the individual.

[0042] In certain embodiments of this specification, a method for treating cancer in an individual as needed is disclosed, the method comprising administering to the individual a therapeutically effective dose of an antibody or recombinant antibody described in any of the embodiments described above, thereby increasing T cell-mediated tumor cell death in the individual.

[0043] In certain embodiments of this specification, a method is disclosed for reducing the tumor-promoting activity of tumor-associated macrophages in an individual where a reduction is desired, the method comprising CD4 in the tumor microenvironment. + T cell activation, CD4 + T cell proliferation, CD8 + T cell activation, CD8 + The method includes administering a certain amount of the pharmaceutical composition described in any one of the aforementioned embodiments, which is effective in modulating T cell proliferation, or any combination thereof, to an individual.

[0044] In certain embodiments of this specification, a method is disclosed for promoting lymphocyte-mediated tumor cell death in an individual in which such a method is desired, comprising administering to the individual an effective amount of the antibody or recombinant step described in any one of the embodiments described above, or the pharmaceutical composition described in any one of the embodiments described above.

[0045] In some embodiments, the method further includes a step of promoting tumor cell death in the tumor microenvironment. In some embodiments, the cancer is lung cancer or sarcoma. In some embodiments, the lung cancer is carcinoma or adenocarcinoma. In some embodiments, the method further includes a step of administering an additional anticancer drug or anticancer therapy to the individual. In some embodiments, the additional anticancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, and cytokine therapy, as well as combinations thereof. In some embodiments, the additional anticancer therapy is immunotherapy. In some embodiments, the immunotherapy is a composition comprising a checkpoint inhibitor.

[0046] In certain embodiments of this specification, a method is disclosed for modulating the activity of tumor-associated macrophages in the tumor microenvironment, the method comprising the step of contacting tumor-associated macrophages with an antibody or recombinant antibody, the antibody or recombinant antibody binding to CD163-expressing human macrophages, and having the following effects: (a) the binding of the antibody reduces the expression of at least one marker on the macrophages, which is CD16, CD64, TLR2, or Siglec-15; (b) upon binding of the antibody to the CD163 protein, the antibody is internalized by the human macrophages; (c) the binding of the antibody is not cytotoxic to the macrophages; (d) the binding of the antibody or recombinant antibody is CD4 + T cells, CD8 + (e) The binding of an antibody or recombinant antibody to T cells, NK cells, or any combination thereof, which produces IFN-γ, TNF-α, perforin, or any combination thereof, is to CD4 + T cells, CD8 + (f) The binding of an antibody or recombinant antibody promotes the activation of T cells, NK cells, or any combination thereof, CD4 + T cells, CD8 + The binding of an antibody or recombinant antibody promotes the proliferation of T cells, NK cells, or any combination thereof, and (g) promotes cell death in the tumor microenvironment, comprising at least one of these. In some embodiments, the binding results in two or more of (a)-(g), three or more of (a)-(g), four or more of (a)-(g), five or more of (a)-(g), six or more of (a)-(g), or all of (a)-(f).

[0047] In some embodiments, the method is carried out in the tumor microenvironment. In some embodiments, the method is carried out in vivo. In some embodiments, binding of an antibody or recombinant antibody to macrophages modulates the immune function of cells in the tumor microenvironment. In some embodiments, binding of an antibody or recombinant antibody to macrophages promotes anti-tumor immune function. In some embodiments, the antibody or recombinant antibody comprises a constant domain, which binds to an Fc receptor. In some embodiments, the Fc receptor is expressed on macrophages. In some embodiments, the method further includes the step of internalizing the antibody or recombinant antibody by human macrophages upon binding to the CD163 protein. In some embodiments, binding to the CD163 protein is not cytotoxic to human macrophages. In some embodiments, the method involves CD4 + The method further includes a step of promoting the expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by T cells. In some embodiments, the method involves CD8 + The method further includes a step of promoting the expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by T cells. In some embodiments, the method includes a step of reducing immunosuppression in the tumor microenvironment. In some embodiments, the method includes a step of promoting cancer cell death mediated by cytotoxic lymphocytes. In some embodiments, the method includes a step of promoting tumor cell death mediated by NK cells. In some embodiments, the method includes a step of promoting IL-2 expression by T cells. In some embodiments, the method includes CD4 + T cells, CD196 - T cells, CXCR3 + T cells, CCR4 - The process includes increasing T cells, or any combination thereof.

[0048] In certain embodiments of this specification, an antibody is disclosed that specifically binds to the CD163 protein expressed on immunosuppressive human bone marrow cells, wherein the binding of the antibody to the bone marrow cells is (i) CD4 + T cells, CD8+ Activation of T cells, NK cells, or any combination thereof, and (ii) CD4 + T cells, CD8 + It promotes immune cell function as measured by the proliferation of one or both of T cells, NK cells, or any combination thereof. In some embodiments, immunosuppressive human myeloid cells are macrophages or myeloid-derived suppressor cells. In some embodiments, CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof is measured as an increase in the production of IFN-γ, TNF-α, perforin, or any combination thereof.

[0049] In certain embodiments of this specification, an antibody is disclosed that specifically binds to the CD163 protein expressed on human macrophages, wherein the binding of the antibody to the macrophages is (i) CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, and (ii) CD4 + T cells, CD8 + The immune cell function is promoted as measured by the proliferation of one or both of T cells, NK cells, or any combination thereof. In some embodiments, the immune cell function is located in the tumor microenvironment. In some embodiments, the immune cell function is located in vivo. In some embodiments, CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof is measured as an increase in the production of IFN-γ, TNF-α, perforin, or any combination thereof.

[0050] In certain embodiments of this specification, antibodies that specifically bind to the CD163 protein expressed on human macrophages are disclosed, and the binding of the antibody to the macrophages increases immunostimulatory activity in the tumor microenvironment. In some embodiments, the tumor microenvironment is in vivo. In some embodiments, the binding of the antibody to the macrophages reduces the immunosuppressive activity of the macrophages. In some embodiments, the binding of the antibody to the macrophages reduces tumors that promote macrophage activity. In some embodiments, the macrophages are tumor-associated macrophages. In some embodiments, the antibody modifies the expression of at least one marker on the macrophages. In some embodiments, the CD163 protein is the glycoform of CD163. In some embodiments, the CD163 protein is the 150kDa glycoform of CD163. In some embodiments, the antibody does not specifically bind to the 130kDa glycoform of CD163 expressed by human macrophages. In some embodiments, the antibody has a constant domain that enables binding to the 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 triplicate Fab3, a single-chain binding polypeptide, a dAb fragment, or a diabody. In some embodiments, at least one marker on the human macrophage is CD16, CD64, TLR2, or Siglec-15. In some embodiments, the human macrophage is an M2 macrophage or an M2-like macrophage. In some embodiments, the human macrophage is an M2a, M2b, M2c, or M2d macrophage. In some embodiments, the CD163 protein is a component of a cell surface complex comprising at least one other protein expressed by the macrophage.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, upon binding to the CD163 protein, the 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 is CD4. + T cell activation, CD4 + T cell proliferation, or CD4 + It promotes both T cell activation and proliferation. In some embodiments, binding to the CD163 protein is linked to CD4 + It promotes the expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by T cells. In some embodiments, binding to the CD163 protein leads to CD8 + T cell activation, CD8 + T cell proliferation, or CD8 + It promotes both T cell activation and proliferation. In some embodiments, binding to the CD163 protein is linked to CD8 + It promotes the expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by T cells. In some embodiments, binding to the CD163 protein reduces immunosuppression in the tumor microenvironment. In some embodiments, binding to the CD163 protein promotes tumor cell death in the tumor microenvironment. In some embodiments, binding to the CD163 protein promotes cancer cell death mediated by cytotoxic lymphocytes. In some embodiments, binding to the CD163 protein promotes tumor cell death mediated by NK cells. In some embodiments, binding to the CD163 protein promotes IL-2 expression by T cells. In some embodiments, binding to the CD163 protein promotes CD4 + T cells, CD196-T cells, CXCR3 + T cells, CCR4 -Increase T cells, or any combination thereof. In some embodiments, binding to CD163 reduces immunosuppression in the tumor microenvironment caused by macrophages.

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

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

[0053] In certain embodiments of the present specification, for use in a method of treating cancer, an antibody that specifically binds to the CD163 protein expressed on human tumor-associated macrophages and reduces the expression of CD16, CD64, TLR2, Siglec-15, or any combination thereof by macrophages is disclosed. In some embodiments, the binding of the antibody to macrophages modulates the immune function of cells in the tumor microenvironment. In some embodiments, the binding of the antibody to macrophages promotes anti-tumor immune function.

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

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

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

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

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

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

[0060] In certain embodiments of this specification, a method for treating cancer in an individual as needed is disclosed, the method comprising administering to the individual a therapeutically effective dose of an antibody described in any one of the embodiments disclosed herein, thereby treating the individual's cancer.

[0061] In certain embodiments of this specification, a method for treating cancer in an individual as needed is disclosed, the method comprising administering to the individual a therapeutically effective dose of an antibody described in any one of the embodiments disclosed herein, thereby reducing immunosuppression by tumor-associated macrophages in the individual.

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

[0063] In certain embodiments of this specification, a method is disclosed for reducing the tumor-promoting activity of tumor-associated macrophages in an individual where a reduction is desired, the method comprising CD4 in the tumor microenvironment. + T cell activation, CD4 + T cell proliferation, CD8 + T cell activation, CD8 +The method comprises administering a certain amount of the pharmaceutical composition according to claim 43, which is effective in modulating T cell proliferation, or any combination thereof, to an individual. In some embodiments, the method further comprises the step of promoting tumor cell death in the tumor microenvironment.

[0064] In certain embodiments of this specification, a method is disclosed for promoting lymphocyte-mediated tumor cell death in an individual in which such a method is desired, the method comprising administering to the individual an effective amount of a pharmaceutical composition described in any one of the embodiments disclosed herein.

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

[0066] Novel features of this disclosure are described in particular in the appended claims. The features and advantages of this disclosure will be better understood by referring to the following detailed description, which describes exemplary embodiments in which the principles of this disclosure are utilized, and to the following appended drawings. [Figure 1] This figure shows the AB101 antibody that binds to the human MDSC population. [Figure 2] This figure shows the binding of AB101 to CD163Hi cells. [Figure 3] This figure compares AB101 binding to human M2C, M1, and M0 cells with isotype controls. [Figure 4] This figure shows AB101 binding to human peripheral blood T cells, B cells, NKT cells, neutrophils, monocytes, and dendritic cells, with isotype controls shown in gray and AB101 binding in black. [Figure 5] This figure shows no binding of AB101 to a panel of human primary cells, including small airway epithelial cells (SAECs), renal proximal tubular epithelial cells (RPTECs), pulmonary microvascular endothelial cells (HMVECs), umbilical vein endothelial cells (HUVECs), aortic smooth muscle cells (AOSMCs), and keratinocytes. [Figure 6A] This figure shows the top 20 targets for AB102 based on mass spectrometry of samples after immunoprecipitation. [Figure 6B] This figure shows the superior cell surface targets for AB102, based on mass spectrometry of the sample after immunoprecipitation. [Figure 6C] This figure shows the superior cell surface targets of AB102 compared to ISO (isotype-negative control), based on mass spectrometry of AB102 and ISO samples after immunoprecipitation. [Figure 7] This figure shows that AB102 co-immunoprecipitations a separate high molecular weight glycotype of CD163. [Figure 8] This figure shows that AB101, AB102, and the control CD163 antibody bound to huCD163, while the isotype control did not show any clear binding. [Figure 9]This figure shows that neither AB101 nor the control anti-huCD163 bound to recombinant mouse CD163, compared to commercially available anti-muCD163 antibodies that did not bind to mouse CD163. [Figure 10] This figure shows that pretreatment of M2c macrophages with a polyclonal anti-CD163 antibody blocked the binding of AB101 antibody compared to treatment with a goat control polyclonal antibody that did not block AB101 binding. [Figure 11] This figure shows that pretreatment of M2c macrophages with a polyclonal anti-CD163 antibody blocked the binding of the control monoclonal anti-huCD163 antibody compared to treatment with a goat control polyclonal antibody that did not block the binding of the control monoclonal anti-huCD163 antibody. [Figure 12] This figure shows that treatment of polarized M2C macrophages with siRNA against CD163 substantially reduced AB102 antibody binding compared to M2C macrophages treated with scrambled siRNA (siScramb) or without siRNA, and represents three replicates. [Figure 13] This figure shows that siRNA knockdown of CD163 reduced AB102 antibody binding, with a slight decrease in AB102 antibody binding observed after siRNA knockdown of FCGR2A+FCGR3A (1 out of 3 donors), FCGR2C, or FCGR3A, and no decrease in AB102 binding observed after knockdown of siCD206, siCD163L1, siPPIA, siLGALS1, siLGALS3, siLILRB2, and siUPAR. [Figure 14] This figure shows that treatment of cultured M2 macrophages with LPS resulted in loss of binding to both the AB101 antibody and the control anti-CD163 antibody. [Figure 15] This figure shows the increase in IL-2 production after treatment of bone marrow cells with AB101 antibody. [Figure 16] This figure shows the treatment of AB101 antibody during polarization, which promoted CD4+ T cell proliferation. [Figure 17]This figure shows the treatment of AB101 antibody during polarization, which promoted CD8+ T cell proliferation. [Figure 18] This figure shows that treatment with AB101 antibody during polarization (labeled "pre" on the graph), after polarization (labeled "post" on the graph), or a combination of during and after polarization (labeled "pre and post" on the graph) enhanced IL-2 production compared to isotype antibody treatment. [Figure 19] This figure shows that treatment of M2 macrophages with AB101 antibody reduced the expression of CD16, CD64, calreticulin, and Siglec-15. [Figure 20] This figure shows that treatment of M2c cells in AB101 increased the Th1 / Th2 ratio compared to isotype controls. [Figure 21] This figure shows that treatment of M2c cells with AB101 increased CD69 expression on CD4 T cells compared to isotype controls. [Figure 22] This figure shows that treatment of M2c cells with AB101 increased ICOS expression on CD4 T cells compared to isotype controls. [Figure 23] This figure shows that treatment of M2c cells with AB101 increased OX40 expression on CD4 T cells compared to isotype controls. [Figure 24] This figure shows that increased CTLs in the presence of Bite resulted in increased Raji tumor cell death compared to isotype controls. [Figure 25] This figure shows that the AB102 antibody was internalized in much the same way as the commercially available anti-CD163 antibody (R&D Systems MAB1607-100), and that approximately twice as much AB101 antibody was internalized compared to the commercially available CD163 antibody. [Figure 26]This figure plots tumor volume over 30 days for A549 tumors. Arrows indicate injections due to antibody therapy. Each point represents the average measurement from 7 mice. Error bars indicate the mean standard error (SEM). Statistical significance was calculated using the Mann-Whitney U test. [Figure 27] This figure plots tumor volume over 30 days for H1975 tumors. Arrows indicate injections due to antibody therapy. Each point represents the average measurement from 7 mice. Error bars indicate the mean standard error (SEM). Statistical significance was calculated using the Mann-Whitney U test. [Figure 28] This figure shows the experimental design for an M2c / T cell co-culture assay to evaluate the effects of AB101 treatment on T cell proliferation and IL-2 production. [Figure 29] This figure shows that treatment with AB101 during M2c macrophage polarization restored T cell proliferation in an M2c / T cell co-culture assay. [Figure 30] This figure shows that treatment with AB101 during M2c macrophage polarization enhanced IL-2 secretion by OKT3-activated T cells in an M2c / T cell co-culture assay. [Figure 31] This figure shows that treatment with AB101 before, before / after, and after the regimen increased CD8+ T cell proliferation in the M2c / T cell co-culture assay. [Figure 32] This figure shows that treatment with AB101 before, before / after, and after the regimen increased CD8+ T cell proliferation in M2c / T cell co-culture assays for individual subjects. [Figure 33] This figure shows that treatment with AB101 enhanced CD8+ T cell proliferation in M2c / co-cultures of multiple subjects. [Figure 34] This figure shows that treatment with AB101 enhanced CD4+ T cell proliferation in M2c / co-cultures of multiple subjects. [Figure 35]This figure shows that treatment with AB101 enhanced IL-2 production by multiple human T cells during M2c / co-culture. [Figure 36] This figure shows that AB101 is more potent than the AB104 and AB102 isotypes in enhancing T cell proliferation in M2c / T cell co-culture assays. [Figure 37] This figure shows that AB101, not AB104, rescues CD8+ T cell proliferation from M2c-mediated immunosuppression both before and after the regimen. [Figure 38] This figure shows that AB101 restored the CD8+ T cell cytokine response in an M2c / T cell co-culture assay. [Figure 39] This figure shows that AB101 rescued CD4+ T cells, IFN-γ, TNF-α, and perforin responses from M2c macrophage-mediated immunosuppression. [Figure 40] This figure shows that treatment with AB101 enhanced the cytotoxic activity of CD8+ T cells. [Figure 41] This figure shows that treatment with AB101 enhanced the BiTE®-assisted cytotoxic activity of CD8+ T cells. [Figure 42] This figure shows that treatment with AB101 enhanced the cytotoxic activity of non-HLA-restricted CD8+ T cells. [Figure 43] This figure shows that treatment with AB101 alleviates M2c cell-mediated immunosuppression and induces a unique expression pattern by activated CD4+ T cells. [Figure 44] This figure shows that treatment with AB101 alleviates M2c macrophage immunosuppression and enhances the activation of CD4+ and CD8+ T cells. [Figure 45] This figure shows CXCR3 expression by activated CD4+ T cells. [Figure 46] This figure shows that treatment with AB101 during the polarization of M2c macrophages reduced the expression of CD16, CD64, Siglex-15, and TLR2 by M2c macrophages. [Figure 47] This figure shows an overview of the changes in AB101 bound to huCD163 based on pepsin digestion. [Figure 48] This figure outlines the changes in A B101 bound to huCD163 based on nepenthesin II digestion. [Figure 49] This figure shows an overview of AB101 binding to human CD163 ECD based on HDX-MS testing. [Figure 50] This diagram shows that AB101 binds to a truncated CD163 ECD composed of SRCR domains 1-5. [Figure 51] This figure shows the alignment of human CD163 to cynomolgus monkey CD163. Signal sequence. The sequence below the black bar shows the nine SRCR domains, and the gray line above the sequence shows the consensus sequence. Protected and exposed regions are indicated based on the AB101 binding epitope, as determined by the protection observed in HDX-MS. The solid line below the sequence shows the nepenthesin II protected region. The open box below the sequence shows the pepsin protected region. The shaded box below the sequence shows the pepsin exposed region. The box with a vertical line below the sequence shows the nepenthesin II exposed region. Lysine (K) at position 323 of human CD163 and glutamate (E) of cynomolgus monkey CD163 are indicated by boxes. [Figure 52] This figure shows that AB101 binds to human and cynomolgus monkey E323K mutants, but not to wild-type cynomolgus monkey CD163 ECD. [Figure 53] This figure shows the detection of SPR (Surface-to-Phase Reaction) of AB101 binding to human CD163. AB101 was serially diluted to various concentrations using (A) EDTA or (B) calcium-containing electrophoresis buffer. Then, GHI / 61 was injected into flow cell 2 at a flow rate of 30 μl / min, concentrations of 6.25 / 12.5 / 25 / 50 / 100 / 200 μg / ml, a contact time of 300 s, and a dissociation time of 600 s. [Figure 54]This figure shows the detection of SPR (Surface-to-Symptom Reaction) of GHI / 61 binding to human CD163. Anti-CD163 clone GHI / 61 was serially diluted to various concentrations using (A) EDTA or (B) calcium-containing electrophoresis buffer. GHI / 61 was then injected into flow cell 2 at a flow rate of 30 μl / min, concentrations 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. [Figure 55] This figure shows the SPR detection of CD163 binding to AB101. Human CD163 protein was serially diluted to various concentrations using calcium-containing electrophoresis buffer. The CD163 protein was then injected into flow cell 2 at a flow rate of 30 μl / min, concentrations of 1.25 / 2.5 / 5.0 / 10.0 / 20.0 / 40.0 μg / ml, a contact time of 300 s, and a dissociation time of 600 s. [Figure 56] This figure shows the binding of AB101 to soluble CD163 in the AlphaLisa assay. AB101 (circular) or isotype control (triangle) was incubated with 750 nM CD163-His at the indicated concentrations for 1 hour. Binding was quantified by AlphaLisa using biotinylated anti-hIgG1 mAb, streptavidin receptor, and nickel donor beads. The symbols represent the mean ± standard error of five independent measurements. Curve fitting was performed using 1- and 2-site saturated binding models (Graphpad Prism). (R²=0.92). (A) Linear, and (B) Logarithmic x-axis scale. [Figure 57]This figure shows the binding of AB101 to M1c macrophages. M2c macrophages were blocked with tight FACS blocking buffer containing 0.5 mg / ml of human IgG1 and then stained with AB101 (circular) or isotype control (triangle) at the indicated concentrations for 30 minutes. Binding of AlexaFluor647-labeled AB101 and isotype control to M2c macrophages was quantified by fluorescence intensity and reported as gMFI. The symbols represent the mean ± standard error of the four test subjects. Curve fitting was performed using a two-site saturated binding model (Graphpad Prism) (R²=0.99). (A) Linear, and (B) Logarithmic x-axis scale. [Modes for carrying out the invention]

[0067] This specification includes CD163 + Antibodies that specifically bind to cells are disclosed. In some embodiments, CD163 + The cells are immunosuppressive myeloid cells. In some embodiments, CD163 + The cells are human CD163 expressing myeloid. In some embodiments, CD163 + The cells are tumor cells. In some embodiments, CD163 + Immunosuppressive myeloid cells are human macrophages. In some embodiments, human CD163 + Immunosuppressive macrophages are M2 or M2-like macrophages. In some embodiments, immunosuppressive myeloid cells are myeloid-derived suppressor cells (MDSCs). In some embodiments, human macrophages have high levels of CD163 (CD163 Hi ) expresses CD163. In contrast, other human hematopoietic cells or primary non-immune human cells do not express CD163. For example, M1 and M1-like macrophages do not express CD163.

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

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

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

[0071] In some embodiments, the antibodies of this disclosure have high binding activity and specific binding to immunosuppressive myeloid cells, specifically to tumor-associated macrophages such as M2 and M2-like macrophages. In some embodiments, the antibodies specifically bind to M2 and M2-like TAMs derived from human primary lung tumors. In some embodiments, the antibodies disclosed herein do not have significant binding to M1 or M1-like macrophages. M1-activated macrophages express transcription factors such as interferon regulator (IRF5), kappa-photopolymer gene enhancer nuclear factor (NF-κB), activating protein (AP-1), and STAT1. M1 macrophages secrete pro-inflammatory cytokines such as IFN-γ, IL-1, IL-6, IL-12, IL-23, and TNFα. M1 macrophages have the function and phenotype corresponding to M1 macrophages. M1-like macrophages are in vivo or ex vivo macrophages that have a subset of the functional or phenotypic features of M1 macrophages.

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

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

[0074] Direct tumor cell death by cytotoxic T cells between tumor-infiltrating lymphocytes (TILs) plays a major role in the immune system's antitumor function. However, tumor-mediated mycoplasma (TAMs) in the tumor microenvironment (TME) suppress the T cell-mediated antitumor immune response. TAMs have an immunosuppressive transcriptional profile and express factors including IL-10 and transforming growth factor β (TGFβ). In humans, TAMs have been shown to directly suppress T cell function through the surface presentation of programmed cell death ligand 1 (PD-L1) in hepatocellular carcinoma and B7 homolog in ovarian cancer, respectively, which activate programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) on T cells. Inhibitory signals to PD-1 and CTLA4 are immune checkpoints, and ligand binding to these inhibitory receptors inhibits T cell receptor signaling and T cell toxicity, promoting T cell apoptosis. HIF-1α induces TAMs and suppresses 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.

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

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

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

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

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

[0080] CD163 mRNA expression is generally limited to myeloid cells but is also expressed by certain human cancers. CD163 expression on TAMs has been associated with an immunosuppressive M2-like phenotype that is recognized to correlate with poor clinical outcomes in cancer. CD163 is required for the tumorigenic activation of macrophages in human and mouse sarcomas. CD163 is also a macrophage scavenger receptor and has been reported to promote immunosuppression. In some embodiments, the interaction between the hemoglobin-haptoglobin complex and 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 metabolites Fe 2+ , CO, and bilirubin.

[0081] Soluble CD163 is produced in humans via echitodomain elimination and has been reported to possess anti-inflammatory properties, including downregulating T cell responses, such as lymphocyte proliferation, stimulated by phytohemaglutinin (PHA) or 12-O-tetradecanoylphorbol-13-acetate (TPA).

[0082] Antibodies targeting CD163 have been shown to modulate the innate immune response of CD163 expressed by macrophages. For example, the RM3 / 1 antibody, an antibody against CD163, is a mouse monoclonal Igg1 (κ light chain) produced against human monocytes. The RM3 / 1 antibody binds to the cysteine-rich domain 9 of human CD163, reducing LPS-induced TNFα and enhancing IL-10 secretion by macrophages.

[0083] Specific terms Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the claimed subject matter pertains. In general, the nomenclature and techniques used in relation to immunology, oncology, cell and tissue culture, molecular biology, and protein and oligo or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. The above general descriptions and the following detailed descriptions are illustrative and descriptive only and should not be understood as being limited to the claimed subject matter. The chapter headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein.

[0084] As used herein, the singular forms "a," "and," and "the" refer to multiple objects unless otherwise specified in the context. For example, a reference to "antibody" includes multiple antibodies, and in some embodiments, a reference to "antibody" includes a large number of antibodies.

[0085] As used herein, all numbers or numerical ranges include all integers and fractions that are within or encompass such a range, or integers that are within or encompass such a range, unless otherwise specified in the context. For example, a reference to the range 90-100% includes not only 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., but also 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., as well as 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc. In another example, references to the range of 1 to 5,000 times include not only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, but also 1.1, 1.2, 1.3, 1.4, 1.5 times, and 2.1, 2.2, 2.3, 2.4, 2.5 times.

[0086] When used in this specification, numbers preceded by "approximately" include the number itself and refer to a range that extends from 10% below that number to 10% above it. The range preceded by "approximately" is 10% below the lower limit of that range and 10% above the upper limit of that range.

[0087] "Percent identity" and "% identity" refer to the degree to which two sequences (nucleotides or amino acids) have the same residues at the same positions in their alignment. For example, "the amino acid sequence is X% identical to sequence number Y" refers to the % identity of the amino acid sequence to sequence number Y, and more specifically, it means that X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in sequence number Y. Generally, computer programs are used for such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(1):11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci USA. 1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98), and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387-95).

[0088] As used herein, “antibody” refers to a protein that binds to an antigen. Antibodies often contain variable and constant domains in both the heavy and light chains. Therefore, most antibodies have a heavy chain variable domain (VH) and a light chain variable domain (VL) that together form the antibody portion that binds to the antigen. Within each variable domain, there are three complementarity-determining regions (CDRs) that form a loop within the heavy chain variable domain (VH) and the light chain variable domain (VL) and come into contact with the surface of the antigen. “Antibodies” include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific (e.g., bispecific antibodies), natural, humanized, human, chimeric, synthetic, recombinant, hybrid, mutant, graft, antibody fragments (e.g., parts of a full-length antibody, generally the antigen-binding or its variable region, e.g., Fab, Fab', F(ab')2, and Fv fragments), and antibodies produced in vitro that have antigen-binding activity. This term also includes single-chain antibodies, such as single-chain FV (sFv or scFv) antibodies in which a variable heavy chain and a variable light chain are bound together (directly or via a peptide linker) to form a continuous polypeptide.

[0089] As used herein, “complementarity-determining regions (CDRs)” refer to the variable chain portion in an antibody that binds to a specific antigen. Multiple methods may be used to define CDRs. Current technologies utilize various numbering schemes that employ different definitions of CDR length and position. For example, the Kabat numbering scheme is sequence alignment-based and predicts CDRs using a “variable parameter” (the number of different amino acids at a given position divided by the frequency of the most frequently occurring amino acid at that position). On the other hand, the Chothia numbering scheme is a structure-based numbering scheme in which the crystal structure of the antibody is aligned so that it defines a loop structure as the CDR. The Martin numbering scheme focuses on the structural alignment of atypical framework regions of varying lengths. The IMGT numbering scheme is a standardized numbering system based on sequence alignment from a complete reference gene database that includes 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 estimates the framework and CDR length using structurally preserved Cα positions. Those skilled in the art should note that the definition of CDR varies depending on the method used. Any method of defining CDR is contemplated using the sequences disclosed herein.

[0090] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to a mammalian subject, specifically a human, to whom diagnosis, treatment, or therapy is desired. For treatment purposes, “mammal” means humans, domesticated animals, and livestock, as well as animals classified as mammals, including laboratory, zoo, sport, or pet animals such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and monkeys. In some embodiments, the mammal is a human.

[0091] As used herein, terms such as “treatment” and “to treat” may, depending on the context, mean administering a drug or performing a treatment for the purpose of obtaining an effect. In some embodiments, the effect is prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it results in a partial or complete cure of the disease and / or its symptoms. As used herein, “treatment” includes treatment of a disease or disorder (e.g., cancer) in a mammal, specifically a human, and includes (a) preventing the occurrence of the disease or its symptoms in a subject who is prone to the disease (e.g., including diseases associated with or caused by a primary disease) but has not yet been diagnosed as having the disease, (b) inhibiting the disease, i.e., stopping its progression, and (c) alleviating the disease, i.e., causing the regression of the disease. In some embodiments, “to treat” means that the treatment, improvement, or prevention of cancer has been successful and includes objective or subjective parameters such as reduction, remission, decrease in symptoms or making the condition more tolerable to the patient, slowing the rate of degeneration or decay, or debilitating the final stage of degeneration. Treatment or improvement of symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Therefore, the term “treatment” includes the administration of the compounds or agents of this disclosure to prevent, delay, alleviate, or prevent or inhibit the onset 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, symptoms of a disease, or side effects of a disease in a subject.If, after administering a therapeutic dose of the antibodies of this disclosure, the patient exhibits observable and / or measurable changes in the parameters or symptoms of the disease or disorder, for example, in the case of cancer treatment, increased tumor cell death activity as assessed ex vivo, decreased levels of immunosuppressive secretory factors in the blood, decreased tumor volume or mass, increased number of cytotoxic lymphocytes and Th1-like T cells in tumor biopsy, decreased pathological condition or mortality, improved quality of life factors, or improvement in any objective indication related to the parameters or symptoms of the disease or disorder, the subject is considered “treated” for the disease or disorder. In some embodiments, the parameters include converting a cold immunotumor to a warm immunotumor by, for example, increasing the number of cytotoxic lymphocytes and markers of T cell activation (such as CD69, ICOS, OX40) in tumor biopsy, or by decreasing the expression of CD16, CD64, TLR2, and Siglec-15 on TAMs in tumor biopsy.

[0092] In some embodiments, “inducing a response” means alleviating or reducing the signs or symptoms of the disease in question, and specifically includes, but is not limited to, extending survival.

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

[0094] In some embodiments, the antibody "effector function" refers to the biological activity that originates from the antibody's Fc region (either the natural sequence FC region or the amino acid sequence variant Fc region) and varies depending on the antibody isotype.

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

[0096] "Human effector cells", as used herein, refers to leukocytes that express one or more FcRs and perform effector functions. For example, the cells express at least Fc γRIII 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.

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

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

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

[0100] The antigen-binding region of an antibody is called an "epitope," which is the antigenic determinant of the antigen, or a "paratope," which is a portion of the antigen molecule that can be bound by the antibody. In some embodiments, an antigenic substance has one or more parts that can be recognized by an antibody, i.e., more than one epitope, so that a single antigenic substance can be specifically bound by various antibodies, each having specificity for a different epitope. In some embodiments, epitopes include non-proximal parts of the antigen. For example, in a polypeptide, amino acid residues that are not proximal in the polypeptide's primary sequence 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.

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

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

[0103] When used herein, "functional antibody fragment" refers to an antibody fragment that, in the context of the description, not only binds to an antigen but also possesses functional attributes that characterize the intact antibody. For example, if the function of an antibody depends on having an Fc domain that enables effector functions such as ADCC, the functional fragment has such a function. In some embodiments, it is assumed that when the antibodies of this disclosure contain an Fc moiety that binds to a macrophage Fc receptor such as CD16(FcγRIIIa) or CD64(FcγRI), they are effective in modulating the functional state of tumor-associated macrophages or reorienting or attenuating M2-state macrophages.

[0104] The term "functional fragment or analog" of an antibody refers to a compound that possesses qualitative biological activity common to the full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody might bind to IgE immunoglobulin, thereby preventing or substantially reducing its ability to bind to the high-affinity receptor FcγRI.

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

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

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

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

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

[0110] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two drugs, K D It is represented as follows: In one embodiment, the antibody or its antigen-binding fragment is 10 -6 A range of M or less, or 10 -16 A range up to M (for example, about 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 Below M, K for CD163 D It exhibits binding affinity as measured by the (equilibrium dissociation constant). In one embodiment, the antibody described herein is 10 -4 M or less, about 10 -5 M or less, about 10 -6 M or less, 10 -7 M or less, or 10 -8 K below M D It specifically binds to the huCD163 polypeptide.

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

[0112] The term "specific" refers to a situation where an antibody preferentially binds to molecules other than the antigen containing the epitope recognized by the antibody. This term also applies, for example, to a situation where an antigen-binding domain is specific to a particular epitope carried by multiple antigens, in which case the antibody or its antigen-binding fragment carrying the antigen-binding domain can bind to various antigens carrying the epitope.

[0113] When used herein, the antibody is preferably at a level detectable by the antigen, preferably about 10 4 M -1 The above is approximately 10 5 M -1 The above is approximately 10 6 M -1 The above is approximately 10 7 M -1 or more, or 10 9 M -1 The affinity constant K is as described above. a When an antibody reacts to an antigen, it is said to be "immunely specific," "specific," or "specifically binding" to that antigen.

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

[0115] The term "polypeptide" is used in its conventional sense, i.e., as a sequence of amino acids. Polypeptides are not limited to a specific length of product. Peptides, oligopeptides, and proteins are included within the definition of polypeptide; these terms are used interchangeably herein unless otherwise specified. This term also does not represent, nor exclude, post-expression modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, or other modifications known in the art, both spontaneous and unspontaneous. In some embodiments, the polypeptide is an entire protein or a subsequence thereof. The specific polypeptide of interest in light of the antibodies of this disclosure is an amino acid subsequence containing a CDR and capable of binding to human M2 macrophage or CD163 protein expressed by such cells.

[0116] As used herein, “substantially pure” and “substantially free” mean, for example, solutions or suspensions containing less than or equal to about 20% foreign substances, less than or equal to about 10% foreign substances, less than or equal to about 5% foreign substances, less than or equal to about 4% foreign substances, less than or equal to about 3% foreign substances, less than or equal to about 2% foreign substances, or less than or equal to about 1% foreign substances.

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

[0118] When applied to polypeptides, "isolated" generally means a polypeptide that has been separated from other naturally occurring proteins and nucleic acids. Preferably, the polypeptide is also separated from substances such as antibodies or gel matrices (polyacrylamide) used to purify it. In some cases, the term means a polypeptide or a part thereof that, due to its origin or manipulation, (i) exists in a host cell as an expression product of part of an expression vector, (ii) is bound to a protein or other chemical moiety other than those that are naturally bound, or (iii) does not occur naturally, and is chemically manipulated by adding or adding at least one hydrophobic moiety to a protein so that the protein is in a form not found naturally. "Isolated" further means a protein that is (i) chemically synthesized, or (ii) expressed in a host cell and purified and isolated from related proteins and contaminant proteins.

[0119] The term "effective dose," as used herein, refers to an amount of the antibody or its antigen-binding moiety described herein that is sufficient to induce a reaction, for example, when administered to a subject, to achieve the treatment, prognosis, or diagnosis of a disease related to macrophage activity or TAM activity as described herein. The therapeutically effective dose of the antibodies provided herein will vary depending on the relative activity of the antibody and the combination (for example, in inhibiting tumor cell growth) when used alone or in combination, as well as on the subject and condition being treated, the subject's weight and age, the severity of the condition, and, if applicable, the mode of administration as readily determined by those skilled in the art.

[0120] The term “therapeutically effective amount” generally refers to the amount of antibody or drug effective in “treating” a disease or disorder in a subject or mammal. In some embodiments, the compositions described herein are administered to a subject in an amount effective in producing some desired therapeutic effect by inhibiting the disease or disorder described herein with a reasonable benefit-risk ratio applicable to any medical treatment. A therapeutically effective amount is an amount that achieves at least partially the desired therapeutic or preventive effect in an organ or tissue. The amount of antibody required to perform preventive and / or therapeutic treatment for a disease or disorder is not fixed in itself. In some embodiments, the amount of antibody administered varies depending on the type of disease, the scalability of the disease, and the size of the mammal suffering from the disease or disorder. When the term “therapeutically effective” is used in conjunction with a treatment method that requires the administration of a therapeutic agent after the subject has developed symptoms of the disease or disorder, it means that after treatment, one or more signs or symptoms of the disease or disorder are improved or eliminated.

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

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

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

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

[0125] In some embodiments, the antibody disclosed herein is human CD163 + The antibodies bind to immunosuppressive myeloid cells and alter the expression of specific cellular markers that characterize M2 or M2-like immunosuppressive macrophages (such as M2c macrophages). This indicates functional differentiation of macrophages into non-immunosuppressive, low-immunosuppressive, or more antitumor states. In some embodiments, the antibodies disclosed herein bind to M2 or M2-like immunosuppressive macrophages and reduce the expression of specific cellular markers that characterize M2 or M2-like immunosuppressive macrophages. This indicates functional differentiation of macrophages into a modified differentiated state. In some embodiments, the antibodies disclosed herein are CD163 + It reduces the expression of one or more of CD16, CD64, TLR2, and Siglec-15 in immunosuppressive myeloid cells.

[0126] In some embodiments, the CD163 of the antibody disclosed herein+ Binding to immunosuppressive myeloid cells is CD163 + It induces functional changes in immunosuppressive myeloid cells. In some embodiments, the CD163 antibody disclosed herein + Binding to immunosuppressive myeloid cells leads to alterations in M2 or M2-like immunosuppressive macrophages. In some embodiments, antibody-bound CD163 + Functional changes in immunosuppressive myeloid cells lead to modifications in their interactions with other cells, such as effector T cells, which in turn modify their effects and interactions with target tumor cells.

[0127] In some embodiments, the antibodies of this disclosure reduce immunosuppression induced by tumor-associated macrophages in the tumor microenvironment. In some embodiments, the reduction in immunosuppression by tumor-associated macrophages in the tumor microenvironment corresponds to an increase in the facilitative production of immune stimulation, such as T cell activation, T cell proliferation, NK cell activation, NK cell proliferation, or any combination thereof. In some embodiments, T cell activation and / or NK cell activation results in increased production of IFN-γ, TNF-α, perforin, or combinations thereof by T cells and / or NK cells. In some embodiments, the antibodies of this disclosure increase the facilitative production of immune stimulation, such as T cell activation, T cell proliferation, NK cell activation, NK cell proliferation, or any combination thereof. In some embodiments, T cell activation and / or NK cell activation results in increased production of IFN-γ, TNF-α, perforin, or combinations thereof by T cells and / or NK cells. In some embodiments, the antibodies of this disclosure specifically bind to the CD163 protein expressed on human macrophages, and the human macrophages have a first immunosuppressive activity before binding to the antibody and a second immunosuppressive activity after binding to the antibody, the second immunosuppressive activity being lower than the first immunosuppressive activity. In various embodiments, the first and second immunosuppressive activities are not zero, respectively.

[0128] In some embodiments, the antibodies of this disclosure promote T cell activation and proliferation. In some embodiments, the antibodies distort the T cell population into an antitumor T cell phenotype. In some embodiments, the antibodies reduce or block myelocyte suppression of T cell activation. In some embodiments, the antibodies reduce the ability of TAM to suppress T cell activation, resulting in greater T cell stimulation and IL-2 production. In some embodiments, the antibodies block the ability of TAM to suppress T cell activation, resulting in greater T cell stimulation and IL-2 production.

[0129] In some embodiments, the antibodies disclosed herein reduce myelosuppression of T cell proliferation. In some embodiments, the antibodies are TAMs that are CD4 + and CD8 + It reduces the ability to suppress T cell activation and proliferation. In some embodiments, the antibody reduces TAM suppression of Th1 cell proliferation. Proliferated T cells are CD4 + This shows enhanced expression of activation markers on T cells.

[0130] In some embodiments, the antibodies of this disclosure modify M2-polarized macrophages so that they exhibit an M1-like phenotype that reduces the immunosuppressive effect of M2 macrophages. In some embodiments, the antibodies described herein affect monocyte-derived macrophages to differentiate them into a less immunosuppressive and more antitumor-like differentiated state.

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

[0132] In some embodiments, the antibodies disclosed herein bind to the CD163 protein expressed by macrophages as 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.

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

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

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

[0136] In some embodiments, the antibody disclosed herein is CD8 + It promotes T cell activity or proliferation. In some embodiments, the antibody is CD8 + It promotes the expression of ICOS, OX40, PD1, LAG3, or CTLA4 by T cells.

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

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

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

[0140] In certain embodiments of this specification, antibodies that specifically bind to the CD163 protein expressed on human M2 and M2-like macrophages are disclosed, and such binding has the following effects: (a) Decreased expression of at least one marker, which is CD16, CD64, TLR2, or Siglec-15, by human macrophages. (b) Internalization of antibodies by human macrophages, (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, (d)CD4 + T cells, CD8 + The proliferation of T cells, NK cells, or any combination thereof, as (e) Promotion of tumor cell death in the tumor microenvironment It brings about at least one of the following.

[0141] In certain embodiments of this specification, antibodies that specifically bind to the CD163 protein expressed on human M2 and M2-like macrophages are disclosed, and such binding has the following effects: (a) Decreased expression of at least one marker, which is CD16, CD64, TLR2, or Siglec-15, by human macrophages. (b) Internalization of antibodies by human macrophages, (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, (d)CD4 + T cells, CD8 + The proliferation of T cells, NK cells, or any combination thereof, as (e) Promotion of tumor cell death in the tumor microenvironment It brings about at least two of these.

[0142] In certain embodiments of this specification, antibodies that specifically bind to the CD163 protein expressed on human M2 and M2-like macrophages are disclosed, and such binding has the following effects: (a) Decreased expression of at least one marker, which is CD16, CD64, TLR2, or Siglec-15, by human macrophages. (b) Internalization of antibodies by human macrophages, (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, (d)CD4 + T cells, CD8 + The proliferation of T cells, NK cells, or any combination thereof, as (e) Promotion of tumor cell death in the tumor microenvironment It brings about at least three of these.

[0143] In certain embodiments of this specification, antibodies that specifically bind to the CD163 protein expressed on human M2 and M2-like macrophages are disclosed, and such binding has the following effects: (a) Decreased expression of at least one marker, which is CD16, CD64, TLR2, or Siglec-15, by human macrophages. (b) Internalization of antibodies by human macrophages, (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, (d)CD4 + T cells, CD8 + The proliferation of T cells, NK cells, or any combination thereof, as (e) Promotion of tumor cell death in the tumor microenvironment It brings about at least four of these.

[0144] In certain embodiments of this specification, antibodies that specifically bind to the CD163 protein expressed on human M2 and M2-like macrophages are disclosed, and such binding has the following effects: (a) Decreased expression of at least one marker, which is CD16, CD64, TLR2, or Siglec-15, by human macrophages. (b) Internalization of antibodies by human macrophages, (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, (d)CD4 + T cells, CD8 + The proliferation of T cells, NK cells, or any combination thereof, as (e) Promotion of tumor cell death in the tumor microenvironment It brings about at least five of these.

[0145] In some embodiments, the antibodies disclosed herein are used in a tumor-associated macrophage (TAM) population to control human CD163 + The antibody selectively binds to immunosuppressive myeloid cells, specifically binding to the CD163 protein expressed on M2 macrophages, thereby reducing the immunosuppressive activity of the TAM population.

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

[0147] In some embodiments, the antibodies of this disclosure include intact immunoglobulin molecules, such as human antibodies, as well as portions of humanized Ig molecules containing antigen-binding sites (i.e., paratopes) or single heavy and single light chains, which include portions known in the art, such as Fab, Fab', F(ab)', F(ab')2, Fd, scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, bispecific Fab2, triplicate Fab3, single-chain binding polypeptide, dAb fragment, diabody, and other antigen-binding fragments. When constructing an immunoglobulin molecule or fragment thereof, various regions or portions are, in some embodiments, fused, ligated, or otherwise conjugated to one or more constant regions or portions to produce any of the antibodies or fragments described herein. Therefore, in some embodiments, one of the antigen-binding fragments among the antibodies described above is Fab, Fab', Fd, F(ab')2, Fv, scFv, a single-chain binding polypeptide (e.g., scFv having an Fc portion), or other functional fragments as described herein.

[0148] In some embodiments, the antibody of this disclosure is of any immunoglobulin class and, therefore, in some embodiments, has a heavy chain of γ, μ, α, δ, or ε. In some embodiments, the γ chain is γ1, γ2, γ3, or γ4. In some embodiments, the α chain is α1 or α2.

[0149] In some embodiments, the antibody of the Disclosure is an IgG immunoglobulin. In some embodiments, the antibody of the Disclosure is an antibody of any IgG subclass. In some embodiments, the antibody is IgG1.

[0150] In some embodiments, the antibodies of this disclosure include a variable light chain that is either κ or λ. In some embodiments, the λ chain is one of the subtypes, for example, λ1, λ2, λ3, and λ4. In some embodiments, the light chain is κ.

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

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

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

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

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

[0156] In some embodiments, the antibody of the Disclosure comprises a human variable framework region and a mouse constant region. In some embodiments, the antibody of the Disclosure comprises a human heavy chain variable framework region and a mouse heavy chain constant region. In some embodiments, the antibody of the Disclosure comprises 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.

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

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

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

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

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

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

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

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

[0165] In some embodiments, the antibodies of this disclosure are bispecific or polyspecific. A bispecific antibody is an antibody that has binding specificity to at least two different epitopes. In some embodiments, an exemplary bispecific antibody binds to two different epitopes of a single antigen. Other such antibodies, in some embodiments, combine a first antigen-binding site with a binding site for a second antigen. In some embodiments, a bispecific antibody binds to at least two different epitopes and has a constant domain that binds to an Fc receptor. In some embodiments, the binding of one or more epitopes of a bispecific antibody occurs simultaneously with the binding of the constant domain of the bispecific antibody to an Fc receptor.

[0166] In some embodiments, the antibodies of this disclosure have two or more valencies, also referred to as polyvalent. In some embodiments, the antibodies of this disclosure are triply specific. In some embodiments, polyvalent antibodies are internalized (and / or catabolized) more rapidly than bivalent antibodies by cells expressing the antigen to which the antibody is bound. In some embodiments, the antibodies of this disclosure are polyvalent antibodies (e.g., tetravalent antibodies) having three or more antigen-binding sites. In some embodiments, the polyvalent antibodies of this disclosure are produced by recombinant expression of the nucleic acid encoding the polypeptide chain of the antibody. In some embodiments, the polyvalent antibody comprises a dimerization domain and three or more antigen-binding sites. In some embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody comprises an Fc region and three or more antigen-binding sites at the amino terminus of the Fc region. In some embodiments, the polyvalent antibodies of this specification comprise about 3 to about 8, preferably 4, antigen-binding sites. A polyvalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), and each polypeptide chain comprises two or more variable regions. For example, the polypeptide chain may be VD1-(X1) n -VD2-(X2) n-Includes Fc, where VD1 is the first variable region, VD2 is the second variable region, Fc is one polypeptide chain in the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. In some embodiments, the polypeptide chains are each independently V H -C H 1-Flexible Linker-V H -C H 1-Fc region chain, or V H -C H 1-V H -C H It includes a 1-Fc region chain. In some embodiments, the polyvalent antibody according to this specification further comprises at least two (preferably four) light chain variable region polypeptides. In some embodiments, the polyvalent antibody according to this specification comprises about two to about eight light chain variable region polypeptides. In some embodiments, the light chain variable region polypeptide described herein comprises a light chain variable region. In some embodiments, the light chain variable region polypeptide described herein comprises C L Includes domains as well.

[0167] In some embodiments, the antibodies of this disclosure are constructed to fold into a multivalent form, thereby improving, in some embodiments, binding affinity, specificity, and / or blood half-life. The multivalent form of the antibody is prepared, for example, by techniques known in the art.

[0168] In some embodiments, the antibodies of this disclosure are SMIP or binding domain immunoglobulin fusion proteins specific to a target protein. These constructs are single-chain polypeptides containing an antigen-binding domain that is fused to an immunoglobulin domain necessary to perform antibody effector function.

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

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

[0171] In some embodiments, the antibodies disclosed herein are monoclonal antibodies. In some embodiments, the antibodies disclosed herein are antigen-binding fragments. In some embodiments, the antibodies disclosed herein are selected from whole immunoglobulin, scFv, Fab, F(ab')2, or disulfide-bonded Fv. 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 human, humanized, or chimeric.

[0172] [Table 1]

[0173] In certain embodiments of this specification, antibodies comprising a light chain variable domain (VL) having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as SEQ ID NO: 7 are disclosed. 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 described as SEQ ID NO: 7. In some embodiments, the VL has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 7.

[0174] In certain embodiments of this specification, antibodies comprising a heavy chain variable domain (VH) having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as SEQ ID NO: 8 are disclosed. In some embodiments, 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 described as SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 8.

[0175] In certain embodiments of this specification, an antibody is disclosed comprising a light chain variable domain (VL) having an amino acid sequence at least about 70% identical to the amino acid sequence described as SEQ ID NO: 7, and a heavy chain variable domain (VH) having an amino acid sequence at least about 70% identical to the amino acid sequence described as SEQ ID NO: 8. In some embodiments, 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 described as SEQ ID NO: 7, and 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 described as SEQ ID NO: 8. In some embodiments, VL has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 7, and VH has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 8.

[0176] [Table 2-1]

[0177] [Table 2-2]

[0178] In certain embodiments of this specification, antibodies comprising a light chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as SEQ ID NO: 9 are disclosed. 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 described as SEQ ID NO: 9. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 9.

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

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

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

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

[0183] In certain embodiments of this specification, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as SEQ ID NO: 9, and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 10. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 10.

[0184] In certain embodiments of this specification, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as SEQ ID NO: 9, and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 11. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 11.

[0185] In certain embodiments of this specification, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as SEQ ID NO: 9, and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 12. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 12.

[0186] In certain embodiments of this specification, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as SEQ ID NO: 9, and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 13. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 13.

[0187] In certain embodiments of this specification, antibodies comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as SEQ ID NO: 14 are disclosed. 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 described as SEQ ID NO: 14. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 14.

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

[0189] In certain embodiments of this specification, antibodies comprising a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as SEQ ID NO: 16 are disclosed. 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 described as SEQ ID NO: 16. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 16.

[0190] In certain embodiments of this specification, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as SEQ ID NO: 14, and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 15. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 15.

[0191] In certain embodiments of this specification, an antibody is disclosed comprising a light chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as SEQ ID NO: 14, and a heavy chain having an amino acid sequence at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 16. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 16.

[0192] [Table 3]

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

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

[0195] In certain embodiments of this specification, an antibody is disclosed comprising at least one of the following: a light chain CDR1 having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as SEQ ID NO: 1; a light chain CDR2 having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as SEQ ID NO: 2; a light chain CDR3 having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as SEQ ID NO: 3; a heavy chain CDR1 having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 6.In some embodiments, the antibody that binds to CD163 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 described as SEQ ID NO: 1, and at least about 75%, 80%, 81%, 82%, 83%, 84% identical to the amino acid sequence described as SEQ ID NO: 2. Light chain CDR2 having an amino acid sequence identical to 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence, and light chain having an amino acid sequence identical to at least approximately 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described as SEQ ID NO: 3. Heavy chain CDR1 has an amino acid sequence that is at least approximately 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 described as CDR3, SEQ ID NO: 4. The material comprises at least one of the following: a heavy chain CDR2 having an amino acid sequence identical to 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; and a heavy chain CDR3 having an amino acid sequence identical to at least approximately 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described as SEQ ID NO: 6.In some embodiments, the antibody that binds to CD163 includes at least one of the following: a light chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence described as SEQ ID NO: 1; a light chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence described as SEQ ID NO: 2; a light chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence described as SEQ ID NO: 3; a heavy chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence described as SEQ ID NO: 4; a heavy chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence described as SEQ ID NO: 5; and a heavy chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence described as SEQ ID NO: 6.

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

[0197] The binding interaction is revealed as an intermolecular contact with one or more amino acid residues of one or more CDRs in some embodiments. Antigen binding is, for example, with a CDR or a CDR pair, or optionally with V H Chain and V L It requires the interaction of all six CDRs in the chain.

[0198] The binding affinity and binding activity of an antibody or antigen-binding fragment can be determined by surface plasmon resonance (SPR) assay, AlphaLisa assay, or equilibrium dissociation constant (K).D It can be measured by flow cytometry.

[0199] This specification includes K for concentrations from 0.1 nM to 1000 nM. D Antibodies that specifically bind to human CD163 are disclosed. This specification includes antibodies with concentrations of 0.1 to approximately 500 nM, approximately 0.1 to approximately 100 nM, approximately 0.1 to approximately 50 nM, approximately 0.1 to approximately 20 nM, approximately 0.1 to approximately 10 nM, approximately 0.1 to approximately 5 nM, approximately 0.1 to approximately 2 nM, approximately 0.1 to approximately 1 nM, approximately 0.1 to approximately 0.5 nM, approximately 0.5 to approximately 1000 nM, approximately 0.5 to approximately 500 nM, approximately 0.5 to approximately 100 nM, and approximately 0.5 to about 50nM, about 0.5 to about 20nM, about 0.5 to about 10nM, about 0.5 to about 5nM, about 0.5 to about 2nM, about 0.5 to about 1nM, about 1 to about 1000nM , about 1 to about 500 nM, about 1 to about 100 nM, about 1 to about 50 nM, about 1 to about 20 nM, about 1 to about 10 nM, about 1 to about 5 nM, about 1 to about 2 nM, about 2 to about 1000 nM, about 2 to about 500 nM, about 2 to about 100 nM, about 2 to about 50 nM, about 2 to about 20 nM, about 2 to about 10 nM, about 2 to about 5 nM, about 5 to about 1000 nM, about 5 ~about 500nM, about 5 to about 100nM, about 5 to about 50nM, about 5 to about 20nM, about 5 to about 10nM, about 10 to about 1000nM, about 10 to about 500nM, about 10 to about K in approximately 100 nM, 10 to 50 nM, 10 to 20 nM, 20 to 1000 nM, 20 to 500 nM, 20 to 100 nM, 20 to 50 nM, 50 to 1000 nM, 50 to 500 nM, 50 to 100 nM, 50 to 100 nM, 100 to 500 nM, 100 to 1000 nM, and 500 to 1000 nM D An antibody that specifically binds to human CD163 is disclosed. In some embodiments, the antibody is K163 at concentrations of 1.8 nM, 12 nM, 45 nM, or 89 nM. D Therefore, it specifically binds to human CD163.

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

[0201] This specification includes K for concentrations from 0.1 nM to 1000 nM. D Antibodies that specifically bind to M2c macrophages are disclosed. This specification includes antibodies with concentrations of 0.1 to approximately 500 nM, approximately 0.1 to approximately 100 nM, approximately 0.1 to approximately 50 nM, approximately 0.1 to approximately 20 nM, approximately 0.1 to approximately 10 nM, approximately 0.1 to approximately 5 nM, approximately 0.1 to approximately 2 nM, approximately 0.1 to approximately 1 nM, approximately 0.1 to approximately 0.5 nM, approximately 0.5 to approximately 1000 nM, approximately 0.5 to approximately 500 nM, approximately 0.5 to approximately 100 nM, and approximately 0.5 to about 50nM, about 0.5 to about 20nM, about 0.5 to about 10nM, about 0.5 to about 5nM, about 0.5 to about 2nM, about 0.5 to about 1nM, about 1 to about 1000nM , about 1 to about 500 nM, about 1 to about 100 nM, about 1 to about 50 nM, about 1 to about 20 nM, about 1 to about 10 nM, about 1 to about 5 nM, about 1 to about 2 nM, about 2 to about 1000 nM, about 2 to about 500 nM, about 2 to about 100 nM, about 2 to about 50 nM, about 2 to about 20 nM, about 2 to about 10 nM, about 2 to about 5 nM, about 5 to about 1000 nM, about 5 ~about 500nM, about 5 to about 100nM, about 5 to about 50nM, about 5 to about 20nM, about 5 to about 10nM, about 10 to about 1000nM, about 10 to about 500nM, about 10 to about K in approximately 100 nM, 10 to 50 nM, 10 to 20 nM, 20 to 1000 nM, 20 to 500 nM, 20 to 100 nM, 20 to 50 nM, 50 to 1000 nM, 50 to 500 nM, 50 to 100 nM, 50 to 100 nM, 100 to 500 nM, 100 to 1000 nM, and 500 to 1000 nM D An antibody that specifically binds to M2c macrophages is disclosed. In some embodiments, the antibody is 7.7 nM K D It specifically binds to M2c macrophages.

[0202] Binding epitope Antibody epitopes may consist of linear peptide sequences (i.e., "continuous") or discontinuous amino acid sequences (i.e., "conformations" or "discontinuous"). In some embodiments, the antibody recognizes one or more amino acid sequences, and therefore the epitope defines more than one distinct amino acid sequence. Epitopes recognized by antibodies are determined, for example, by peptide mapping and sequence analysis techniques well known to those skilled in the art. Binding interactions are revealed as intermolecular contacts with one or more amino acid residues of the CDR.

[0203]

[0204] This specification discloses antibodies that specifically bind to epitopes in human CD163. In some embodiments, the antibodies disclosed herein bind to epitopes comprising discontinuous amino acid sequences. In some embodiments, the antibody binds to a human CD163 epitope comprising the amino acid sequence IGRNVNASKGFGHIWLDSVSCQGHEPAI (SEQ ID NO: 18). In some embodiments, the antibody binds to a human CD163 epitope comprising the amino acid sequence VVCRQLGCGSA (SEQ ID NO: 19). In some embodiments, the antibody binds to a human CD163 epitope comprising the amino acid sequence WDCKNWQWGGLTCD (SEQ ID NO: 20). In some embodiments, the antibody binds to a human CD163 epitope comprising the amino acid sequences of SEQ ID NOs: 18-20.

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

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

[0207] In some cases, if the antigen-binding fragment does not contain an Fc moiety, the Fc moiety is added to the fragment (by recombination) to increase the half-life of the antigen-binding fragment in blood circulation, for example, when administered to a subject. The selection of an appropriate Fc region and methods for incorporating such fragments are known in the art. Incorporating the Fc region of IgG into a target polypeptide in a way that increases its circulating half-life without loss of its biological activity is achieved, for example, using conventional techniques in the art. In some embodiments, the Fc moiety of an antibody is further modified to increase the half-life of the antigen-binding fragment in blood circulation when administered to a subject. Modifications are determined, for example, using conventional means in the art.

[0208] In addition, in some embodiments, antibodies and their antigen-binding fragments are produced or expressed in such a way that they do not contain fucose on the N-glycosidic glycans that constitute their complex. Removing fucose from the N-glycosidic glycans of the complex is known to enhance the effector functions of the antibodies and antigen-binding fragments, including but not limited to antibody-dependent cytotoxicity (ADCC activity) and complement-dependent cytotoxicity (CDC activity). Similarly, antibodies or their antigen-binding fragments that bind to an epitope may optionally be attached at their C-terminus to all or part of an immunoglobulin heavy chain derived from an antibody isotype, e.g., IgG, IgA, IgE, IgD, and IgM, and an isotype subclass, specifically IgG1, IgG2, IgG3, and IgG4.

[0209] In addition, the antibodies or antigen-binding fragments described herein are modified in some embodiments to enable them to cross the blood-brain barrier. Such modifications of antibodies or antigen-binding fragments described herein enable the treatment of brain diseases such as glioblastoma multiforme (GBM). Exemplary modifications for enabling proteins such as antibodies or antigen-binding fragments to cross the blood-brain barrier are described in U.S. Patent Application Publication 2007 / 0082380.

[0210] Glycosylation of immunoglobulins has been shown to have a significant effect on their effector function, structural stability, and secretion rate from antibody-producing cells. The carbohydrate groups responsible for these properties are generally attached to the constant (C) region of antibodies. For example, C H Glycosylation of IgG at asparagine 297 within the domain is required for the full capacity of IgG to activate the classical pathway of complement-dependent cell lysis (Tao and Morrison, J Immunol 143:2595 (1989)). H Glycosylation of IgM at asparagine 402 in the 3 domains is required for proper antibody assembly and cytolytic activity (Muraoka and Shulman, J Immunol 142:695 (1989)). C of IgA antibody H 1 and C HRemoval of glycosylation sites at positions 162 and 419 in the three domains resulted in at least 90% inhibition of intracellular degradation and secretion (Taylor and Wall, Mol Cell Biol 8:4197 (1988)). In addition, in some embodiments, antibodies and their antigen-binding fragments are produced or expressed in such a way as not to contain fucose on the N-glycosidic glycans that form their complex. Removing fucose from the N-glycosidic glycans of the complex is known to increase the effector function of the antibody and antigen-binding fragment, including but not limited to antibody-dependent cytotoxicity (ADCC activity) and complement-dependent cytotoxicity (CDC activity). These "defucosylated" antibodies and antigen-binding fragments are produced in some embodiments by various systems utilizing molecular cloning techniques known in the art, including transgenic animals, transgenic plants, or cell lines (also known as fucosyltransferase knockout animals, plants, or cells) that have been genetically engineered to no longer contain the enzymes and biochemical pathways necessary to include fucose in the complex N-glycosidic glycans. Non-limiting examples of cells engineered to become fucosyltransferase knockout cells include CHO cells, SP2 / 0 cells, NS0 cells, and YB2 / 0 cells.

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

[0212] Glycosylation at variable domain framework residues may, in some cases, modify the antibody-antigen binding interaction. This disclosure includes criteria for selecting amino acids that are limited in number in the framework or CDR of a humanized immunoglobulin chain to be mutated (e.g., by residue substitution, deletion, or addition) to increase antibody affinity.

[0213] In some embodiments, cysteine ​​residues are removed or introduced into the Fc region of the antibody or Fc-containing polypeptide, thereby eliminating or increasing interchain disulfide bond formation in that region. Homodimer-specific binders or antibodies produced using such methods exhibit, in some embodiments, improved internalization ability, as well as / or increased complement-mediated cell death and antibody-dependent cytotoxicity (ADCC).

[0214] Sequences within the CDR have been shown to bind antibodies to MHC class II cells, potentially inducing undesirable helper T cell responses. In some embodiments, conservative substitutions allow the antibody to retain its binding activity, but reduce its ability to induce undesirable T cell responses. In one embodiment, one or more of the N-terminal 20 amino acids of the heavy or light chain are removed.

[0215] In some embodiments, antibody molecules are produced by modification of the carbohydrate structure resulting in improved effector activity, including antibody molecules with absent or reduced fucosylation that exhibits improved ADCC activity. Various methods for achieving this are known in the art. For example, ADCC effector activity is mediated by the binding of antibody molecules to the FcγRIII receptor, which is C HIt has been observed that the N-linked glycosylation at Asn-297 in the two domains is dependent on the carbohydrate structure. Non-fucosylated antibodies bind to this receptor with increased affinity and induce FcγRIII-mediated effector function more efficiently than naturally occurring fucosylated antibodies. Some host cell lines, such as Lec13 or the rat hybridoma YB2 / 0 cell line, spontaneously produce antibodies with low levels of fucosylation. For example, increasing the concentration of biconjugates by recombinant production of antibodies in cells overexpressing the GnTIII enzyme has also been shown to increase ADCC activity. In some embodiments, the absence of only one of the two fucose residues is sufficient to increase ADCC activity.

[0216] Covalent modifications of antibodies are also included herein. In some embodiments, covalent modifications are prepared, where applicable, by chemical synthesis or by enzymatic or chemical cleavage of antibodies. In some embodiments, other types of covalent modifications are introduced by reacting a targeted amino acid residue with an organic derivatizing agent that reacts with a selected side chain or N-terminal or C-terminal residue.

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

[0218] In some embodiments, the histidyl residue is derivatized by reaction with diethyl pyrocarbonate at pH 5.5–7.0 because this agent is relatively specific to the histidyl side chain. In some embodiments, para-bromophenacyl bromide is also useful. In some embodiments, the reaction is carried out at pH 6.0 in 0.1 M sodium cacodylate.

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

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

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

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

[0223] In some embodiments, glutaminyl and alparaginyl residues are deamidated to their corresponding glutamyl and alparthyl residues, respectively. These residues are deamidated under neutral or basic conditions.

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

[0225] Another type of covalent modification involves chemically or enzymatically attaching a glycoside to a specific binder or antibody. These procedures do not require the production of polypeptides or antibodies in host cells that have glycosylation ability for N-linked or O-linked glycosylation. Depending on the mode of attachment used, in some embodiments the sugar is attached to (a) arginine and histidine, (b) a free carboxyl group, (c) a free sulfhydryl group such as that of cysteine, (d) a free hydroxyl group such as that of serine, threonine, or hydroxyproline, (e) an aromatic residue such as that of phenylalanine, tyrosine, or tryptophan, or (f) an amide group of glutamine.

[0226] In some embodiments, the removal of any carbohydrate moieties present on polypeptides or antibodies is achieved chemically or enzymatically. Chemical deglycosylation involves exposure of the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This procedure leaves the antibody intact while resulting in the cleavage of most or all sugars except linking sugars (N-acetylglucosamine or N-acetylgalactosamine). In some embodiments, enzymatic cleavage of carbohydrate moieties on antibodies is achieved by the use of various endoglycosidases and exoglycosidases.

[0227] Another type of covalent modification involves attaching an antibody to one of various non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyethylated polyol, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylene, or polysaccharide polymers such as dextran. Such methods are known in the art.

[0228] The pre-determined binding affinity to polypeptide antigens is generally regulated by introducing one or more mutations into the V-region framework, typically in regions adjacent to one or more CDRs and / or one or more framework regions. Typically, such mutations involve the introduction of conserved amino acid substitutions that disrupt or create glycosylation site sequences but substantially do not affect the hydrophobic structural properties of the polypeptide. Typically, mutations introducing proline residues are avoided.

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

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

[0231] In one embodiment, the FcR is a naturally occurring human FcR. Furthermore, preferred FcRs are those that bind to IgG antibodies (gamma ("γ") receptors) and include the FcγI, FcγII, and FcγIII subclass receptors, as well as allele variants and alternatively spliced ​​forms of these receptors. The FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) within its cytoplasmic domain. The inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) within its cytoplasmic domain.

[0232] Antibody-dependent cell-mediated cytotoxicity, or ADCC, refers to a form of cytotoxicity in which secreted immunoglobulin (Ig) bound to Fc receptors (FcR) present on specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-containing target cells, subsequently killing them through cytotoxicity. Antibodies "arm" the cytotoxic cells and are necessary for such death. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. In some embodiments, in vitro ADCC assays are performed to evaluate the ADCC activity of a molecule of interest. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0233] Alternatively, or additionally, in some embodiments, the ADCC activity of the target molecule is evaluated in vivo, for example, in animal models.

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

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

[0236] In some embodiments, the antibodies or antigen-binding fragments provided herein are conjugated or bound to a therapeutic portion, an imaging or detectable portion, or an affinity tag. Methods for conjugating or binding polypeptides are well known in the art. Association (binding) of compounds with labels includes, but is not limited to, any means known in the art, including covalent and non-covalent interactions, chemical bonding, 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 manipulated by recombinantly using such a tag. Conventional tags in the art include, for example, polyhistidine (e.g., His6) tags.

[0237] In some embodiments, the antibody or antigen-binding fragment further comprises a detectable portion. Detection is achieved, for example, in vitro, in vivo, or ex vivo. For example, in vitro assays (quantification, qualification, etc.) for the detection and / or determination of the huCD163 protein expressed by macrophages using an antibody or its antigen-binding fragment include, but are not limited to, ELISA, RIA, and Western blotting. In some embodiments, in vitro detection, diagnosis, or monitoring of an antibody antigen is performed, for example, by taking a sample (e.g., a blood sample) from a subject in a standard ELISA assay and testing this sample.

[0238] In certain embodiments of this specification, compositions comprising the antibodies and carriers disclosed herein are also disclosed.

[0239] Pharmaceutical composition In certain embodiments of this specification, a pharmaceutical composition comprising an antibody disclosed herein and a pharmaceutically acceptable excipient is disclosed.

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

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

[0242] Pharmaceutical formulations comprising antibodies or antigen-binding fragments as specified herein are, in some embodiments, prepared for storage by mixing the protein of desired purity with an optional physiologically acceptable carrier, excipient, or stabilizer (e.g., Remington's Pharmaceutical Sciences, 16) th It is prepared in the form of a cryopreservation or aqueous solution (see edition, Osol, A. Ed. (1980)). Acceptable carriers or stabilizers are nontoxic to the recipient at the dose and concentration used, and include buffers such as phosphates, citrates, and other organic acids, antioxidants such as ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens such as methyl and propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum albumin, gelatin, or immunoglobulins. The composition may include proteins such as robulin, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as TWEEN®, PLURONIC®, or polyethylene glycol (PEG). In some embodiments, the pharmaceutical composition contains antibody at a concentration of 5 to 200 mg / mL, preferably 10 to 100 mg / mL.

[0243] An acceptable carrier is physiologically acceptable to the target to which it is administered and preserves the therapeutic properties of the compound being administered. Acceptable carriers and their formulations are generally described, for example, in Remington's Pharmaceutical Sciences mentioned above. One exemplary carrier is saline. The term "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or solvent, such as a liquid or solid filler, diluent, excipient, solvent, or mounting material, required for the transport or delivery of the compound of interest from one organ or body part to another, or for an in vitro assay system. Each carrier is acceptable in the sense that it is compatible with components of other formulations and is not harmful to the target to which it is administered. An acceptable carrier does not modify the specific activity of the compound of interest.

[0244] In another embodiment, the pharmaceutical compositions disclosed herein further include acceptable additives to improve the stability of compounds in the composition and / or to control the release rate of the composition. The acceptable additives do not modify the specific activity of the compound of interest. Exemplary acceptable additives include, but are not limited to, mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, and mixtures thereof. In some embodiments, the acceptable 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 and polysorbate 80 to increase peptide stability and reduce 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 during storage.

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

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

[0247] In another embodiment, the pH of the pharmaceutical composition disclosed herein is in the range of 4.5 to 6.5 or 4.5 to 5.5.

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

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

[0250] Antibody suspensions and crystalline forms are also intended herein. Methods for preparing suspensions and crystalline forms are known to those skilled in the art.

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

[0252] In some embodiments, freeze-drying is used to stabilize polypeptides for long-term storage, such as when polypeptides are relatively unstable in liquid compositions.

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

[0254] In some embodiments, for injection, the pharmaceutical compositions disclosed herein are powders suitable for reconstitution with suitable solutions as described above. Examples of these include, but are not limited to, lyophilized, tumble-dried, or spray-dried powders, amorphous powders, granules, precipitates, or fine particles. For injection, the compositions optionally contain stabilizers, pH adjusters, surfactants, bioavailability adjusters, and combinations thereof.

[0255] In some embodiments, sustained-release preparations are prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of molded articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (e.g., see U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow for molecular release over periods of 100 days or more, while certain hydrogels release proteins over shorter periods. In some embodiments, encapsulated antibodies remain in the body for extended periods, but exposure to water at 37°C leads to denaturation or aggregation, resulting in loss of biological activity and, in some cases, alteration of immunogenicity. Reasonable strategies devised for stabilization depend, in some cases, on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular SS bond formation via thio-disulfide exchange, stabilization may be achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, adjusting water content, using appropriate additives, and developing specific polymer matrix compositions.

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

[0257] Pharmaceutical compositions are administered by injection, including but not limited to subcutaneous, intravitreous, intradermal, intravenous, intra-arterial, intraperitoneal, intracerebrospinal, or intramuscular injection. Excipients and carriers used in the formulation of each type of injectable composition are contemplated herein. The following description is merely illustrative and not intended to limit the range of compositions. Injectable compositions include, but are not limited to, aqueous solutions (if water-soluble) or dispersions, as well as sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In some embodiments, the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Fluidity is maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Examples of antimicrobial and antifungal agents include parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In some embodiments, isotonic agents, such as sugars, mannitol, sorbitol, and polyalcohols such as sodium chloride, are included in the composition. In some embodiments, the resulting solution is packaged for immediate use or lyophilized. In some embodiments, the lyophilized preparation is later combined with a sterile solution before administration. For intravenous injection or injection at a painful site, the active ingredient is in the form of a parenterally-tolerable aqueous solution that is pyrogenic and has appropriate pH, isotonicity, and stability. Those skilled in the art can prepare a suitable solution using an isotonic vehicle, such as sodium chloride injection, Ringer's injection, and lactate Ringer's injection. In some embodiments, preservatives, stabilizers, buffers, antioxidants, and / or other additives are included as needed.In some embodiments, sterile injection solutions are prepared by incorporating the required amount of active ingredient in a suitable solvent along with one or a combination of the components listed above, followed by sterile filtration as necessary. Generally, dispersions are prepared by incorporating the active ingredient into a sterile solvent containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield a powder of the active ingredient with the desired additional components added to the previously sterile-filtered solution.

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

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

[0260] In one embodiment, the pharmaceutical compositions disclosed herein are freeze-dried, for example, to increase their shelf life during storage. When the compositions are considered for use in the pharmaceuticals or methods provided herein, in some embodiments the compositions are substantially free of pyrogens so as not to cause inflammatory or dangerous allergic reactions when administered to human subjects. Testing of compositions for pyrogens and the preparation of compositions substantially free of pyrogens are well understood by those skilled in the art and, in some embodiments, are achieved using commercially available kits.

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

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

[0263] In some embodiments, this disclosure provides the use of compositions described herein for producing agents for treating diseases, disorders, or disabilities described herein. In some embodiments, the agents are formulated based on the physical characteristics of the subject requiring treatment and are formulated in one or more formulations based on the stage of the disease, disorder, or disability. In some embodiments, the agents are packaged in appropriate packaging with appropriate labels for distribution to hospitals and clinics, the labels relating to instructions for treatment of subjects suffering from the diseases described herein. In some embodiments, the agents are packaged as one or more units. Dosage and administration instructions for the compositions are included in some embodiments with the packaging as described below. This disclosure further covers agents comprising antibodies or antigen-binding fragments described herein and pharmaceutically acceptable carriers.

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

[0265] In some embodiments, when formulated for administration to human subjects, the composition is formulated to be pyrogenic. Testing of compositions for pyrogenic substances and the preparation of pyrogenic-free pharmaceutical compositions are well understood by those skilled in the art.

[0266] In some embodiments, the antibody or its antigen-binding fragment is formulated for any suitable route of administration to the subject, including but not limited to injection. Injections include, for example, subcutaneous injection, peritoneal injection, intravenous injection, intramuscular injection, or spinal injection into cerebrospinal fluid (CSF). In some embodiments, administration is carried out at one, two, three, four, five, six, seven, or more injection sites. In one embodiment, administration is carried out via six injection sites.

[0267] In in vivo applications, contact is achieved, for example, by administration of a composition (as described herein) to a subject by any suitable means. In some embodiments, the antibodies described herein are administered by suitable means such as systemic or topical administration, including parenteral administration, subcutaneous administration, intraperitoneal administration, intracerebrospinal administration, intrapulmonary administration, and intranasal administration, and, if necessary, by topical treatment, intrafocal administration. Parenteral routes include, for example, intravenous administration, intra-arterial administration, intraperitoneal administration, epidural administration, intramuscular administration, and intrathecal administration. In some embodiments, such administration is performed as a bolus injection, continuous injection, or pulse injection. In some embodiments, the composition is administered by injection, depending in part whether the administration is short-term or chronic. Other methods of administration are considered, including topical administration, particularly transdermal administration, transmucosal administration, rectal administration, oral administration, or topical administration, such as administration via a catheter placed near the desired site.

[0268] Treatment and Usage Instructions In certain embodiments of this specification, a method for treating cancer in an individual as needed is disclosed, the method comprising the step of administering an antibody disclosed herein to the individual. In some embodiments, this disclosure provides the use of an antibody as disclosed herein in the manufacture of a drug for treating cancer in a human subject. In some embodiments, the antibody specifically binds to the CD163 protein expressed on human tumor-associated macrophages, thereby reducing the expression of at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophages.

[0269] In certain embodiments of this specification, a method for modulating the immune activity of a target subject of interest is disclosed, the method comprising the step of administering an antibody described herein to the target subject. In some embodiments, the antibody specifically binds to the CD163 protein expressed on human tumor-associated macrophages, thereby reducing the expression of at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophages.

[0270] In certain embodiments of this specification, a method is disclosed for treating a subject in which the level of M2 macrophages is pathologically or inappropriately elevated (e.g., inappropriately elevated compared to a level useful for promoting immune-mediated tumor cell death in the subject), the method comprising the step of 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 the macrophages.

[0271] In some embodiments, the antibody comprises at least one of the following: light chain CDR1 having an amino acid sequence identical to at least about 70% of the amino acid sequence described as SEQ ID NO: 1; light chain CDR2 having an amino acid sequence identical to at least about 70% of the amino acid sequence described as SEQ ID NO: 2; and light chain CDR3 having an amino acid sequence identical to at least about 70% of the amino acid sequence described as SEQ ID NO: 3. In some embodiments, the antibody comprises light chain CDR1 having an amino acid sequence identical to 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% of the amino acid sequence described as SEQ ID NO: 2; and light chain CDR1 having an amino acid sequence identical to at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, The antibody comprises at least one of the following: a light chain CDR2 having an amino acid sequence identical to 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; and a light chain CDR3 having an amino acid sequence identical to 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% of the amino acid sequence described as SEQ ID NO: 3. In some embodiments, the antibody comprises at least one of the following: a light chain CDR1 having an amino acid sequence identical to 100% of the amino acid sequence described as SEQ ID NO: 1; a light chain CDR2 having an amino acid sequence identical to 200% of the amino acid sequence described as SEQ ID NO: 2; and a light chain CDR3 having an amino acid sequence identical to 300% of the amino acid sequence described as SEQ ID NO: 3.

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

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

[0274] In some embodiments, the antibody includes a light chain variable domain (VL) having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as SEQ ID NO: 7. 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 described as SEQ ID NO: 7. In some embodiments, the VL has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 7.

[0275] In some embodiments, the antibody includes a heavy chain variable domain (VH) having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as SEQ ID NO: 8. In some embodiments, 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 described as SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 8.

[0276] In some embodiments, the antibody comprises a light chain variable domain (VL) having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as SEQ ID NO: 7, and a heavy chain variable domain (VH) having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as SEQ ID NO: 8. In some embodiments, 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 described as SEQ ID NO: 7, and 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 described as SEQ ID NO: 8. In some embodiments, VL has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 7, and VH has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 8.

[0277] In some embodiments, the antibody includes a light chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as 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 described as SEQ ID NO: 9. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 9.

[0278] 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 described as 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 described as SEQ ID NO: 10. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 10.

[0279] 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 described as 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 described as SEQ ID NO: 11. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 11.

[0280] 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 described as 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 described as SEQ ID NO: 12. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 12.

[0281] 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 described as 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 described as SEQ ID NO: 13. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 13.

[0282] 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 described as SEQ ID NO: 9, and a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 10. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 10.

[0283] 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 described as SEQ ID NO: 9, and a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 11. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 11.

[0284] 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 described as SEQ ID NO: 9, and a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 12. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 12.

[0285] 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 described as SEQ ID NO: 9, and a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 13. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 9, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 13.

[0286] In some embodiments, the antibody includes a light chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as 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 described as SEQ ID NO: 14. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 14.

[0287] 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 described as 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 described as SEQ ID NO: 15. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 15.

[0288] 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 described as 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 described as SEQ ID NO: 16. In some embodiments, the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 16.

[0289] 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 described as SEQ ID NO: 14, and a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 15. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 15.

[0290] 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 described as SEQ ID NO: 14, and a heavy chain having an amino acid sequence that is at least about 70% identical to the amino acid sequence described as 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 described as 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 described as SEQ ID NO: 16. In some embodiments, the light chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 14, and the heavy chain has an amino acid sequence that is 100% identical to the amino acid sequence described as SEQ ID NO: 16.

[0291] In some embodiments, this disclosure provides the use of antibodies described herein in the manufacture of agents that reduce immunosuppression by tumor-associated macrophages in human cancer subjects.

[0292] In some embodiments, this disclosure provides the use of the antibodies described herein in the manufacture of agents that promote T cell-mediated tumor cell death in human subjects with cancer.

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

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

[0295] In certain embodiments of this specification, a method for functionally reorienting tumor-associated macrophages to reduce immunosuppression in cancer patients is disclosed, the method being used to reorient CD4 in the tumor microenvironment. + or CD8 + The process includes administering a certain amount of a pharmaceutical composition containing an antibody described herein that is effective in improving T cell activity or proliferation to a patient.

[0296] In certain embodiments of this specification, a method is disclosed for promoting lymphocyte-mediated tumor cell death in a human subject of interest, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody described herein.

[0297] In certain embodiments of this specification, a method is disclosed for modulating the activity of tumor-associated macrophages in the tumor microenvironment, the method comprising the step of contacting tumor-associated macrophages with an antibody disclosed herein, the method having the following effects: (a) Decreased expression of at least one marker, which is CD16, CD64, TLR2, or Siglec-15, by human macrophages. (b) Internalization of antibodies by human macrophages, (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, (d)CD4 + T cells, CD8 + The proliferation of T cells, NK cells, or any combination thereof, as (e) Promotion of tumor cell death in the tumor microenvironment It brings about at least one of the following.

[0298] In certain embodiments of this specification, a method is disclosed for modulating the activity of tumor-associated macrophages in the tumor microenvironment, the method comprising the step of contacting tumor-associated macrophages with an antibody disclosed herein, the method having the following effects: (a) Decreased expression of at least one marker, which is CD16, CD64, TLR2, or Siglec-15, by human macrophages. (b) Internalization of antibodies by human macrophages, (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, (d)CD4 + T cells, CD8 + The proliferation of T cells, NK cells, or any combination thereof, as (e) Promotion of tumor cell death in the tumor microenvironment It brings about at least two of these.

[0299] In certain embodiments of this specification, a method is disclosed for modulating the activity of tumor-associated macrophages in the tumor microenvironment, the method comprising the step of contacting tumor-associated macrophages with an antibody disclosed herein, the method having the following effects: (a) Decreased expression of at least one marker, which is CD16, CD64, TLR2, or Siglec-15, by human macrophages. (b) Internalization of antibodies by human macrophages, (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, (d)CD4 + T cells, CD8 + The proliferation of T cells, NK cells, or any combination thereof, as (e) Promotion of tumor cell death in the tumor microenvironment It brings about at least three of these.

[0300] In certain embodiments of this specification, a method is disclosed for modulating the activity of tumor-associated macrophages in the tumor microenvironment, the method comprising the step of contacting tumor-associated macrophages with an antibody disclosed herein, the method having the following effects: (a) Decreased expression of at least one marker, which is CD16, CD64, TLR2, or Siglec-15, by human macrophages. (b) Internalization of antibodies by human macrophages, (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, (d)CD4 + T cells, CD8 + The proliferation of T cells, NK cells, or any combination thereof, as (e) Promotion of tumor cell death in the tumor microenvironment It brings about at least four of these.

[0301] In certain embodiments of this specification, a method is disclosed for modulating the activity of tumor-associated macrophages in the tumor microenvironment, the method comprising the step of contacting tumor-associated macrophages with an antibody disclosed herein, the method having the following effects: (a) Decreased expression of at least one marker, which is CD16, CD64, TLR2, or Siglec-15, by human macrophages. (b) Internalization of antibodies by human macrophages, (c)CD4 + T cells, CD8 + Activation of T cells, NK cells, or any combination thereof, (d)CD4 + T cells, CD8 + The proliferation of T cells, NK cells, or any combination thereof, as (e) Promotion of tumor cell death in the tumor microenvironment It brings about at least five of these.

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

[0303] Macrophage polarization from M2 to M1 refers to the process by which an M2 or M2-like macrophage is modified such that the resulting macrophage has certain functional or phenotypic characteristics 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.

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

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

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

[0307] Any of the methods disclosed herein further include, in some examples, the step of administering additional anticancer therapy to the subject. Anticancer therapy includes, but is not limited to, surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, and cytokine therapy, as well as combinations thereof. In one embodiment, the antibody or its antigen-binding fragment and the anticancer therapy are administered simultaneously or sequentially.

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

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

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

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

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

[0313] In some embodiments, the Disclosure provides a method for reducing myelocyte suppression of T cell activation. In some embodiments, the Method induces increased IL-2 production by T cells. In some embodiments, the Disclosure provides a method for increasing Th1 cell proliferation. In some embodiments, the Disclosure provides a method for increasing the proportion of Th1 cells in T cell proliferation. In each of these embodiments, the Method comprises the steps of administering an antibody described herein in vivo, or contacting an antibody with a combined immune cell system in vivo or ex vivo, wherein macrophages, effector T cells, and optionally selected tumor target cells are enabled to interact in a manner that replicates or models in vivo interactions.

[0314] In some embodiments, the present disclosure provides a method for reducing myelocyte suppression of T cell proliferation. In some embodiments, the present disclosure provides a method for CD4 + Cell proliferation or CD8 + This invention provides a method for increasing T cell proliferation, or both.

[0315] In some embodiments, the disclosure provides a method for increasing the expression of at least one T cell from among CD69, ICOS, OX40, PD-1, LAG3, and CTLA4 in patients who require it. In some embodiments, the method involves CD4 + The method increases the expression of at least one of the following T cells: CD69, ICOS, OX40, PD-1, LAG3, and CTLA4. In some embodiments, the method increases the expression of CD8 +This increases the expression of at least one of ICOS, OX40, PD-1, LAG3, and CTLA4 by T cells.

[0316] In some embodiments, the disclosure provides methods for increasing cancer cell death. In some embodiments, cancer cell death by cytotoxic lymphocytes (CTLs) is increased. In some embodiments, T cell-mediated death of MHC-mismatched cancer cells is increased.

[0317] In some embodiments, this disclosure relates to CD8 + T cell activation or CD8 + A method is provided for contacting bone marrow cells with the antibody described herein in order to reduce macrophage-mediated suppression of T cell proliferation. In some embodiments, the antibody is contacted with bone marrow cells containing M0 macrophages. In some embodiments, the antibody is contacted with bone marrow cells containing M2 macrophages. In some embodiments, the antibody is contacted with bone marrow cells containing both M0 and M2 macrophages.

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

[0319] An effective response in this disclosure is achieved when the subject experiences partial or overall relief or reduction of the signs or symptoms of the disease, and in the case of cancer treatment, specifically includes, but is not limited to, cure, remission, extended survival, or other objective responses. In some embodiments, expected progression-free survival is measured over several months to several years, depending on prognostic factors including the number of relapses, disease stage, and other factors. Extended survival includes, but is not limited to, times such as at least one month (mo.), at least about two months, at least about three months, at least about four months, at least about six months, at least one year, at least two years, or at least three years. Overall survival is also measured over several months to several years, for example. Alternatively, in some embodiments, an effective response is when the subject's symptoms remain static. Further indicators of treatment for indications are detailed below.

[0320] In some embodiments, the administration of therapeutic agents in preventive methods is carried out before the symptoms of the undesirable disease or disorder appear, thereby preventing the disease or disorder or, alternatively, delaying its progression. For this reason, when used in conjunction with preventive methods, the term “therapeutically effective” means that, on average, fewer subjects develop the undesirable disease or disorder or experience a progression in the severity of their symptoms after the treatment.

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

[0322] In some embodiments, the antibodies and antigen-binding fragments described herein are administered to a subject in various doses over various timeframes. Non-limiting doses include approximately 0.01 mg / kg, approximately 0.05 mg / kg, approximately 0.1 mg / kg, approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 20 mg / kg, approximately 30 mg / kg, approximately 40 mg / kg, approximately 50 mg / kg, approximately 60 mg / kg, approximately 70 mg / kg, approximately 80 mg / kg, approximately 90 mg / kg, approximately 100 mg / kg, approximately 125 mg / kg, approximately 150 mg / kg, approximately 175 mg / kg, approximately 200 mg / kg, or any integer between these. In addition, in some embodiments, the administration of antibodies or antigen-binding fragments is carried out twice a week, weekly, every two weeks, every three weeks, every four weeks, every six weeks, every eight weeks, every twelve weeks, or a combination of these weekly intervals. For example, a treatment cycle in which an antibody or its antigen-binding fragment is administered once or twice a week for four weeks, followed by a two-week break from treatment, is also envisioned. Additional treatment cycles, including different combinations of doses and weekly cycles as described herein, are also envisioned within the scope of this disclosure.

[0323] In some embodiments, the therapeutically effective dose of the composition varies and depends on the severity of the disease, as well as the body weight and general condition of the subject being treated, but is generally in the range of approximately 1.0 μg / kg to approximately 100 mg / kg, approximately 10 μg / kg to approximately 30 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, or approximately 1 mg / kg to approximately 10 mg / kg per application. Dosage may be daily, every other day, weekly, twice a month, monthly, or more or less frequently, depending on the response to the disorder or condition and the subject's tolerance to treatment. In some embodiments, maintenance administration for longer periods, such as 4, 5, 6, 7, 8, 10, or 12 weeks or more, is required until suppression of the desired disorder symptoms occurs, and the dose is adjusted as needed. The progress of this therapy is readily monitored by conventional techniques and assays.

[0324] In some embodiments, the antibodies of the present disclosure are administered intravenously in a physiological solution at a frequency of daily, weekly, or monthly (e.g., daily, every other day, every three days, or two, three, four, five, or six times a week) in doses ranging from 0.01 mg / kg to 100 mg / kg, preferably two or three times a week in doses ranging from 0.1 to 45 mg / kg, 0.1 to 15 mg / kg, or 0.1 to 10 mg / kg, or once a month at a maximum of 45 mg / kg.

[0325] A response is achieved when the subject experiences partial or complete relief or reduction of the signs or symptoms of the disease, specifically including, but not limited to, an extension of survival. Expected progression-free survival is measured over several months to several years, depending on prognostic factors, including, for example, the number of relapses, the stage of the disease, and other factors. An extension of survival may include, but is not limited to, at least one month (mo), at least about two months (mos.), at least about three mos., at least about four mos., at least about six mos., at least one year, at least two years, at least three years, or longer. In some embodiments, overall survival is also measured over several months to several years. In some embodiments, the subject's symptoms remain static or decrease.

[0326] Depending on the circumstances, a physician or veterinarian, including those skilled in the art, may determine the effective amount (ED) of the required composition. 50 This allows for easy determination and prescription. For example, a physician or veterinarian can start administering a compound utilized in the composition at a lower level than necessary to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. Alternatively, in some embodiments, the dose remains constant.

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

[0328] In some embodiments, the toxicity and therapeutic efficacy of such components are, for example, LD 50 (Lethal dose for 50% of the population) and ED 50 To determine the therapeutically effective dose (50% of the population), standard pharmaceutical procedures involving cell cultures or experimental animals are used. In some embodiments, the dose-to-toxicity ratio is the therapeutic index, which is the LD50. 50 / ED 50 It is expressed as a ratio. In some embodiments, compounds that exhibit toxic side effects are used, but care must be taken to design a delivery system that targets such compounds to the affected tissue site in order to minimize the damage that may occur to healthy cells and thereby reduce side effects.

[0329] Data obtained from cell culture assays and animal studies are used, in some embodiments, to formulate dose ranges for human use. The doses of such compounds are based on EDs with little to no toxicity. 50 It is preferable that the circulating concentration is within the range of the compound. In some embodiments, the dose varies within this range depending on the dosage form used and the route of administration used. For any compound used in the methods of this disclosure, the therapeutically effective dose is first estimated from a cell culture assay in some embodiments. In some embodiments, the dose is determined in the cell culture as IC 50 The drug is formulated in animal models to achieve a circulating plasma concentration sequence (i.e., the concentration of the test compound that achieves maximum half-volume inhibition). Plasma levels are measured, for example, by high-performance liquid chromatography. In some cases, such information is used to more accurately determine an effective dosage in humans.

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

[0331] Diagnostic products and methods In some embodiments, methods for detecting huCD163 protein or M2 macrophages in a sample or subject are disclosed for evaluating the treatment status of a patient or for diagnosing a disease or disorder associated with or correlated with the activity of M2 macrophages or TAMs, such as the diseases and disorders described herein.

[0332] In in vivo detection, diagnosis, or monitoring of soluble huCD163 protein, expression of huCD163 protein by cells or tissues, or the presence or activity of M2 macrophages, the subject is administered an antibody or antigen-binding fragment described herein, the antibody or antigen-binding fragment conjugated to the detectable portion. In some embodiments, the detectable portion is visualized using methods recognized in the art, including but not limited to magnetic resonance imaging (MRI), fluorescence, radioimaging, endoscopy, laparoscopy, or intravascular catheterization (i.e., via detection of a photoactivator), light sources, optical scanning, positron emission tomography (PET) scanning, whole-body magnetic resonance (NMR), radioscintigraphy, single-photon emission computed tomography (SPECT), targeted near-infrared (NIR) scanning, X-ray, and ultrasound. Labels for detecting compounds using such methods are also known in the art. In some embodiments, visualization of the detectable portion enables the detection, diagnosis, and / or monitoring of diseases or disorders related to M2 macrophage activity or the activity of other cells expressing huCD163 protein. Additional diagnostic assays utilizing antibodies specific to a desired target protein, namely the huCD163 protein, are known in the art and are also intended herein.

[0333] In in vitro detection methods, samples obtained from the target include, but are not limited to, blood, tissue biopsy samples, and fluids derived therefrom.

[0334] Therefore, this disclosure provides antibodies and antigen-binding fragments thereof that are useful for detecting or diagnosing the level of M2 macrophages or TAM macrophages associated with a disease or disorder, and which may indicate the need for therapeutic intervention. In other embodiments, the antibody further comprises a second agent. In some embodiments, such agent is a molecule or part, such as a reporter molecule or a detectable label. Detectable labels / parts for such detection methods are known in the art and are described in detail below. A reporter molecule is any part that is detected using, for example, an assay. Non-limiting examples of reporter molecules conjugated to polypeptides include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, biotin, and other colored particles or ligands. In some embodiments, a detectable label comprises a compound and / or element that is detected due to its specific functional and / or chemical properties, and its use allows the polypeptide to which they are attached to be detected and / or further quantified, if desired. Many suitable detectable (imaging) agents are known in the art, as are methods for their attachment to polypeptides.

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

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

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

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

[0339] The antibody and antigen-binding fragment compositions described herein may also be used as non-therapeutic agents (e.g., affinity purifiers) in some embodiments.

[0340] antibody technology As will be understood by those skilled in the art, the overall description herein of antibodies, as well as methods for preparing and using them, also applies to individual antibody polypeptide components and antibody fragments.

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

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

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

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

[0345] In some embodiments, human antibodies are produced in transgenic animals (e.g., mice) capable of producing the entire repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, the antibody heavy chain binding region (J) of chimeric and germline mutant mice. HIt has been reported that homozygous deletion of the gene results in complete inhibition of endogenous antibody production. Transfer of a human germline immunoglobulin gene array to such germline mutant mice results in the production of human antibodies upon antigen challenge. See, for example, 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. Patents 5,545,806, 5,569,825, 5,591,669, 5,545,807, and WO97 / 17852. In some embodiments, such animals are genetically engineered to produce human antibodies comprising the polypeptides of this disclosure.

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

[0347] As described above, this disclosure further provides antibody fragments. In certain situations, there are advantages to using antibody fragments rather than the whole antibody. For example, smaller fragments allow for faster clearance, which in some embodiments improves access to specific 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 polyspecific antibodies formed from antibody fragments.

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

[0349] In other embodiments, the antibody of choice is a single-stranded Fv fragment (scFv). See WO93 / 16185, U.S. Patents 5,571,894 and 5,587,458. Fv and sFv are the only species that possess an intact binding site lacking a constant region. For this reason, they are suitable for reduced nonspecific binding during in vivo use. In some embodiments, the sFv fusion protein is constructed to result in the fusion of an effector protein at either the amino or carboxyl terminus of the sFv. See Antibody Engineering, ed. Borrebaeck, mentioned above. In some embodiments, the antibody fragment is also a “linear antibody,” such as described in U.S. Patent 5,641,870, for example. In some embodiments, such a linear antibody fragment is monospecific or bispecific.

[0350] Methods for producing bispecific or other multispecific antibodies are known in the art and include chemical crosslinking, the 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)).

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

[0352] According to another method, an antibody variable region (antibody-antigen binding site) with desired binding specificity is fused to the immunoglobulin constant domain sequence. Preferably, this fusion is at least a portion of the hinge, C H 2, and C H This is performed on the Ig heavy chain constant domain, which includes 3 regions. The first heavy chain constant region (C) contains the site necessary for light chain binding. H 1) is preferably present in at least one of the fusions. The immunoglobulin heavy chain fusion and, optionally, the DNA encoding the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into suitable host cells. This provides greater flexibility in adjusting the ratios of the four polypeptide fragments to each other in embodiments where an unequal ratio of the four polypeptide chains used in the construct yields the desired bispecific antibody at optimal yield. However, when the expression of at least two polypeptide chains at equal ratio yields a high yield, or when this ratio has no significant effect on the yield of the desired chain combination, it is possible to insert coding sequences for two or all four polypeptide chains into a single expression vector.

[0353] A bispecific antibody is composed, for example, of a hybrid immunoglobulin heavy chain with primary bispecificity on one arm and a hybrid immunoglobulin heavy-light chain pair (resulting in secondary binding specificity) on the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain on only half of the bispecific molecule provides an easy separation method. This technique is disclosed in WO94 / 04690. For further details on bispecific antibody production, see, for example, Suresh et al., Methods Enzymol 121:210 (1986).

[0354] According to another method described in U.S. Patent No. 5,731,168, in some embodiments, the interface between antibody molecule pairs is manipulated to maximize the proportion of heterodimers recovered from recombinant cell cultures. A preferred interface is C H This method involves at least a portion of three domains. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). At the interface of the second antibody molecule, a compensatory "cavity" of the same or similar size as the larger side chain is formed by replacing the larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.

[0355] Bispecific antibodies include crosslinked or "heteroconjugated" antibodies, for example, in which one antibody in the heteroconjugation is bound to avidin and the other to biotin. Such antibodies have been proposed, for example, for targeting immune system cells against unwanted cells (U.S. Patent No. 4,676,980) and for treating HIV infection (WO91 / 00360, WO92 / 20373, and EP03089). In some embodiments, heteroconjugated antibodies are prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 along with many crosslinking techniques. Another method is designed to prepare a tetramer by adding a streptavidin coding sequence at the C-terminus of scFv. Since streptavidin is composed of four subunits, when scFv-streptavidin folds, the four subunits associate to form a tetramer (Kipriyanov et al., Hum Antibodies Hybridomas 6(3):93-101(1995)).

[0356] According to another method for producing bispecific antibodies, in some embodiments, the interface between antibody molecule pairs is manipulated to maximize the proportion of heterodimers recovered from recombinant cell cultures. One interface is the C of the antibody constant domain. H It comprises at least a portion of three domains. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). At the interface of the second antibody molecule, a compensatory "cavity" of the same or similar size as the larger side chain is formed by replacing the larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). This provides a mechanism to increase the yield of heterodimers over other unwanted end products such as homodimers. See WO96 / 27011.

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

[0358] Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically bound to form bispecific antibodies. Shalaby et al., J Exp Med 175:217-25 (1992) describe the production of the humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was secreted separately from E. coli and subjected to targeted chemical binding in vitro to form bispecific antibodies. These bispecific antibodies bound to the ErbB2 receptor and cells overexpressing normal human T cells, and were also able to induce lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0359] Various techniques for directly producing and isolating bispecific antibody fragments from recombinant cell cultures are also described. For example, bispecific antibodies have been produced using leucine zippers. See Kostelny et al., J Immunol 148(5):1547-53 (1992). Leucine zipper peptides derived from Fos and Jun proteins were conjugated to the Fab' portion of two different antibodies by gene fusion. The antibody homodimer was reduced at the hinge region to form a monomer, which was then reoxidized to form an antibody heterodimer. This method is used in some embodiments to produce antibody homodimers.

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

[0361] In certain embodiments of the method described herein, B cells isolated from peripheral blood or lymph nodes are sorted, for example, based on being CD19-positive and seeded in configurations of, for example, 96, 384, or 1536 wells, at a level comparable to, for example, single-cell specificity per well. The cells are induced to differentiate into antibody-producing cells, such as plasma cells, and the culture supernatant is collected and tested for binding to cells expressing a target polypeptide on its surface, for example, using FMAT or FACS analysis. The positive wells are then subjected to whole-well RT-PCR to amplify the heavy and light chain variable regions of the IgG molecule expressed by the cloned daughter plasma cells. The resulting PCR product, encoding the heavy and light chain variable regions, or a portion thereof, is subcloned into a human antibody expression vector for recombinant expression. The resulting recombinant antibody is then tested to confirm its intrinsic binding specificity and, in some embodiments, is further tested for cross-reactivity to other cells or proteins.

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

[0363] In some embodiments, polynucleotides encoding the antibodies or portions thereof of the present disclosure are isolated from antibody-expressing cells by methods available in the art and described herein. These methods include amplification by polymerase chain reaction using primers specific to the conserved regions of the human antibody polypeptide. For example, the light and heavy chain variable regions are cloned from B cells by molecular biological techniques described in WO92 / 02551, U.S. Patent No. 5,627,052, or Babcook et al., Proc Natl Acad Sci USA 93:7843-48 (1996). In some embodiments, a polynucleotide encoding all or one region of both the heavy and light chain variable regions of an IgG molecule expressed by a cloned daughter plasma cell expressing the antibody is subcloned and sequenced. In some embodiments, the sequence of the encoded polypeptide is readily determined from the polynucleotide sequence.

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

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

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

[0367] Once isolated, B cells are induced to produce antibodies by culturing them under conditions that support the proliferation or development of B cells into, for example, plasma cells, plasmablasts, or plasma cells. The antibodies are then screened, typically using high-throughput techniques, to identify antibodies that specifically bind to target antigens, such as specific tissues, cells, or polypeptides. In some embodiments, the specific antigen, such as an antibody-bound cell surface polypeptide, is unknown, while in other embodiments, an antigen specifically bound to the antibody is known.

[0368] According to this disclosure, B cells are isolated in some embodiments from biological samples, e.g., tumors, tissues, peripheral blood, or lymph node samples, by any means known and available in the art. B cells are generally sorted by FACS based on the presence of B cell-specific markers on their surface, e.g., CD19, CD138, and / or surface IgG. However, in some embodiments, other methods known in the art are utilized, e.g., column purification using CD19 magnetic beads or IgG-specific magnetic beads, followed by elution from the column. However, in some embodiments, magnetic isolation of B cells using any marker results in the loss of specific B cells. Therefore, in some embodiments, the isolated cells are not sorted, and instead, Ficoll-purified mononuclear cells isolated from tumors are directly seeded in appropriate or desired specificities in each well.

[0369] To identify antibody-producing B cells, B cells are typically seeded at low densities (e.g., single-cell specificity per well, 1–10 cells per well, 10–100 cells per well, 1–100 cells per well, 1–100 cells per well, less than 10 cells per well, or less than 100 cells per well) in multi-well plates or microtiter plates, for example, in 96, 384, or 1536 well configurations. Once B cells are initially seeded at a cell density of more than 1 per well, the method of this disclosure includes the step of further diluting the cells in the wells identified as producing antigen-specific antibodies until single-cell specificity per well is achieved, thereby facilitating the identification of antigen-specific antibody-producing B cells in some embodiments. In some embodiments, the cell supernatant or a portion thereof, and / or the cells are cryopreserved for future testing and subsequent recovery of antibody polynucleotides.

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

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

[0372] When comparing the binding of an antibody to a specific target antigen (e.g., a biological sample such as cancer tissue or cells, or infectious material) to the binding of an antibody to a control sample (e.g., a biological sample such as normal cells, comparison cells from a different species, different cancer tissue or cells, or different infectious material), in some embodiments, an antibody is considered to preferentially bind to a specific target antigen if at least twice, at least three times, at least five times, or at least ten times more of the antibody binds to the specific target antigen compared to the amount that binds to the control sample.

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

[0374] In some embodiments, one or more replicable expression vectors containing polynucleotides encoding variable and / or constant regions are prepared and used to transform suitable cell lines, such as non-producing bone marrow cell lines like mouse NSO strain, or bacteria such as Escherichia coli, in which antibody production occurs. To obtain efficient transcription and translation, the polynucleotide sequences in each vector must include appropriate regulatory sequences, specifically promoter and leader sequences that are operably bound to the variable region sequence.

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

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

[0377] Packages, kits, and pre-filled containers This specification also provides kits containing one or more of the compounds described above. In some embodiments, the kit contains the antibody or antigen-binding fragment described herein in a suitable container.

[0378] In some embodiments, a container means containing the compositions described herein is provided. In some embodiments, this container means is any suitable container for containing, for example, a liquid or lyophilized composition, and includes, but is not limited to, vials, syringes, bottles, intravenous (IV) bags, or ampoules. In some embodiments, the syringe holds a liquid suitable for injection into a subject in any volume containing, but not limited to, 0.5 cc, 1 cc, 2 cc, 5 cc, 10 cc, or more.

[0379] This specification provides kits comprising one or more compositions described herein. In some embodiments, this specification provides kits for treating a cancer subject comprising antibodies and anticancer therapeutic agents described herein.

[0380] In some embodiments, this specification provides a kit for cancer treatment comprising the antibody described herein and a label attached to or packaged in a container, the label indicating a combination of the antibody and an anti-cancer drug.

[0381] In some embodiments, this specification provides a cancer treatment kit comprising an anticancer therapy agent and a label attached to or packaged with the container, the label indicating a combination of the anticancer therapy agent and an antibody described herein.

[0382] In some embodiments, the kit container means 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 disposed and / or preferably appropriately divided. This specification provides container means containing the compositions described herein.

[0383] In some embodiments, the kit contains, for commercial purposes, at least one fusion protein, a detectable portion, a reporter molecule, and / or any other reagents, tightly sealed. In some embodiments, such a container includes an injection bottle and / or a spray-molded plastic container that holds the desired vial. In some embodiments, the kit also includes printed material relating to the use of the materials in the kit.

[0384] The packages and kits further, in some embodiments, include buffering agents, preservatives, and / or stabilizers in the pharmaceutical formulation. In some embodiments, each component of the kit is sealed in an individual container, and all the various containers may be in a single package. In some embodiments, the kits of this disclosure are designed for cold storage or room temperature storage.

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

[0386] In some embodiments, the package and kit further include one or more components for assays, such as ELISA assays. Examples of samples to be tested in this application include blood, plasma, tissue sections, and secretions, urine, lymph, and their products. In some embodiments, the package and kit further include one or more components for sample collection (e.g., syringes, cups, cotton swabs).

[0387] In some embodiments, the packages and kits include labels that identify information required by the US FDA or similar regulatory authority, such as a product description, dosage, dosage form, and / or indications for treatment. The packages provided herein may include any of the compositions described herein.

[0388] The term “packaging material” refers to the physical structure that contains the components of the kit. In some embodiments, the packaging material is intended to maintain the components in a sterile state and is made of materials commonly used for this purpose (e.g., paper, corrugated cardboard, glass, plastic, foil, ampoules, etc.). In some embodiments, the label or package insert includes appropriate written instructions. For this reason, the kit further includes, in some embodiments, a label or instructions for using the kit components in any manner of this disclosure. In some embodiments, the kit contains the compound in a pack or dispenser, along with instructions for administering the compound in the manner described herein.

[0389] In further embodiments, the kit further includes container means for anticancer drugs.

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

[0391] In some embodiments, the instructions are present on “printed material,” for example, on paper or cardboard attached to or within the kit, on a label attached to the kit or packaging material, or attached to a vial or tube containing the components of the kit. In some embodiments, the instructions are further included in a computer-readable medium, such as a CD-ROM, DVD, flash memory device, solid-state memory, magnetic disk and disk device, magnetic tape, cloud computing device and service. Depending on the circumstances, the programs and instructions are coded permanently, almost permanently, semi-permanently, or non-temporarily on the medium.

[0392] This specification provides container means for comprising the compositions described herein. In some embodiments, this container means is any suitable container for containing a liquid or lyophilized composition, including but not limited to vials, syringes, bottles, intravenous (IV) bags, or ampoules. A syringe holds a liquid suitable for injection into a subject in any volume, including but not limited to 0.5 cc, 1 cc, 2 cc, 5 cc, 10 cc, or more.

[0393] This specification provides kits comprising the compositions described herein. In some embodiments, this specification provides kits for treating cancer, comprising the antibodies described herein in combination with anticancer therapeutic agents.

[0394] In some embodiments, the Specified herein provides a kit for cancer treatment comprising the antibody described herein and a label attached to or packaged in a container, the label describing the use of the antibody or its antigen-binding fragment with an anticancer therapeutic agent.

[0395] In some embodiments, this specification provides a cancer treatment kit comprising an anticancer therapy agent and a label attached to or packaged with the container, the label indicating a combination of the anticancer therapy agent and an antibody described herein. [Examples]

[0396] This disclosure is provided for illustrative purposes only and is not intended to limit this disclosure in any way, as further illustrated in the following embodiments.

[0397] Example 1 - Identification and Cloning Antibodies that specifically bind to human bone marrow-derived suppressor cells (MDSCs) produced by patients responding to the checkpoint inhibitor anti-PD-1 treatment were isolated and cloned. Further investigation of the immunomodulatory properties of these monoclonal antibodies was conducted with the aim of identifying antibodies that target and reverse the immunosuppressive effect of MDSCs, thereby improving tumor removal.

[0398] We identified cancer patients who achieved a partial or complete response to immune checkpoint inhibitors for at least six months and selected them for memory B cell repertoire analysis via the I-STAR platform. This platform utilized a short-term B cell culture system to examine the memory B cell repertoire. Over 15,000 memory B cells were isolated from 10 million peripheral blood mononuclear cells (PBMCs) from each donor patient based on CD19 and IgG surface expression. These memory B cells were then seeded at approximately 1 cell / well in 40 384-well microtiter plates under conditions that promoted B cell activation, proliferation, terminal differentiation, and antibody secretion. The 1-cell / well seeding density allowed for the expansion of single B cell clones, enabling the reconstruction of genuine antibody heavy- and light-chain pairs from each culture well. Secreted IgG antibodies in each well were screened for binding to MDSCs using high-throughput and miniaturized multiplex flow cytometry assays. 49 positive B cell clones were identified. A selected subset of antibodies, prioritized based on MDSC binding profiles and antibody variable region sequences, was sequenced, cloned, and expressed as recombinant IgG1 for further in vitro characterization.

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

[0400] A single B-cell clone (Germline ID for heavy chain VH3.30-3 / IGHG1 and light chain VK1.O12) was identified from MDSC screening and named AB101, containing a light chain with SEQ ID NO: 9 and a heavy chain with SEQ ID NO: 10. The donor from which the clone was obtained was a patient diagnosed with non-small cell lung cancer (NSCLC). This patient had progressive disease despite chemotherapy and had achieved complete remission with anti-PD-1 treatment, but was still receiving treatment at the time of blood collection. Sequencing confirmed that the B-cell clone well contained only one heavy chain and one light chain. As observed with IgG antibodies secreted from the single B-cell clone, the reproduced antibody further exhibited distinct bimodal binding on MDSCs, indicating that the antibody target was highly expressed on a selective subpopulation of MDSCs. See Figure 1. Results from reproduction screens on two MDSC donors under relaxed block (recombinant Fc block 10 μg / mL (Abcam)) and strict block (recombinant Fc block 10 μg / mL + 1 μg / mL anti-CD16, anti-CD32, and anti-CD64) conditions revealed dose-dependent saturation binding of AB101 to human MDSCs at an IC50 of approximately 10 nm under relaxed block conditions. Under strict block conditions, overall AB101 binding was reduced, as supported by a decrease in MFI.

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

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

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

[0404] Follow the manufacturer's instructions, each containing human CD3 + T cell isolation kit (StemCell 19051), EasySep® Human CD4 + T cell isolation kit (StemCell No. 17952), EasySep® Human CD8 + All CD3, CD4, or CD8 T cells were isolated using a T cell isolation kit (StemCell No. 17953). These negative selection kits utilize antibodies to label undesirable cell types to be removed, allowing for the isolation of desired target cells from the sample.

[0405] Example 3 - Specific binding of AB101 to immunosuppressive myeloid cells To evaluate specificity, the binding of the antibody of the present invention, antibody AB101, conjugated to the far-infrared fluorescent dye AF647, was tested against different cell types, including MDSCs, immunosuppressive M2c and pro-inflammatory M1 macrophages generated as described in Example 11 and below. In addition, a separate immune population obtained from PBMCs of healthy donors was evaluated for antibody binding. At least three individual donors were used in each of these tests. PBMCs were isolated from blood using a Ficoll gradient with a standard procedure. Next, to differentiate the immune cell populations, PBMC cells were stained with hematopoietic lineage markers (CD45-BV421 (BD642275), CD3-BV510 (BD563109), CD11c-PE-Cy7 (BD561356), CD14-FITC (BD347493), CD20-APC-Cy7 (BD562643), CD56-PE (BD347747), and CD66c (BD551478)). The distinct lineages were further characterized by the following expression patterns, which were evaluated for binding to AB101: T cells CD45 + CD3 + , B cell CD45 + CD20 + , single-tube CD45 + CD14 + , NK cell CD45 + SSC low CD14 - CD3 - CD56 + , granulocyte CD45 + SSC Hi CD14 - CD66 + , and dendritic cell CD45 + CD14 - CD66 - CD11c +Antibodies binding to primary human non-immune cells (Lonza), including small airway epithelial cells (SAEC, #CC-2547), renal proximal tubular 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), were also evaluated. These cells were cultured in cell type-specific media and under conditions according to the manufacturer's instructions until the concentration reached 60-70%, and then harvested and tested for antibody binding by flow cytometry (Figures 4 and 5).

[0406] In vitro monocyte MDSCs were generated from monocytes isolated by standard methods. On day 0, monocytes were placed in RPMI1640 (Hyclone SH30027.02, serum-free) in a 1.5 × 10⁶ solution. 5 / cm 2The cells were seeded, incubated at 5% CO2 and 37°C for 1 hour, then washed with pre-warmed RPMI, and finally added to MDSC medium (RMPI + 10% FBS (Hyclone SH30070.03) + 50 ng / mL GM-CSF (R&D Systems 215-GM-010) + 50 ng / mL IL-6 (R&D Systems 206-IL-010), 20 mL per T75 flask). The cells were then cultured at 5% CO2 and 37°C for 7 days without changing the medium. After 7 days, the cells were washed twice with PBS (Hyclone SH30028.03) + 2 mM EDTA, then 15 mL of cold macrophage detachment reagent (PromoCell C-41330) was added to each T75 flask, followed by incubation at 2-8°C for 40 minutes to collect the cells. The cells were removed by gently tapping the flask with the palm of the hand, collected, and diluted 1:1 with PBS + 2mM EDTA. The cells were pelletized in a conical tube by centrifugation at 450xg for 15 minutes, washed once with PBS + 2mM EDTA, counted, and then 1 × 10⁶ cells were placed in FACS blocking buffer (PBS + 1% FBS + 0.1 μg / mL Fc block (Abcam Ab90285) under relaxed staining conditions, or PBS + 1% FBS + Fc block and + 0.01 μg / mL CDR block (antibodies against FcR CD16, CD32, and CD64, respectively, BD Biosciences 556617, 557333, and 555525) under strict staining conditions). 7 The cells were resuspended in / mL. The cells were incubated in FACS blocking buffer at room temperature (RT) for 20 minutes (min), then at 4°C for 30 minutes. Subsequently, the cells were sterilized in FACS buffer + 5% BSA (Sigma A3059) at a rate of 1 × 10⁻¹⁶. 6 Dilute to cells / mL and prepare 40 μL of cells (4 × 10⁶). 4Cells were divided equally into wells and stained. Primary antibodies (AB101 at 20, 6, 2, 0.75, 0.25, 0.08, and 0.02 μg / mL, or human IgG1 isotype control at 6 μg / mL) were added to the cells and incubated at room temperature for 90 minutes. Cells were washed three times with 250 μL / well of FACS buffer + 5% BSA. Secondary APC goat anti-human IgG (Jackson IR109-136-097) antibody was prepared at a dilution of 1:1000 in FACS buffer + BSA + e780 bio-dye at a ratio of 1:250 and added to the cells (50 μL per well). After incubation at 4°C for 45 minutes, cells were washed three times with 250 μL of FACS buffer. Next, the cells were fixed in 100 μL of 4% PFA in each well at room temperature for 10–15 minutes, washed once with 250 μL of FACS buffer, pelletized at 650xg for 5 minutes, and then resuspended in 100 μL of FACS buffer for analysis by flow cytometry.

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

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

[0409] In an IP including cross-linking using the PFA method, monocytes were isolated from human blood and then polarized to M2 cells using the protocols of Example 2 and Example 11. The M2 macrophages were detached from the plate after incubation at 37°C for up to 10 minutes using a macrophage detachment reagent (Macrophage Detachment Solution DXF, PromoCell, No. C-41330), during which time the cells began to cluster and detach. The flask was vigorously tapped to promote cell detachment. After detachment, the macrophage detachment reagent was rapidly cooled by adding FACS buffer to the cells. The cells were pelletized at 300xg for 10 minutes, and the supernatant was removed. The cell pellet was resuspended in 30 mL of PBS containing 5% BSA (w / v) and 1 mM EDTA pH 8.0 and incubated on ice for 30 minutes.

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

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

[0412] The beads were analyzed by mass spectrometry. This analysis was generally performed according to the method described in Yan et al., Mol Cell Proteomics 10(3):M110.005611 (2011). In this method, the reversal of formaldehyde crosslinking and protein elution from streptavidin beads were performed at 60°C for 3 hours with constant stirring using 6M guanidine and 150mM Tris buffer (pH 8.3). The supernatant was denatured, reduced, and alkylated at 95°C for 10 minutes in the same buffer with 10mM tris(2-carboxyethyl)-phosphine (TCEP) and 50mM chloroacetamide (CAA). The sample was then diluted 10-fold and digested with 1.3 μg of trypsin overnight at 37°C. The peptide was purified using a C18 cartridge. One replica of AB102IP and one replica of anti-CD163IP were eluted from a C18 cartridge for direct MS / MS analysis. Both replicas were incubated in PBS buffer pH 7.5 with 0.1 M formaldehyde-d2 and 0.4 M sodium cyanohydride and labeled with dedimethylation (d4). Both replicas of ISO2(Fc null)IP were incubated in PBS buffer pH 7.5 with 0.1 M formaldehyde and 0.4 M sodium cyanohydride for 1 hour and labeled with light dimethylation (d0) on a C18 cartridge. The C18 cartridge was then washed wit...

Claims

1. An antibody that specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells, wherein the antibody is An antibody comprising (a) a light chain variable region (VL) containing a complementation-determining region (CDR) having an amino acid sequence that is 100% identical to the respective amino acid sequences of SEQ ID NOs. 1 (CDRL1), 2 (CDRL2), and 3 (CDRL3); and (b) a heavy chain variable region (VH) containing a CDR having an amino acid sequence that is 100% identical to the respective amino acid sequences of SEQ ID NOs. 4 (CDRH1), 5 (CDRH2), and 6 (CDRH3).

2. The antibody according to claim 1, wherein the VL has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

7.

3. The antibody according to claim 1, wherein the VL has an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:

7.

4. The antibody according to claim 1, wherein the VH has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

8.

5. The antibody according to claim 1, wherein the VH has an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:

8.

6. An antibody that specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells, wherein the antibody is An antibody comprising: (a) a light chain variable region (VL) containing a complementation-determining region (CDR) having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, and having an amino acid sequence that is 100% identical to the amino acid sequences of SEQ ID NO: 1 (CDRL1), 2 (CDRL2), and 3 (CDRL3); and (b) a heavy chain variable region (VH) containing a CDR having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8, and having an amino acid sequence that is 100% identical to the amino acid sequences of SEQ ID NO: 4 (CDRH1), 5 (CDRH2), and 6 (CDRH3).

7. The antibody according to claim 6, wherein VL has an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 7, and VH has an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:

8.

8. An antibody that specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells, wherein the antibody is An antibody comprising (a) a light chain variable region (VL) having an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 7, and (b) a heavy chain variable region (VH) having an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:

8.

9. An antibody that specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells, wherein the antibody is An antibody comprising (i) a light chain (LC) having an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 9, and (ii) a heavy chain (HC) having an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:

10.

10. Use of an antibody that specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells in the manufacture of a drug for use in the treatment of cancer in human subjects, wherein the antibody comprises (i) a light chain variable region (VL) containing a complementation-determining region (CDR) having an amino acid sequence having 100% identity with the respective amino acid sequences of SEQ ID NOs. 1 (CDRL1), 2 (CDRL2), and 3 (CDRL3), and (ii) a heavy chain variable region (VH) containing a CDR having an amino acid sequence having 100% identity with the respective amino acid sequences of SEQ ID NOs. 4 (CDRH1), 5 (CDRH2), and 6 (CDRH3).

11. Use of an antibody that specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells in the manufacture of a drug for use in the treatment of cancer in human subjects, wherein the antibody comprises (i) a light chain variable region (VL) containing a complementation determining region (CDR) having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 7 and having an amino acid sequence having 100% identity with the respective amino acid sequences of SEQ ID NO: 1 (CDRL1), 2 (CDRL2), and 3 (CDRL3); and (ii) a heavy chain variable region (VH) containing a CDR having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 8 and having an amino acid sequence having 100% identity with the respective amino acid sequences of SEQ ID NO: 4 (CDRH1), 5 (CDRH2), and 6 (CDRH3).

12. The use according to claim 10 or 11, wherein the antibody is formulated to administer to the subject additional anticancer therapies selected from surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, and cytokine therapy, as well as combinations thereof.

13. The use according to claim 12, wherein the additional anti-cancer therapy is immunotherapy.

14. The use according to claim 13, wherein the immunotherapy is a composition comprising a checkpoint inhibitor.

15. The use according to any one of claims 10 to 14, wherein the cancer is lung cancer, adenocarcinoma, or sarcoma.

16. An antibody according to any one of claims 1 to 9, comprising a human heavy chain constant region or a human light chain constant region.

17. The antibody according to claim 16, wherein the human heavy chain constant region is IgG1 or IgG4, or a fragment thereof.

18. The antibody according to any one of claims 1 to 9 or 16 to 17, comprising a human variable framework region and a mouse constant region.

19. An antibody according to any one of claims 1 to 9 or 16 to 18, which binds to an epitope comprising the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, and / or SEQ ID NO:

20.

20. An antibody according to any one of claims 1 to 9 or 16 to 19, formulated for use against additional anticancer therapies selected from surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, and cytokine therapy, as well as combinations thereof.

21. The antibody according to claim 20, wherein the additional anti-cancer therapy is immunotherapy.

22. The antibody according to claim 21, wherein the immunotherapy is a composition comprising a checkpoint inhibitor.

23. The antibody according to claim 21 or 22, wherein the cancer is lung cancer, adenocarcinoma, or sarcoma.

24. Use of an antibody according to any one of claims 1 to 9 in the manufacture of a drug for use in the treatment of cancer in human subjects, wherein the antibody (a) specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells, and (i) CD4 ＋ T cells, CD8 ＋ Activate T cells, NK cells, or any combination thereof, and / or (ii) CD4 ＋ T cells, CD8 ＋ Uses that promote immune cell function by increasing T cells, NK cells, or any combination thereof, thereby (b) reducing immunosuppression by tumor-associated macrophages in human subjects with cancer, and / or (c) promoting T cell-mediated tumor cell death in human subjects with cancer.

25. The use according to claim 24, wherein the enhancement of immune cell function is measured as an increase in the level of IFN-γ, TNF-α, or perforin, or any combination thereof.

26. The use according to claim 24, wherein the immunosuppressive human bone marrow cells are macrophages or bone marrow-derived suppressor cells.

27. The antibody according to claim 1, comprising VL of SEQ ID NO: 7 having one or more conservative amino acid substitutions, and VH of SEQ ID NO: 8 having one or more conservative amino acid substitutions.

28. A pharmaceutical composition comprising (a) an antibody comprising (i) a light chain variable region (VL) containing a complementarity-determining region (CDR) having an amino acid sequence that is 100% identical to the respective amino acid sequences of SEQ ID NOs. 1 (CDRL1), 2 (CDRL2), and 3 (CDRL3), and (ii) a heavy chain variable region (VH) containing a CDR having an amino acid sequence that is 100% identical to the respective amino acid sequences of SEQ ID NOs. 4 (CDRH1), 5 (CDRH2), and 6 (CDRH3), and (b) one or more pharmaceutically acceptable excipients.

29. The pharmaceutical composition according to claim 28, wherein the VL has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

7.

30. The pharmaceutical composition according to claim 28, wherein the VH has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

8.

31. (a) an antibody that specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells, comprising (a) (i) a light chain variable region (VL) containing a complementation-determining region (CDR) having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 7 and having an amino acid sequence having 100% identity with each of the amino acid sequences of SEQ ID NO: 1 (CDRL1), 2 (CDRL2), and 3 (CDRL3); and (ii) a heavy chain variable region (VH) containing a CDR having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 8 and having an amino acid sequence having 100% identity with each of the amino acid sequences of SEQ ID NO: 4 (CDRH1), 5 (CDRH2), and 6 (CDRH3); and (b) one or more pharmaceutically acceptable excipients.

32. The pharmaceutical composition according to claim 31, wherein VL has an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 7, and VH has an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:

8.

33. A pharmaceutical composition comprising (a) an antibody that specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells, comprising (i) a light chain variable region (VL) having an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 7, and (ii) a heavy chain variable region (VH) having an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 8, and (b) one or more pharmaceutically acceptable excipients.

34. A pharmaceutical composition comprising (a) an antibody that specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells, comprising (i) a light chain (LC) having an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 9, and (ii) a heavy chain (HC) having an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 10, and (b) one or more pharmaceutically acceptable excipients.

35. The pharmaceutical composition according to any one of claims 28 to 34, wherein the one or more pharmaceutically acceptable excipients are a surfactant, a buffer, water, one or more excipients that improve the stability of the antibody, or a combination thereof.

36. Use of a pharmaceutical composition in the manufacture of a drug for use in the treatment of cancer in human subjects, wherein the pharmaceutical composition comprises (a) an antibody that specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells, and (b) one or more pharmaceutically acceptable excipients.

37. Use of a pharmaceutical composition in the manufacture of a drug for use in the treatment of cancer in human subjects, wherein the pharmaceutical composition comprises (a) (i) a light chain variable region (VL) containing a complementation-determining region (CDR) having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 7 and having an amino acid sequence having 100% identity with each of the amino acid sequences of SEQ ID NO: 1 (CDRL1), 2 (CDRL2), and 3 (CDRL3); and (ii) a heavy chain variable region (VH) containing a CDR having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 8 and having an amino acid sequence having 100% identity with each of the amino acid sequences of SEQ ID NO: 4 (CDRH1), 5 (CDRH2), and 6 (CDRH3), and an antibody that specifically binds to human CD163 expressed on immunosuppressive human bone marrow cells; and (b) one or more pharmaceutically acceptable excipients.

38. The use according to claim 36 or 37, wherein the cancer is lung cancer, adenocarcinoma, or sarcoma.

39. The use according to claim 36 or 37, wherein the one or more pharmaceutically acceptable excipients are a surfactant, a buffer, water, one or more excipients that improve the stability of the antibody, or a combination thereof.

40. The use according to any one of claims 36 to 39, wherein the pharmaceutical composition is formulated to administer to the subject an additional anticancer therapy selected from surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, and cytokine therapy, and combinations thereof.

41. The use according to claim 40, wherein the additional anti-cancer therapy is immunotherapy.

42. The use according to claim 41, wherein the immunotherapy is a composition comprising a checkpoint inhibitor.

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