CN117062835A - Immunomodulatory antibodies and uses thereof - Google Patents

Immunomodulatory antibodies and uses thereof Download PDF

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Publication number
CN117062835A
CN117062835A CN202280022616.1A CN202280022616A CN117062835A CN 117062835 A CN117062835 A CN 117062835A CN 202280022616 A CN202280022616 A CN 202280022616A CN 117062835 A CN117062835 A CN 117062835A
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seq
antibody
amino acid
acid sequence
group
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卡默尔·D·普里
张惊雷
王萍萍
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Oncorhys Ponce
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Oncorhys Ponce
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Priority claimed from PCT/US2022/012598 external-priority patent/WO2022159349A1/en
Publication of CN117062835A publication Critical patent/CN117062835A/en
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Abstract

Antibodies and methods of use thereof are provided herein. Antibodies as disclosed herein and CD163 on cells (such as on macrophages) + And (5) combining. These antibodies can be used in therapeutic methods, such as methods of treating cancer and fibrosis.

Description

Immunomodulatory antibodies and uses thereof
Sequence listing
The present application comprises a sequence listing that is electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created at 2022, 1/14, named "ONR-006WO_SL.txt" and is 37,676 bytes in size.
Cross Reference to Related Applications
The present application claims the benefit of U.S. provisional patent application No. 63/199,732 filed on 1 month 20 of 2021 and U.S. provisional patent application No. 63/200,897 filed on 4 month 1 of 2021, both of which are incorporated herein by reference in their entireties.
Summary of the disclosure
Provided herein are antibodies, including antigen binding fragments and other antigen binding polypeptides, useful for treating cancer and fibrosis.
In certain embodiments, disclosed herein are antibodies comprising: (a) A heavy chain variable region (VH) having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and (b) a light chain variable region (VL) having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of: 28, 30, 32, 34, 36, 38 and 40 SEQ ID NO; with the proviso that the antibody does not comprise a light chain variable region (VL) having the sequence set forth in SEQ ID NO. 40 and a heavy chain variable region (VH) having the sequence set forth in SEQ ID NO. 41. In some embodiments, the light chain variable region (VL) has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40. In some embodiments, the light chain variable region (VL) has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40. In some embodiments, the light chain variable region (VL) has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40. In some embodiments, the light chain variable region (VL) has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40. In some embodiments, the light chain variable region (VL) has a sequence 100% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40. In some embodiments, the heavy chain variable region (VH) has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41. In some embodiments, the heavy chain variable region (VH) has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41. In some embodiments, the heavy chain variable region (VH) has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41. In some embodiments, the heavy chain variable region (VH) has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41. In some embodiments, the heavy chain variable region (VH) has a sequence 100% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41. In some embodiments, the amino acid sequence of the variable heavy chain is 100% identical at CDR H1, CDR H2, CDR H3, and the amino acid sequence of the variable light chain is 100% identical at CDR L1, CDR L2, and CDR L3. In some embodiments, CDR H1 has a sequence as set forth in the amino acid sequence selected from the group consisting of seq id no: SEQ ID NO.4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25. In some embodiments, CDR H2 has a sequence as set forth in the amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26. In some embodiments, CDR H3 has a sequence as set forth in the amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27. In some embodiments, CDR L1 has a sequence as set forth in the amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13. In some embodiments, CDR L2 has a sequence as set forth in the amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14. In some embodiments, CDR L3 has a sequence as set forth in the amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15.
In certain embodiments, disclosed herein are antibodies comprising: (a) Light chain CDR1 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id nos: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; and (b) a heavy chain CDR1 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; heavy chain CDR3 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise at least the sequences as set forth in seq id no: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. In some embodiments, CDR L1 has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; CDR L2 has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and CDR L3 has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15. In some embodiments, CDR L1 has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; CDR L2 has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and CDR L3 has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15. In some embodiments, CDR L1 has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; CDR L2 has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and CDR L3 has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15. In some embodiments, CDR L1 has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; CDR L2 has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and CDR L3 has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15. In some embodiments, CDR L1 has a sequence 100% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; CDR L2 has a sequence 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and CDR L3 has a sequence at least 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15. In some embodiments, CDR H1 has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; CDR H2 has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; and CDR H3 has a sequence at least 85% identical to an amino acid selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27. In some embodiments, CDR H1 has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; CDR H2 has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; and CDR H3 has a sequence at least 90% identical to an amino acid selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27. In some embodiments, CDR H1 has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; CDR H2 has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; and CDR H3 has a sequence at least 95% identical to an amino acid selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27. In some embodiments, CDR H1 has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; CDR H2 has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; and CDR H3 has a sequence at least 99% identical to an amino acid selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27. In some embodiments, CDR H1 has a sequence at least 100% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; CDR H2 has a sequence at least 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; and CDR H3 has a sequence at least 100% identical to an amino acid selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27. In some embodiments, the antibody comprises: (a) A heavy chain variable region (VH) having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and (b) a light chain variable region (VL) having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40. In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) having a sequence at least 85% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and (b) a light chain variable region (VL) having a sequence at least 85% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40. In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) having a sequence at least 90% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and (b) a light chain variable region (VL) having a sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40. In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) having a sequence at least 95% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and (b) a light chain variable region (VL) having a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40. In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) having a sequence at least 99% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and (b) a light chain variable region (VL) having a sequence at least 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40. In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) having a sequence 100% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and (b) a light chain variable region (VL) having a sequence 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
In some embodiments, the antibodies disclosed herein comprise a human heavy chain constant region or a human light chain constant region. In some embodiments, the antibodies disclosed herein comprise a human heavy chain constant region that is IgG1 or IgG4 or a fragment thereof. In some embodiments, disclosed hereinIs bound to Fc receptors. In some embodiments, the Fc receptor is expressed on macrophages. In some embodiments, the antibodies disclosed herein are antibody fragments comprising: single heavy chain, single light chain, fab, F (ab') 2 Fd, scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, bispecific Fab 2 Trispecific Fab 3 A single chain binding polypeptide, a dAb fragment or a diabody. In some embodiments, the antibodies disclosed herein are directed to Dendritic Cells (DCs), such as, for example, bone marrow DCs (mdcs) (e.g., CD14 - HLA-DR + CD11c + mDC) are bound. In some embodiments, the antibodies disclosed herein are directed against classical monocytes (e.g., CD14 + HLA-DR + CD16 - Monocytes) are bound. In some embodiments, the antibodies disclosed herein are directed to a monocyte intermediate (intermediate monocyte, e.g., CD14 + HLA-DR + CD16 + Monocytes) are bound. In some embodiments, the antibodies disclosed herein are directed to non-classical monocytes (e.g., CD14 - HLA-DR + CD16 + Monocytes) are bound. In some embodiments, the antibodies disclosed herein bind to cancer cells. In some embodiments, the cancer cell is a lymphoma cell. In some embodiments, the lymphoma cell constitutively expresses CD163. In some embodiments, the antibodies disclosed herein bind to immunosuppressive bone marrow cells. In some embodiments, the immunosuppressive bone marrow cells are in a tumor microenvironment. In some embodiments, the immunosuppressive bone marrow cells are macrophages or bone marrow-derived suppressor cells. 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 macrophage is a tumor-associated macrophage. In some embodiments, an antibody disclosed herein binds to CD163 protein. In some embodiments, the CD163 protein is a glycoform of CD163. In some embodiments, the CD163 protein is a 150kDa glycoform of CD163. In some embodiments, disclosed hereinThe antibodies did not specifically bind to the 130kDa glycoform of CD163 expressed by human macrophages. In some embodiments, the CD163 protein is a component of a cell surface complex comprising at least one other protein expressed by macrophages. In some embodiments, the at least one other protein is a galectin-1 protein, a LILRB2 protein, a casein kinase II protein, or any combination thereof. In some embodiments, the antibodies disclosed herein specifically bind to a CD163 epitope comprising the amino acid sequence SEQ ID NO. 42. In some embodiments, the antibodies disclosed herein specifically bind to a CD163 epitope comprising the amino acid sequence SEQ ID NO. 43. In some embodiments, the antibodies disclosed herein specifically bind to a CD163 epitope comprising the amino acid sequence SEQ ID NO. 44. In some embodiments, the antibodies disclosed herein specifically bind to a CD163 epitope comprising each of the amino acid sequences SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO: 44. In some embodiments, the antibodies disclosed herein alter the expression of at least one marker on macrophages. In some embodiments, the at least one marker on human macrophages is CD16, CD64, TLR2, or Siglec-15.
In some embodiments, the antibodies disclosed herein are raised against a K of 0.5nM to 100nM D Specifically binds to CD 163. In some embodiments, the antibodies disclosed herein are raised against a K of 0.5nM to 50nM D Specifically binds to CD 163. In some embodiments, the antibodies disclosed herein are raised against a K of 0.5nM to 10nM D Specifically binds to CD 163. In some embodiments, the antibodies disclosed herein are raised against a K of 0.5nM to 1.5nM D Specifically binds to CD 163. In some embodiments, an antibody disclosed herein is at a K of 0.5nM to 1.0nM D Specifically binds to CD 163. In some embodiments, the antibodies disclosed herein are raised against a K of 0.5nM to 100nM D Specifically binds to human M2c macrophages. In some embodiments, the antibodies disclosed herein are raised against a K of 0.5nM to 50nM D Specifically binds to human M2c macrophages. In some embodiments, the antibodies disclosed herein are raised against a K of 0.5nM to 10nM D Specifically binds to human M2c macrophages. In some embodiments, the antibodies disclosed herein are at 0.5K of nM to 1.5nM D Specifically binds to human M2c macrophages. In some embodiments, an antibody disclosed herein is at a K of 0.5nM to 1.0nM D Specifically binds to human M2c macrophages.
In certain embodiments, disclosed herein are pharmaceutical compositions comprising (a) an antibody disclosed herein and (b) at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient is selected from the group consisting of: stabilizers, buffers, surfactants, fillers, solvents, tonicity agents and antioxidants. In some embodiments, the pharmaceutical composition comprises two or more pharmaceutically acceptable excipients independently selected from the group consisting of: stabilizers, buffers, surfactants, fillers, solvents, tonicity agents and antioxidants.
In certain embodiments, disclosed herein are methods of treating a cancer or fibrotic disease or disorder associated with the presence of M2-macrophages in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody disclosed herein. In some embodiments, binding of the antibody to the macrophage promotes immune cell function, as measured by one or both of the following parameters: activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof; and proliferation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof. In some embodiments, activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof is measured as an enhanced level of IFN- γ, TNF- α, or perforin, or any combination thereof. In some embodiments, binding of the antibody to the macrophage is not cytotoxic to the macrophage. In some embodiments, binding of the antibody to the macrophage results in at least one of the following effects: reduced expression by the macrophage of at least one marker, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15; internalization of antibodies by macrophages; secretion of IFN-gamma, TNF-alpha and perforin; activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof; proliferation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof; and promoting tumor cell killing in the tumor microenvironment. In some embodiments, the binding results in: two or more of (a) to (e); three or more of (a) to (e); four or more of (a) to (e); or all of (a) to (e). In some embodiments, binding of the antibody to macrophages increases immunostimulatory activity in the tumor microenvironment. In some embodiments, binding of the antibody to the macrophage reduces immunosuppressive activity of the macrophage. In some embodiments, binding of the antibody to the macrophage reduces the tumor promoting activity of the macrophage. In some embodiments, binding of the antibody promotes cd4+ T cell activation, cd4+ T cell proliferation, or both cd4+ T cell activation and cd4+ T cell proliferation. In some embodiments, binding promotes expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by cd4+ T cells. In some embodiments, binding of the antibody promotes cd8+ T cell activation, cd8+ T cell proliferation, or both cd8+ T cell activation and cd8+ T cell proliferation. In some embodiments, binding of the antibody promotes expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by cd8+ T cells. In some embodiments, binding of the antibody protein promotes cytotoxic lymphocyte-mediated killing of cancer cells. In some embodiments, binding of the antibody promotes NK cell-mediated killing of tumor cells. In some embodiments, binding of the antibody promotes expression of IL-2 by T cells. In some embodiments, binding of the antibody increases CD4+ T cells, CD196-T cells, CXCR3+ T cells, CCR 4-T cells, or any combination thereof. In some embodiments, binding of the antibody to the macrophage reduces inhibition of cytotoxic T cell mediated killing of tumor cells in the tumor microenvironment. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is lung epithelial cancer (lung cancer) or lung sarcoma. In some embodiments, the cancer is lung adenocarcinoma. In some embodiments, any one of the methods further comprises administering an anti-cancer therapeutic to the subject. In some embodiments, binding of the antibody to the macrophage reduces the pro-fibrotic function of the macrophage. In some embodiments, the fibrotic disease or disorder is pulmonary fibrosis. In some embodiments, the fibrotic disease or disorder is cardiac fibrosis. In some embodiments, the fibrotic disease or disorder is liver fibrosis. In some embodiments, the fibrotic disease or disorder is renal fibrosis. In some embodiments, the fibrotic disease or disorder is retinal fibrosis. In some embodiments, the fibrosis is a primary fibrotic disease or disorder. In some embodiments, the primary fibrotic disease or disorder is Idiopathic Pulmonary Fibrosis (IPF). In some embodiments, the primary fibrotic disease or disorder is cirrhosis. In some embodiments, the primary fibrotic disease or disorder is systemic sclerosis (SSc). In some embodiments, the primary fibrotic disease or disorder is radiofibrosis. In some embodiments, the primary fibrotic disease or disorder is scar formation associated with mechanical injury. In some embodiments, the fibrosis is a secondary fibrotic disease. In some embodiments, the secondary fibrotic disease is associated with a disease or disorder selected from the group consisting of: infection, autoimmune diseases or disorders, cancer, and inflammatory diseases or disorders. In some embodiments, the secondary fibrotic disease is associated with a disease or disorder selected from the group consisting of: atherosclerosis, atrial fibrillation, chronic heart failure, peripheral arterial disease, acute coronary syndrome, nonalcoholic fatty liver disease (NAFLD), chronic acute liver failure, acute kidney injury, acute tubular necrosis, and chronic kidney disease. In some embodiments, the infection is selected from the group consisting of: sepsis, HIV infection, SARS-CoV-2 infection, acute viral hepatitis, chronic viral hepatitis and malaria. In some embodiments, the autoimmune or inflammatory disease or disorder is selected from the group consisting of: acute Lung Injury (ALI), acute Respiratory Distress Syndrome (ARDS), allergic pneumonia, alcoholic hepatitis, non-alcoholic steatohepatitis, viral hepatitis, sickle cell disease, type 1 diabetes, type 2 diabetes, crohn's disease, celiac disease, asthma, sarcoidosis, glomerulonephritis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis, sjogren's syndrome, scleroderma, cystic Fibrosis (CF), graft versus host disease, allograft rejection, renal allograft rejection, sarcoidosis, pulmonary sarcoidosis, hemophagocytic Lymphocytosis (HLH), inflammatory arthritis, chronic Obstructive Pulmonary Disease (COPD), asthma, osteoarthritis, fibromas and multiple sclerosis. In some embodiments, any one of the methods further comprises administering an anti-inflammatory therapy to the subject.
In certain embodiments, disclosed herein are antibodies according to the foregoing aspects and embodiments for use as a medicament.
In certain embodiments, disclosed herein are antibodies according to the foregoing aspects and embodiments for use in treating a cancer or fibrotic disease or disorder associated with the presence of M2-macrophages in a subject in need thereof. In some embodiments, binding of the antibody to the macrophage promotes immune cell function, as measured by one or both of the following parameters: (a) Activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof; and (b) proliferation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof. In some embodiments, activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof is measured as an enhanced level of IFN- γ, TNF- α, or perforin, or any combination thereof. In some embodiments, binding of the antibody to the macrophage is not cytotoxic to the macrophage. In some embodiments, binding of the antibody to the macrophage results in at least one of the following effects: (a) Reduced expression by the macrophage of at least one marker, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15; (b) internalization of the antibody by macrophages; (c) secretion of IFN-gamma, TNF-alpha and perforin; (d) Activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof; (e) Proliferation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof; and (f) promoting tumor cell killing in the tumor microenvironment. In some embodiments, the binding results in: two or more of (a) to (e); three or more of (a) to (e); four or more of (a) to (e); or all of (a) to (e). In some embodiments, binding of the antibody to macrophages increases immunostimulatory activity in the tumor microenvironment. In some embodiments, binding of the antibody to the macrophage reduces immunosuppressive activity of the macrophage. In some embodiments, binding of the antibody to the macrophage reduces the tumor promoting activity of the macrophage. In some embodiments, binding of the antibody promotes cd4+ T cell activation, cd4+ T cell proliferation, or both cd4+ T cell activation and cd4+ T cell proliferation. In some embodiments, binding promotes expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by cd4+ T cells. In some embodiments, binding of the antibody promotes cd8+ T cell activation, cd8+ T cell proliferation, or both cd8+ T cell activation and cd8+ T cell proliferation. In some embodiments, binding of the antibody promotes expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by cd8+ T cells. In some embodiments, binding of the antibody protein promotes cytotoxic lymphocyte-mediated killing of cancer cells. In some embodiments, binding of the antibody promotes NK cell-mediated killing of tumor cells. In some embodiments, binding of the antibody promotes expression of IL-2 by T cells. In some embodiments, binding of the antibody increases CD4+ T cells, CD 196-T cells, CXCR3+ T cells, CCR 4-T cells, or any combination thereof. In some embodiments, binding of the antibody to the macrophage reduces inhibition of cytotoxic T cell mediated killing of tumor cells in the tumor microenvironment. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is lung epithelial cancer or lung sarcoma. In some embodiments, the cancer is lung adenocarcinoma. In some embodiments, the antibody is formulated for administration to a subject in combination with an anti-cancer therapeutic. In some embodiments, binding of the antibody to the macrophage reduces the pro-fibrotic function of the macrophage. In some embodiments, the fibrotic disease or disorder is pulmonary fibrosis. In some embodiments, the fibrotic disease or disorder is cardiac fibrosis. In some embodiments, the fibrotic disease or disorder is liver fibrosis. In some embodiments, the fibrotic disease or disorder is renal fibrosis. In some embodiments, the fibrotic disease or disorder is retinal fibrosis. In some embodiments, the fibrosis is a primary fibrotic disease or disorder. In some embodiments, the primary fibrotic disease or disorder is Idiopathic Pulmonary Fibrosis (IPF). In some embodiments, the primary fibrotic disease or disorder is cirrhosis. In some embodiments, the primary fibrotic disease or disorder is systemic sclerosis. In some embodiments, the primary fibrotic disease or disorder is radiofibrosis. In some embodiments, the primary fibrotic disease or disorder is scar formation associated with mechanical injury. In some embodiments, the fibrosis is a secondary fibrotic disease. In some embodiments, the secondary fibrotic disease is associated with a disease or disorder selected from the group consisting of: infection, autoimmune diseases or disorders, cancer, and inflammatory diseases or disorders. In some embodiments, the secondary fibrotic disease is associated with a disease or disorder selected from the group consisting of: atherosclerosis, atrial fibrillation, chronic heart failure, peripheral arterial disease, acute coronary syndrome, nonalcoholic fatty liver disease (NAFLD), chronic acute liver failure, acute kidney injury, acute tubular necrosis, and chronic kidney disease. In some embodiments, the infection is selected from the group consisting of: sepsis, HIV infection, SARS-CoV-2 infection, acute viral hepatitis, chronic viral hepatitis and malaria. In some embodiments, the autoimmune or inflammatory disease or disorder is selected from the group consisting of: acute Lung Injury (ALI), acute Respiratory Distress Syndrome (ARDS), allergic pneumonia, alcoholic hepatitis, non-alcoholic steatohepatitis, viral hepatitis, sickle cell disease, type 1 diabetes, type 2 diabetes, crohn's disease, celiac disease, asthma, sarcoidosis, glomerulonephritis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis, sjogren's syndrome, scleroderma, cystic Fibrosis (CF), graft versus host disease, allograft rejection, renal allograft rejection, sarcoidosis, pulmonary sarcoidosis, hemophagocytic Lymphocytosis (HLH), inflammatory arthritis, chronic Obstructive Pulmonary Disease (COPD), asthma, osteoarthritis, fibromas and multiple sclerosis. In some embodiments, the antibody is formulated for administration to a subject in combination with an anti-inflammatory therapy.
Brief Description of Drawings
FIGS. 1A-1D are a series of graphs showing binding of anti-CD 163 antibodies to SU-DHL-1 lymphoma cells. A graph showing binding of AB101, human IgG1 isotype control (hig 1) and representative variant of AB101 (V3) to wild-type SU-DHL-1 cells (SU-DHL-1-WT) as a function of antibody concentration (fig. 1A). A graph showing binding of AB101, human IgG1 isotype control (hIgG 1) and V3 to CD 64-co-expressing SU-DHL-1 cells (FcgammaRI; SU-DHL-1-CD 64) as a function of antibody concentration (FIG. 1B). A graph showing binding of AB101, human IgG1 isotype control (hIgG 1) and V3 to CD 16-coexpressing SU-DHL-1 cells (FcgammaRIII; SU-DHL-1-CD 16) as a function of antibody concentration (FIG. 1C). A graph showing binding of AB101, human IgG1 isotype control (hIgG 1) and V3 to CD 32-coexpressing SU-DHL-1 cells (FcgammaRII; SU-DHL-1-CD 32) as a function of antibody concentration (FIG. 1D).
FIGS. 2A-2F are absorbance series plots showing the binding of anti-CD 163 antibodies to recombinant CD163 protein as measured by ELISA. Absorbance plots showing binding of AB101 parent antibody, V1 and hIgG1 isotype control to recombinant CD163 protein (fig. 2A). Absorbance plots showing binding of AB101 parent antibody, V2 and hIgG1 isotype controls to recombinant CD163 protein (fig. 2B). Absorbance plots showing binding of AB101 parent antibody, V3 and hIgG1 isotype control to recombinant CD163 protein (fig. 2C). Absorbance plots showing binding of AB101 parent antibody, V4 and hIgG1 isotype control to recombinant CD163 protein (fig. 2D). Absorbance plots showing binding of AB101 parent antibody, V5 and hIgG1 isotype control to recombinant CD163 protein (fig. 2E). Absorbance plots showing binding of AB101 parent antibody, V6 and hIgG1 isotype control to recombinant CD163 protein (fig. 2F).
FIG. 3 shows the binding of anti-CD 163 antibodies to M2c macrophages, measured as geometric median fluorescence intensity (gMFI) using Fluorescence Activated Cell Sorting (FACS). The figure shows improved binding of V1 and V4 compared to the parent AB101 antibody. The hIgG1 isotype control showed no binding in this assay.
Fig. 4A-4C are a series of bar graphs showing the binding of anti-CD 163 antibodies to CD163 positive cells (dendritic cells (DCs), classical monocytes, intermediate monocytes, and non-classical monocytes) isolated from whole blood. The antibodies tested included the commercially available anti-CD 163 antibody (R20), the parent AB101 antibody, V3, the hIgG1 isotype control (IgG 1) and the murine IgG1 isotype control (mIgG 1). V3 showed improved binding to CD163 positive cells in whole blood. anti-CD 163 antibodies were administered at three different concentrations: 1 μg/mL (FIG. 4A), 3 μg/mL (FIG. 4B), and 10 μg/mL (FIG. 4C).
FIGS. 5A-5C are a series of graphs showing the alleviation of M2C macrophage mediated immunosuppression in M2C/CD8+ T cell co-cultures. A graph showing recovery of cd8+ T cell counts (proliferation) after treatment with different concentrations of anti-CD 163 antibody (fig. 5A). The antibodies tested included the parent AB101 antibody, V3 and human IgG1 isotype controls. V3 shows about 40-fold higher potency compared to AB101 in alleviating M2c macrophage-mediated inhibition of cd8+ T cell proliferation. A graph showing recovery of cd8+ T cell perforin secretion following treatment with different concentrations of anti-CD 163 antibody (fig. 5B). V3 showed significantly higher efficacy than AB101 in alleviating M2c macrophage mediated inhibition of cd8+ T cell perforin secretion. A graph showing recovery of cd8+ T cell cytokine (ifnγ) secretion following treatment with different concentrations of anti-CD 163 antibody (fig. 5C). V3 showed significantly higher efficacy than AB101 in alleviating M2c macrophage mediated inhibition of cd8+ T cell ifnγ secretion.
Detailed description of the disclosure
Disclosed herein are antibodies directed to CD163 + Antibodies that cell specifically bind. In some embodiments, CD163 + The cells are immunosuppressive bone marrow cells. In some embodiments, CD163 + The cells are bone marrow cells expressing human CD163. In some embodiments, CD163 + The immunosuppressive bone marrow cells are human macrophages. In some embodiments, human CD163 + Immunosuppressive macrophages are M2 or M2-like macrophages. In some embodiments, the immunosuppressive myeloid cells are myeloid-derived suppressor cells (MDSCs). In some embodiments, the human macrophages express high levels of CD163 (CD 163 High height ). 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.
In some embodiments, the macrophage is a lung macrophage. In some embodiments, the macrophage is an Alveolar Macrophage (AM). In some embodiments, the macrophage is a mesenchymal macrophage.
Monocytes and macrophages exposed to certain inflammatory cytokines or microorganism-related 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 activated classically with IFN- γ and lipopolysaccharide) or anti-inflammatory (when activated in lieu of IL-4 or IL-10), respectively, whereas macrophages with M1 or M2 phenotype in vivo or ex vivo are defined as M1-like or M2-like macrophages. In some embodiments, M2 macrophages are produced by their exposure to certain cytokines. In some embodiments, M2 macrophages differentiate through IL-4, IL-10, IL-13, or a combination thereof.
M2-like macrophages have functions and phenotypes corresponding to M2 macrophages and subtypes thereof. M2-like macrophages are any macrophages that have a subset of the functional or phenotypic characteristics of M2 macrophages in vivo or ex vivo.
In some embodiments, the antibodies of the present disclosure have high avidity and specific binding to immunosuppressive bone marrow cells (particularly macrophages, such as M2 and M2-like macrophages). In some embodiments, the antibodies specifically bind to M2 and M2-like macrophages residing, infiltrating, or recruiting into the fibrotic tissue. In some embodiments, the antibodies as disclosed herein do not bind detectably to M1 or M1-like macrophages. M1 activated macrophages express transcription factors such as interferon regulatory factor (IRF 5), nuclear factor of kappa light polypeptide gene enhancer (NF-. Kappa.B), activator protein (AP-1) and STAT 1. M1 macrophages secrete pro-inflammatory cytokines such as IFN-gamma, IL-1, IL-6, IL-12, IL-23 and TNF alpha. M1 macrophages have a function and phenotype corresponding to M1 macrophages. M1-like macrophages are any macrophages that have a subset of the functional or phenotypic characteristics of M1 macrophages in vivo or ex vivo.
In some embodiments, the antibodies of the disclosure do not bind to primary human cells. In some embodiments, the antibodies of the present disclosure do not bind to hematopoietic stem cells, leukocytes, T cells, B cells, NK cells, and granulocytes.
According to the functional characteristics of macrophages, including macrophages and T helper cells (CD 4 + ) The relationship between types Th1 and Th2, macrophages generally fall into two categories, namely M1-like pro-inflammatory macrophages and M2-like immunosuppressive macrophages. M1 macrophages are a "classical" model and can be produced by IFN- γ and innate immune activators such as pathogen-associated molecular patterns (PAMPs) (e.g., lipopolysaccharide (LPS)) or injury-associated molecular patterns (DAMP) as well as inflammatory cytokines (e.g., tumor necrosis factor- α (TNF- α)). In addition, T cell dependent macrophage activation via the CD40-CD40 ligand pathway induces M1 differentiation. M1 macrophages have pro-inflammatory, bactericidal and cytotoxic functions. These macrophages promote antigen-dependent induction of Th1 cells and Th1 and CD8 + Activation of T cells. In some embodiments, the M1-like macrophages are characterized by surface marker expression as measured by flow cytometry and have CD80 + CD86 + CD163 Low/-) Or CD206 Low/-) Phenotype. M1 macrophages secrete IL-12 and low levels of IL-10 and/or TGF-beta.
In contrast, M2-like immunosuppressive macrophages are a "surrogate" or "non-classical" activation model that can be generated in vitro by IL-4 or IL-10, are anti-inflammatory, and promote wound healing and tissue repair. In some embodiments, M2-like immunosuppressive macrophages are polarized from monocyte-derived macrophages and recruited by factors secreted into tissues in need of wound healing and/or other forms of tissue repair. M2-like immunosuppressive macrophages are the predominant macrophage types involved in tissue regeneration such as activation and stimulation of proliferation of fibroblasts. M2-like macrophages express surface markers CD15, CD23, CD64, CD68, CD163 High height 、CD204 High height 、CD206 High height And/or other M2 macrophage markers, as determined by flow cytometry. M2 macrophages secrete high levels of IL-10 and TGF- β1 and low levels of IL-12.
Subtypes of M2 macrophages include the M2a, M2b, M2c and M2d subtypes. M2a macrophages are induced by IL-4 and IL-13, causing up-regulation of expression of CD163, arginase-1, the mannose receptor MRC1 (CD 206), antigen presentation by the MHC II system, and production of IL-10 and TGF- β, leading to tissue regeneration and inhibition of pro-inflammatory molecules to prevent inflammatory responses. M2b macrophages produce IL-1, IL-6, IL-10 and TNF- α as responses to immune complexes. M2c macrophages are induced by IL-10, transforming growth factor beta (TGF- β) and glucocorticoid exposure and produce IL-10 and TGF- β, resulting in inhibition of inflammatory responses. The M2d subtype is activated as a response to IL-6 and adenosine.
The macrophage population may be plastic and differentiate into an M1 or M2 phenotype according to the environment (e.g., tissue environment) such as the cytokine environments described above. Macrophage populations may also shift phenotypes during response. For example, an initial tissue injury or injury (e.g., pathogen, autoimmune, or mechanically mediated injury) may first result in a pro-inflammatory environment that promotes the M1 phenotype, and then transition to the M2 phenotype during a regression/rehabilitation phase that may include wound healing and/or tissue regeneration. However, and without wishing to be bound by theory, excessive wound healing and/or tissue regeneration mediated by M2 or M2-like macrophages may lead to fibrotic pathogenesis (i.e., "fibrosis"), potentially leading to extensive tissue scar formation and organ dysfunction. Such excessive wound healing and/or tissue regeneration may be the result of chronic injury or injury such as chronic inflammation.
Thus, there remains a need to identify compounds and methods that improve immunotherapy for the treatment of fibrosis.
CD163 (scavenger receptor is cysteine-rich protein type 1M 130; hemoglobin scavenger receptor) is a cell surface protein that functions as a scavenger receptor for the hemoglobin-haptoglobin complex and protects tissues from free hemoglobin-mediated oxidative damage. Four subtypes of the CD163 protein have been reported, with molecular weights of 125,457 Da, 125,982Da, 121,319 Da and 124,958Da, respectively. Subtype 1 is the most common subtype of CD163, has a molecular weight of 125,457 da and consists of a 1115 amino acid residue polypeptide comprising an extracellular domain, a transmembrane segment and a cytoplasmic tail. The extracellular domain comprises nine cysteine-rich repeat domains. Subtype 1 of the CD163 protein has four N-linked glycosylation sites and shows two distinct bands at-150 kDa and-130 kDa in SDS-PAGE of the CD163 protein under reducing conditions in M2 macrophages.
CD163 mRNA expression is generally limited to bone marrow cells, but is also expressed by certain human cancers. CD163 has also been reported to be a macrophage scavenger receptor and promote immunosuppression. In some embodiments, the interaction of the hemoglobin-haptoglobin complex with CD163 induces secretion of the immunosuppressive cytokine IL-10 and expression of heme-oxygenase-1 (HO-1). HO-1 production of anti-inflammatory metabolite Fe 2+ CO and bilirubin.
In humans, soluble CD163 occurs via ectodomain shedding and is reported to have anti-inflammatory properties such as down-regulating T cell responses, including lymphocyte proliferation stimulated by Phytohemagglutinin (PHA) or 12-O-tetradecanoyl phorbol-13-acetate (TPA).
Antibodies targeting CD163 have been shown to modulate the innate immune response of CD163 expressing macrophages. For example, the antibody RM3/1 to CD163 is a mouse monoclonal IgG1 (kappa light chain) raised against human monocytes. The RM3/1 antibody binds to cysteine-rich domain 9 of human CD163, reduces LPS-induced TNF alpha, and enhances IL-10 secretion by macrophages.
Certain terms
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which claimed subject matter belongs. In general, the nomenclature and techniques described herein used in connection with immunology, oncology, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization are those well known and commonly employed in the art. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any claimed subject matter. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes more than one antibody, and reference to "an antibody" in some embodiments includes a plurality of antibodies, and so forth.
As used herein, unless the context clearly indicates otherwise, all numbers or ranges of numbers include all integers within or encompassing such ranges as well as fractions of values or integers within or encompassing such ranges. Thus, for example, reference to a range of 90% -100% includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and the like. In another example, references to a range of 1-5,000 times include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5 times, etc., 2.1, 2.2, 2.3, 2.4, 2.5 times, etc., and the like.
As used herein, an "about" number is meant to include the number and ranges from 10% below the number to 10% above the number. "about" a range means 10% below the lower limit of the range, spanning 10% above the upper limit of the range.
"percent identity" and "% identity" refer to the degree to which two sequences (nucleotides or amino acids) have identical residues at identical positions in an alignment. For example, "amino acid sequence is Y X% identical" refers to the% identity of an amino acid sequence to SEQ ID NO: Y and specifies that X% of the residues in the amino acid sequence are identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using a computer program. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, comput Appl biosci.1988Mar;4 (1): 11-7), FASTA (Pearson and Lipman, proc Natl Acad Sci USA.1988Apr;85 (8): 2444-8;Pearson,Methods Enzymol.1990;183:63-98) and gapped BLAST (Altschul et al, nucleic Acids Res.1997Sep 1;25 (17): 3389-40), BLASTP, BLASTN or GCG (Devereux et al, nucleic Acids Res.1984Jan 11;12 (1 Pt 1): 387-95).
As used herein, "antibody" refers to a protein that binds an antigen. Antibodies typically comprise a variable domain and a constant domain in each of the heavy and light chains. Accordingly, most antibodies have heavy chain variable domains (V H ) And a light chain variable domain (V L ) Together they form the part of the antibody that binds to the antigen, sometimes referred to as the "antigen receptor". Three Complementarity Determining Regions (CDRs) exist within each variable domain, which are found within the heavy chain variable domain (V H ) And a light chain variable domain (V L ) Forms a loop and contacts the surface of the antigen. "antibodies" include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), natural antibodies, humanized antibodies, human antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, grafted antibodies, antibody fragments (e.g., a portion of a full-length antibody, typically an antigen-binding fragment or variable region thereof, e.g., a Fab fragment, F (ab') 2 Fragments and Fv fragments) and antibodies produced in vitro having antigen binding activity. The term also includes single chain antibodies, e.g., single chain Fv (sFv or scFv) antibodies, wherein a variable heavy chain and a variable light chain are joined together (either directly or through a peptide linker) to form a continuous polypeptide.
As used herein, "complementarity determining regions", "CDRs" and "hypervariable regions" refer to the portions of the variable domains in an antibody that determine the binding specificity of the antibody to its specific antigen. As mentioned, a single variable region of an antibody polypeptide will typically comprise three CDRs, typically designated CDR1, CDR2 and CDR3. More particularly, the heavy chain variable region may comprise CDRs designated H1, H2, and H3; likewise, the light chain variable region may comprise CDRs L1, L2 and L3. More than one method may be used to define CDRs. The current technology utilizes different numbering schemes with different definitions of CDR length and position. For example, the Kabat numbering scheme is based on sequence alignment, and uses the "variability parameter" of a given amino acid position (the number of different amino acids at a given position divided by the frequency of the most frequently occurring amino acids at that position) to predict CDRs. On the other hand, the Chothia numbering scheme is a structure-based numbering scheme in which the loop structure is defined as CDR for comparison to the antibody crystal structure. The Martin numbering scheme focuses on the structural alignment of different framework regions of non-conventional length. The IMGT numbering scheme is a standardized numbering system based on sequence alignments from complete reference gene databases (including the entire immunoglobulin superfamily). The Honneger numbering scheme (AHo's) is based on structural alignment of the 3D structure of the variable region and uses structurally conserved ca positions to infer framework and CDR lengths. Those skilled in the art will note that the definition of CDR will vary depending on the method used. Any method of defining CDRs is contemplated for the sequences disclosed herein.
The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy. For therapeutic purposes, "mammal" refers to any animal classified as a mammal, including humans, domestic animals, and farm animals, as well as laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human.
As used herein, the terms "treatment", "treatment" and the like, in some cases, refer to administration of an agent or performing a procedure for the purpose of achieving an effect. In some embodiments, the effect is prophylactic in terms of completely or partially preventing a disease or symptom thereof, and/or therapeutic in terms of achieving a partial or complete cure for the disease and/or symptom of the disease. As used herein, "treatment" includes treatment of a disease or disorder (e.g., fibrosis) in a mammal, particularly a human, and includes: (a) Preventing a disease or symptoms of a disease from occurring in a subject susceptible to the disease but not yet diagnosed with the disease (e.g., including a disease associated with or caused by a primary disease); (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease. In some embodiments, treatment refers to any sign of success in treating or ameliorating or preventing fibrosis, including any objective or subjective parameter, such as reduction in symptoms, alleviation, diminishment, or making a disease condition more tolerable to a subject; slowing the rate of degradation or decay; or the end point of the degradation is less debilitating. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Accordingly, the term "treating" includes administration of a compound or agent of the present disclosure to prevent or delay, alleviate or prevent or inhibit the development of symptoms or conditions associated with a disease (e.g., fibrosis). The term "therapeutic effect" refers to reducing, eliminating or preventing a disease, symptom of a disease, or side effect of a disease in a subject. A disease or disorder in a subject is "treated" if, upon receiving a therapeutic amount of an antibody of the present disclosure, the patient exhibits an observable and/or measurable change in a parameter or symptom of the disease or disorder.
In some embodiments, "inducing a response" refers to reducing or lessening the sign or symptom of a disease in a subject, and particularly includes, but is not limited to, an extension of survival.
The term "affinity" refers to the resistance of a complex of two or more agents to dissociation after dilution.
In some embodiments, antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (native sequence Fc region or amino acid sequence variant Fc region) and that vary with antibody isotype.
"Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody.
As used herein, "human effector cells" refers to leukocytes which express one or more fcrs and perform effector functions. For example, the cell expresses at least fcγriii and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include, but are not limited to, peripheral Blood Mononuclear Cells (PBMCs), NK cells, monocytes, macrophages, cytotoxic T cells, and neutrophils.
"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 binding of the first component of the complement system (C1 q) to antibodies (appropriate subclasses of antibodies) that bind to their cognate antigens. To assess complement activation, for example, CDC assays are performed.
An "internalized" antibody is an antibody that is taken up (i.e., entered) by a cell upon binding to an antigen (e.g., a cell surface polypeptide or receptor) on a mammalian cell. Internalizing antibodies include antibody fragments, human or chimeric antibodies, and antibody conjugates. In some cases, internalization of an antibody (e.g., such as disclosed herein) alters the biology of the cell, causing it to alter its function.
An "antigen binding domain", "antigen binding region" or "antigen binding site" is a portion of an antibody that comprises amino acid residues (or other portions) that interact with an antigen and contribute to the specificity and affinity of the antibody for the antigen. For antibodies that specifically bind to their antigen, this will include at least a portion of at least one CDR domain of the antibody.
The antigen binding region of an antibody is referred to as the "paratope" which binds an antigenic determinant, i.e., an "epitope" of an antigen, i.e., the portion of an antigen molecule that is capable of being bound by an antibody. In some embodiments, the antigenic material has one or more portions that can be recognized by the antibody, i.e., more than one epitope, and thus a single antigenic material is specifically bound by different antibodies, each specific for a different epitope. In some embodiments, the epitope comprises a discontinuous portion of the antigen. For example, in a polypeptide, amino acid residues that are discontinuous in the primary sequence of the polypeptide but sufficiently close to each other in the tertiary and quaternary structure of the polypeptide to be bound by an antigen binding protein constitute an epitope.
An "antibody fragment" comprises a portion of an intact antibody. In some embodiments, the antibody fragment comprises the antigen binding or variable regions of an intact antibody.
The terms "antigen binding portion of an antibody", "antigen binding fragment", "antigen binding domain", "antibody fragment" are used interchangeably herein to refer to the retention of an antibody with an antigenOne or more fragments of the ability to specifically bind. Non-limiting examples of antibody fragments included within such terms include, but are not limited to, (i) Fab fragments, defined by V L 、V H 、C L And C H1 A monovalent fragment of a domain; (ii) F (ab') 2 A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) From V H And C H Fd fragments of domain composition; (iv) V comprising antibody single arm L And V H Fv fragments of the domains; (v) Comprising V H The dAb fragment of the domain (Ward et al, nature 341 (6242): 544-6 (1989)); and (vi) isolated CDRs. Also included are "half" antibodies comprising a single heavy chain and a single light chain. Other forms of single chain antibodies, such as diabodies, are also included herein.
"functional antibody fragment" as used herein refers in the context to an antibody fragment that not only binds to an antigen of an antibody but also has functional properties that characterize an intact antibody. For example, if the function of an antibody is dependent on having an Fc domain capable of achieving an effector function (such as ADCC), a functional fragment will have such a function. It is hypothesized that when the antibodies of the present disclosure comprise an Fc moiety that binds to macrophage Fc receptors, such as CD16 (fcγriiia) or CD64 (fcγri) in some embodiments, the antibodies of the present disclosure are effective in modulating the functional state of macrophages, such as tissue resident or infiltrating macrophages, or redirecting or inhibiting M2 state macrophages.
The expression "functional fragment or analog" of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one that binds IgE immunoglobulins to prevent or greatly reduce the ability of such molecules to bind to the high affinity receptor fcyri.
An "antigen binding protein" is a protein that comprises a portion that comprises an antigen binding portion of an antibody, optionally together with a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to an antigen.
An "intact" antibody is a polypeptide comprisingAntigen binding site and C L And at least heavy chain constant domain C H 1、C H 2 and C H 3. In some embodiments, the constant domain is a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof.
The term "recombinant antibody" as used herein refers to a recombinant antibody comprising an antigen binding domain of a first antibody (such as, for example, CDRs, V H Region or complete light chain) and antibodies from domains of one or more other antibodies or proteins. Chimeric, hybrid and humanized antibodies are examples of recombinant antibodies.
A "CDR grafted antibody" is an antibody that comprises one or more CDRs derived from an antibody or isotype of one species and a framework of another antibody or isotype of the same or a different species.
The term "human antibody" includes all antibodies having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all the variable and constant domains of an antibody are derived from a human immunoglobulin sequence (referred to as a "fully human antibody").
As used herein, the term "affinity" refers to the equilibrium constant for reversible binding of two agents, and is expressed as the equilibrium dissociation constant K D . In one embodiment, the antibody or antigen binding fragment thereof exhibits a binding activity as defined by K D Measured at 10 for CD163 -6 M or less in the range or as low as 10 -16 M or less (e.g., about 10 -7 M、10 -8 M、10 -9 M、10 -10 M、10 -11 M、10 -12 M、10 -13 M、10 -14 M、10 -15 M、10 -16 M or less). In certain embodiments, the antibodies as described herein are in the following K D Specifically binds to human CD163 (huCD 163) polypeptide: less than or equal to 10 -4 M, less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, less than or equal to 10 -7 M is less than or equal to 10 -8 M。
The term "preferentially binds" or "specifically binds" means that an antibody or fragment thereof binds to an epitope with greater affinity than it binds to an unrelated amino acid sequence and is non-toxic at the level of administration at which they are formulated for human use if cross-reacted with other polypeptides containing the epitope. In some embodiments, such affinity is at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the antibody or fragment thereof for the unrelated amino acid sequence.
The term "specific" refers to the situation in which an antibody will not preferentially bind to molecules other than an antigen containing an epitope recognized by the antibody. The term also applies, for example, to the case where an antigen binding domain is specific for a particular epitope carried by a number of antigens, in which case an antibody or antigen binding fragment thereof carrying the antigen binding domain will be able to bind to a different antigen carrying the epitope.
As used herein, if the antibody is present at a detectable level (preferably at greater than or equal to about 10 4 M -1 Or greater than or equal to about 10 5 M -1 Greater than or equal to about 10 6 M -1 Greater than or equal to about 10 7 M -1 Or greater than or equal to 10 9 M -1 Affinity constant (association constant) K of (a) a A ) By reacting with an antigen, an antibody is said to have "immunospecificity" or "specificity" for an antigen or to "specifically bind" to an antigen.
As used herein, the term "monospecific" refers to an antibody composition comprising antibodies that exhibit preferential affinity for a particular epitope. In some embodiments, the monospecific antibody preparation consists of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 99.9% antibody with specific binding activity for a particular antigen.
The term "polypeptide" is used in its conventional sense, i.e. in the amino acid sequence. The polypeptide is not limited to a specific length of product. Peptides, oligopeptides and proteins are included within the definition of polypeptide, and these terms are used interchangeably herein unless specifically indicated otherwise. The term also does not refer to or exclude post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. In some embodiments, the polypeptide is an intact protein or a subsequence thereof. In the context of antibodies of the present disclosure, a particular polypeptide of interest is an amino acid subsequence that comprises CDRs and is capable of binding to human M2 macrophages or CD163 protein expressed by such cells.
As used herein, "substantially pure" and "substantially free" refer to a solution or suspension containing less than about 20% or less of foreign material, about 10% or less of foreign material, about 5% or less of foreign material, about 4% or less of foreign material, about 3% or less of foreign material, about 2% or less of foreign material, or about 1% or less of foreign material.
The term "isolated" refers to a protein (e.g., an antibody), nucleic acid, or other substance that is substantially free of other cellular material and/or chemicals. In some embodiments, the antibodies or antigen binding fragments thereof of the present disclosure and the nucleic acid are isolated. In some embodiments, the antibodies or antigen binding fragments thereof and nucleic acids of the present disclosure are substantially pure.
When applied to polypeptides, "isolated" generally means a polypeptide that is separate from other proteins and nucleic acids that naturally occur together. Preferably, the polypeptide is also isolated from a substance used to purify it, such as an antibody or a gel matrix (polyacrylamide). In some cases, the term means a polypeptide or a portion thereof that, in view of its origin or manipulation: (i) An expression product present in the host cell as part of the expression vector; or (ii) to proteins or other chemical moieties other than those to which it is linked in nature; or (iii) are not present in nature, e.g., by chemical manipulation by attaching or adding at least one hydrophobic moiety to the protein such that the protein is in a form not present in nature. "isolated" also means the following proteins: (i) chemically synthesized; or (ii) expressed in a host cell and purified from the protein of interest and contaminating proteins.
The term "effective amount" as used herein refers to an amount of an antibody or antigen-binding portion thereof as described herein that is sufficient to induce a response when administered to a subject, e.g., as described herein, sufficient to effect treatment, prognosis, or diagnosis of a disease associated with macrophage activity. The therapeutically effective amount of an antibody provided herein, when used alone or in combination, will vary depending on the relative activity of the antibody and the composition (e.g., in terms of treatment/reduction/amelioration of fibrosis) and depending on the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the mode of administration, and the like, and in some cases, the therapeutically effective amount of the antibody is readily determined by one of ordinary skill in the art.
The term "therapeutically effective amount" generally refers to an amount of an antibody or drug effective to "treat" a disease or disorder in a subject or mammal. In some embodiments, the compositions described herein are administered to a subject in an amount effective to produce some desired therapeutic effect by inhibiting a disease or disorder as described herein at a reasonable benefit/risk ratio applicable to any medical treatment. A therapeutically effective amount is an amount that at least partially achieves a desired therapeutic or prophylactic effect in an organ or tissue. The amount of antibody required to bring about the prophylactic and/or therapeutic treatment of a disease or disorder is not itself fixed. In some embodiments, the amount of antibody administered varies with the type of disease, the prevalence of the disease, and the size of the mammal afflicted with the disease or disorder. The term "therapeutically effective" when used in connection with a method of treatment involving the administration of a therapeutic agent following the appearance of a symptom of a disease or disorder in a subject means that one or more signs or symptoms of the disease or disorder are ameliorated or eliminated following treatment.
The effective response of the present disclosure is achieved when the subject experiences a partial or complete alleviation or reduction of the signs or symptoms of the disease, and in the case of fibrotic treatment, includes, but is not limited to, amelioration of symptoms, prolonged progression, cure, alleviation, prolonged survival, or other objective response, among others. In some embodiments, the expected progression-free survival time is measured in months to years, depending on prognostic factors including the number of relapses, disease stage, and other factors. Extended survival includes, but is not limited to, a period of at least 1 month (mo.), about at least 2 months, about at least 3 months, about at least 4 months, about at least 6 months, about at least 1 year, about at least 2 years, about at least 3 years, etc. Total survival is also measured, for example, in months to years. Alternatively, in some embodiments, the effective response is that the symptoms of the subject remain static. Further indications of therapeutic indications are described in more detail below.
In some embodiments, the administration of the therapeutic agent is performed in a prophylactic manner prior to the symptomatic manifestation of the undesired disease or disorder, such that the disease or disorder is prevented or, alternatively, its progression is delayed. Thus, when used in conjunction with a prophylactic method, the term "therapeutically effective" means that a smaller number of subjects (on average) develop an undesired disease or disorder or symptom severity progression after treatment.
The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not generally produce allergies or similar adverse reactions (such as gastric discomfort, dizziness, etc.) when administered to humans.
The term "contacting" is defined herein as the manner in which a composition as provided herein is brought into physical proximity with a cell, organ, tissue or fluid as described herein.
As used herein, the term "primary fibrotic disease" means a disease in which the pathology of the disease is driven by fibrosis. Exemplary primary fibrotic diseases include, but are not limited to, cystic fibrosis, idiopathic pulmonary fibrosis, cirrhosis, systemic sclerosis (SSc), scleroderma graft versus host disease (GvHD), nephrogenic systemic fibrosis, and radiofibrosis.
As used herein, the term "secondary fibrotic disease" means a disease in which fibrosis occurs as a sequelae of non-fibrotic disease. In some cases, the secondary fibrosis is cancer-associated fibrosis. Another example of a secondary fibrotic disease is pulmonary fibrosis associated with paracoccidioidosis (paracoccidioidosis).
Antibodies to
In certain embodiments, disclosed herein are methods of treating or preventing human CD163 + Antibodies that specifically bind to CD163 proteins expressed on cells. In some embodiments, CD163 + The cells are immunosuppressive bone marrow cells. In some embodiments, the immunosuppressive bone marrow cell is a human macrophage. In some embodiments, binding of an antibody disclosed herein alters expression of at least one marker on a human macrophage.
In some embodiments, the antibodies disclosed herein bind to huCD163 protein expressed on human M2 or M2-like immunosuppressive macrophages. In some embodiments, the antibody specifically binds to a CD163 protein, which is about 140kDa huCD163 glycoform. In some embodiments, the antibody specifically binds to extracellular domain 3 of huCD 163. In some embodiments, the antibody specifically binds to extracellular domain 4 of huCD 163. In some embodiments, the antibody specifically binds to extracellular domain 3 and extracellular domain 4 of huCD 163. In some embodiments, the antibody specifically binds to huCD163, resulting in a conformational change of huCD 163. In some embodiments, conformational changes of huCD163 expose extracellular domains 2, 5, and 9 of huCD 163. In some embodiments, the antibody does not specifically bind the lower molecular weight (-115 kDa) huCD163 glycoform.
In some embodiments, the antibodies disclosed herein are directed to Dendritic Cells (DCs), such as, for example, bone marrow DCs (mdcs) (e.g., CD14 - HLA-DR + CD11c + mDC) are bound. In some embodiments, the antibodies disclosed herein are directed against classical monocytes (e.g., CD14 + HLA-DR + CD16 - Monocytes) are bound. In some embodiments, the antibodies disclosed herein are directed to a monocyte intermediate (e.g., CD14 + HLA-DR + CD16 + Monocytes) are bound. In some embodimentsIn the case, antibodies and non-classical monocytes disclosed herein (e.g., CD14 - HLA-DR + CD16 + Monocytes) are bound. In some embodiments, the antibodies disclosed herein bind to cancer cells. In some embodiments, the cancer cell is a lymphoma cell.
In some embodiments, the antibodies disclosed herein are directed against human CD163 + Immunosuppressive bone marrow cells bind and cause changes in the expression of certain cellular markers that characterize M2 or M2-like immunosuppressive macrophages (such as M2c macrophages), indicating functional differentiation of macrophages to non-immunosuppressive or less immunosuppressive and less pro-fibrotic states. In some embodiments, the antibodies disclosed herein bind to M2 or M2-like immunosuppressive macrophages and result in reduced expression of certain cellular markers that characterize M2 or M2-like macrophages, indicating functional differentiation of the macrophages to an altered differentiation state. In some embodiments, an antibody disclosed herein reduces CD163 + Expression of one or more of CD16, CD64, TLR2 and Siglec-15 by immunosuppressive bone marrow cells.
In some embodiments, the antibodies disclosed herein are directed against CD163 + Binding of immunosuppressive bone marrow cells results in CD163 + Immunosuppressive bone marrow cells have altered function. In some embodiments, the antibodies disclosed herein are directed against CD163 + Binding of immunosuppressive bone marrow cells results in altered expression of markers in M2 or M2-like immunosuppressive macrophages.
In some embodiments, the antibodies of the disclosure reduce immunosuppression caused by tumor-associated macrophages in a tumor microenvironment. In some embodiments, a decrease in immunosuppression of tumor-associated macrophages in the tumor microenvironment corresponds to an increase in immunostimulation, e.g., promoting production of T cell activation, T cell proliferation, NK cell activation, NK cell proliferation, or any combination thereof. In some embodiments, T cell activation and/or NK cell activation results in increased production of IFN- γ, TNF- α, perforin, or a combination thereof by T cells and/or NK cells. In some embodiments, the antibodies of the present disclosure increase immune stimulation, e.g., promote production of T cell activation, T cell proliferation, NK cell activation, NK cell proliferation, or any combination thereof. In some embodiments, T cell activation and/or NK cell activation results in increased production of IFN- γ, TNF- α, perforin, or a combination thereof by T cells and/or NK cells. In some embodiments, the antibodies of the disclosure specifically bind to CD163 protein expressed on human macrophages, wherein the human macrophages have a first immunosuppressive activity prior to binding the antibodies and a second immunosuppressive activity after binding the antibodies, and wherein the second immunosuppressive activity is lower than the first immunosuppressive activity. In various embodiments, the first and second immunosuppressive activities are each non-zero.
In some embodiments, the antibodies of the disclosure promote T cell activation and proliferation. In some embodiments, the antibodies bias the T cell population towards an anti-tumor T cell phenotype. In some embodiments, the antibody reduces or blocks the inhibition of T cell activation by bone marrow cells. In some embodiments, the antibody reduces the ability of TAMs to inhibit T cell activation, resulting in greater T cell stimulation and IL-2 production. In some embodiments, the antibody blocks the ability of TAMs to inhibit T cell activation, resulting in greater T cell stimulation and IL-2 production.
In some embodiments, the antibodies disclosed herein reduce the inhibition of T cell proliferation by bone marrow. In some embodiments, the antibody reduces TAM to CD4 + T cells and CD8 + Inhibition of both T cell activation and proliferation. In some embodiments, the antibody reduces inhibition of Th1 cell proliferation by TAM. Proliferated T cells showed CD4 + Enhanced expression of activation markers on T cells.
In some embodiments, the antibodies of the disclosure alter M2 polarized macrophages such that the macrophages exhibit an M1-like phenotype that reduces the immunosuppressive effects of the M2 macrophages. In some embodiments, the antibodies described herein affect differentiation of monocyte-derived macrophages to a less immunosuppressive and more antitumor differentiation state.
In some embodiments, the antibodies of the present disclosure reduce the pro-fibrotic function of tissue resident or infiltrating macrophages in the fibrotic tissue. In some embodiments, a decrease in the pro-fibrotic function of tissue resident or infiltrating macrophages in the fibrotic tissue corresponds to a decrease in activation and/or proliferation of fibroblasts. In some embodiments, the antibodies of the disclosure reduce activation and/or proliferation of fibroblasts. In some embodiments, the antibodies of the disclosure specifically bind to a CD163 protein expressed on a human macrophage, such as an M2 macrophage, wherein the human macrophage has a first pro-fibrotic activity prior to binding the antibody and a second pro-fibrotic activity after binding the antibody, and wherein the second pro-fibrotic activity is less than the first pro-fibrotic activity. In various embodiments, the first and second pro-fibrotic activities are each non-zero. In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the fibrosis is a secondary fibrotic disease.
In some embodiments, the antibodies of the disclosure alter M2 polarized macrophages such that the macrophages exhibit an M1-like phenotype that reduces immunosuppressive or pro-fibrotic activity of the M2 macrophages. In some embodiments, the antibodies described herein affect differentiation of monocyte-derived macrophages to a less pro-fibrotic and/or less immunosuppressive differentiated state. For example, the antibodies described herein can disrupt IL-10 signaling axes, including disrupting IL-10 mediated polarization of macrophages to the M2 phenotype and/or disrupting IL-10 production by M2 macrophages. In some embodiments, antibodies of the present disclosure alter M2-polarized macrophages such that the macrophages produce and/or secrete less immunosuppressive or pro-fibrotic cytokines, including, but not limited to, TGF- β, PDGF, VEGF, IGF-1, IL-10, and galectin-3. In some embodiments, the antibodies of the present disclosure alter M2-polarized macrophages such that the macrophages produce and/or secrete less TGF- β, PDGF, VEGF, IGF-1, galectin-3, IL-10, or a combination thereof.
In some embodiments, provided herein are antibodies that specifically bind to huCD163 expressed on human macrophages and reduce the expression of at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophages. In some embodiments, the human macrophage is an immunosuppressive macrophage. In some embodiments, the human macrophage is an M2-like immunosuppressive macrophage. In some embodiments, the human macrophage is a tissue-resident macrophage. In some embodiments, the tissue-resident macrophages reside in the lung, kidney, heart, or liver. In some embodiments, the human macrophage is a lung macrophage. In some embodiments, the human macrophage is an Alveolar Macrophage (AM). In some embodiments, the human macrophage is a stromal macrophage. In some embodiments, the human macrophage is an infiltrating macrophage.
In some embodiments, the antibodies disclosed herein bind to a CD163 protein expressed by a macrophage as a component of a complex comprising at least one other protein expressed by a macrophage. In some embodiments, the complex is a cell surface complex. In some embodiments, the complex comprises at least one other protein selected from the group consisting of galectin-1 protein, LILRB2 protein and casein kinase II protein.
In some embodiments, an antibody disclosed herein promotes CD4 + T cell activity or proliferation. In some embodiments, the antibody promotes CD4 + Expression of CD69, ICOS, OX40, PD1, LAG3 or CTLA4 by T cells.
In some embodiments, an antibody disclosed herein promotes CD8 + T cell activity or proliferation. In some embodiments, the antibody promotes CD8 + Expression of ICOS, OX40, PD1, LAG3 or CTLA4 by T cells.
In some embodiments, the antibodies disclosed herein are produced by promoting CD8 + T cells activate or proliferate to promote tumor cell killing in the tumor microenvironment. In some embodiments, the antibody promotes cytotoxic lymphocyte-mediated killing of cancer cells. In some embodiments, the antibody promotes NK cell-mediated killing of tumor cells.
In some embodiments, an antibody disclosed hereinThe body promotes the expression of IL-2 by T cells. In some embodiments, binding of an antibody of the disclosure to a CD163 protein increases CD4 + T cells, CD196 - T cells, CXCR3 + T cells, CCR4 - T cells or any combination thereof. In some embodiments, the antibody increases CD4 + CD196 - CXCR3 + CCR4 - T cells.
In some embodiments, the antibodies disclosed herein have a constant domain that binds to an Fc receptor expressed on macrophages. In some embodiments, the antibody specifically binds to huCD163 and has a constant domain that binds to an Fc receptor. In some embodiments, the antibody has a binding to CD163 + A constant domain that binds to an Fc receptor expressed on immunosuppressive bone marrow cells, such as CD16 (fcyriiia) or CD64 (fcyri). In some embodiments, huCD163 and Fc receptor are expressed on the same cell. In some embodiments, huCD163 and Fc receptor are expressed on different cells. In some embodiments, the antibody variable domain specifically binds to huCD163 and the antibody constant domain binds to the Fc receptor simultaneously.
In some embodiments, the antibodies disclosed herein bind to CD163 protein on macrophages and are internalized by macrophages.
In some embodiments, the antibodies disclosed herein are not cytotoxic to macrophages to which they bind.
In some embodiments, the antibodies disclosed herein have a constant domain that binds to an Fc receptor expressed on macrophages. In some embodiments, the antibody specifically binds to huCD163 and has a constant domain that binds to an Fc receptor. In some embodiments, the antibody has a binding to CD163 + A constant domain that binds to an Fc receptor expressed on immunosuppressive bone marrow cells, such as CD16 (fcyriiia) or CD64 (fcyri). In some embodiments, huCD163 and Fc receptor are expressed on the same cell. In some embodiments, huCD163 and Fc receptor are expressed on different cells. In some embodiments, the antibody variable domain specifically binds to huCD163 and the antibody constant domain binds to the Fc receptor simultaneously.
In certain embodiments, disclosed herein are antibodies that specifically bind to CD163 protein expressed on human M2 and M2-like macrophages, wherein the binding results in at least one of the following effects:
(a) Reduced expression by a human macrophage of at least one marker, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15;
(b) Internalization of antibodies by human macrophages;
(c) Reducing activation and/or proliferation of fibroblasts; and
(d) Reducing secretion of TGF-beta, PDGF, VEGF, IGF-1, galectin-3, IL-10 or combinations thereof by macrophages.
In some embodiments, the antibodies disclosed herein are directed against human CD163 in a tissue-resident macrophage population + Immunosuppressive bone marrow cells selectively bind, wherein the antibodies specifically bind to CD163 protein expressed on M2 macrophages of the tissue resident population. In some embodiments, the antibodies disclosed herein are directed against human CD163 in the population of infiltrating macrophages + Immunosuppressive bone marrow cells selectively bind, wherein the antibodies specifically bind to CD163 protein expressed on M2 macrophages and reduce immunosuppressive activity of the infiltrating population.
In some embodiments, the antibodies disclosed herein are directed against human CD163 in tumor microenvironment or fibrotic tissue + Immunosuppressive bone marrow cells selectively bind, wherein the antibodies specifically bind to CD163 protein expressed on M2 macrophages and reduce M2 macrophage-mediated pro-fibrotic function or immunosuppression. In some embodiments, the antibodies disclosed herein are human, humanized, or chimeric. In some embodiments, the antibodies disclosed herein are antigen binding fragments to which they bind as described.
In some embodiments, the antibodies of the present disclosure are intact immunoglobulin molecules, such as, for example, human antibodies, and those portions of humanized Ig molecules that contain an antigen binding site (i.e., paratope) or a single heavy chain and a single light chain, including those known in the artParts, e.g. Fab, F (ab') 2 Fd, scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, bispecific Fab 2 Trispecific Fab 3 Single chain binding polypeptides, dAb fragments, diabodies and other moieties also known as antigen binding fragments. In constructing an immunoglobulin molecule or fragment thereof, in some embodiments, a variable region or portion thereof is fused, linked, or otherwise linked to one or more constant regions or portions thereof to produce any of the antibodies or fragments thereof described herein. Thus, in some embodiments, the antigen binding fragment of any one of the antibodies described above is Fab, F (ab '), fd, F (ab') 2 Fv, scFv, single chain binding polypeptide (e.g., scFv with an Fc portion), or any other functional fragment thereof as described herein.
In some embodiments, the antibodies of the present disclosure belong to any immunoglobulin class and, thus, in some embodiments, have a gamma, mu, alpha, delta, or epsilon heavy chain. In some embodiments, the gamma chain is gamma 1, gamma 2, gamma 3 or gamma 4. In some embodiments, the alpha chain is α1 or α2.
In some embodiments, the antibodies of the disclosure are IgG immunoglobulins. In some embodiments, the antibodies of the disclosure are of any IgG subclass. In some embodiments, the antibody is IgG1.
In some embodiments, the antibodies of the disclosure comprise a variable light chain that is kappa or lambda. In some embodiments, the lambda chain belongs to any subtype including, for example, lambda 1, lambda 2, lambda 3 and lambda 4. In some embodiments, the light chain is kappa.
In some embodiments, the antibodies disclosed herein comprise a human variable framework region and a human constant region. In some embodiments, the antibody comprises a human light chain variable framework region and a human light chain constant region. In some embodiments, the antibody comprises a human heavy chain variable framework region and a human heavy chain constant region. In some embodiments, the antibody comprises 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.
In some embodiments, the antibodies of the present disclosure comprise a human variable framework region and a murine constant region. In some embodiments, the antibodies of the present disclosure comprise a human heavy chain variable framework region and a murine heavy chain constant region. In some embodiments, the antibodies of the present disclosure comprise a human light chain variable framework region, a murine light chain constant region, a human heavy chain variable framework region, and a murine heavy chain constant region.
In some embodiments, binding of the antibody or antigen binding fragment to CD163 protein expressed on M2 macrophages partially (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or any number therein) or fully modulates a biological function of such M2 macrophages. The activity of an antibody or antigen binding fragment is determined, for example, using in vitro assays and/or in vivo using assays well known in the art, such as those described herein or otherwise known in the art.
In some embodiments, the antibodies of the present disclosure are further modified, if desired, to alter specific properties of the antibodies while retaining desired functionality. For example, in one embodiment, the antibodies of the present disclosure are modified to alter the pharmacokinetic properties of the antibodies, including, but not limited to, in vivo stability, solubility, bioavailability, or half-life.
In some embodiments, the antibodies described herein have the following dissociation constants (K D ): about 1pM to about 10pM, about 10pM to about 20pM, about 1pM to about 29pM, about 30pM to about 40pM, about 10pM to about 100pM, or about 20pM to about 500pM.
In some embodiments, the antibodies described herein have the following dissociation constants (K D ): less than about 500pM, less than about 400pM, less than about 300pM, less than about 200pM, less than about 100pM, less than about 75pM, less than about 50pM, less than about 30pM, less than about 25pM, less than about 20pM, less than about 18pM, less than about 15pM, less than about 10pM, less than about 75.PM, less than about 5pM, less than about 2.5pM, or less than about 1pM.
In some embodiments, an antibody pair described herein is a huCD163 protein or peptideHas the following affinities: about 10 -9 M to about 10 -14 M, about 10 -10 M to about 10 -14 M, about 10 -11 M to about 10 -14 M, about 10 -12 M to about 10 -14 M, about 10 -13 M to about 10 -14 M, about 10 -10 M to about 10 -11 M, about 10 -11 M to about 10 -12 M, about 10 -12 M to about 10 -13 M or 10 -13 M to about 10 -14 M。
In some embodiments, an antibody described herein has more than one binding site. In some embodiments, the binding sites are identical to each other. In some embodiments, the binding sites are different from each other. Naturally occurring human immunoglobulins typically have two identical binding sites, while engineered antibodies, for example, have two or more different binding sites.
In some embodiments, the antibodies of the disclosure are bispecific or multispecific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. In some embodiments, the exemplary bispecific antibody binds to two different epitopes of a single antigen. In some embodiments, other such antibodies combine a first antigen binding site with a binding site for a second antigen. In some embodiments, the 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 the bispecific antibody to one or more epitopes is simultaneous with the binding of the constant domain of the bispecific antibody to the Fc receptor.
In some embodiments, the antibodies of the disclosure have two or more valencies, which are also referred to as multivalent. In some embodiments, the antibodies of the disclosure are trispecific. In some embodiments, the multivalent antibody is internalized (and/or catabolized) faster than the bivalent antibody by a cell expressing the antigen to which the antibody binds. In some embodiments, the antibodies of the present disclosure are multivalent antibodies (e.g., tetravalent antibodies) having three or more antigen binding sites. In some embodiments, within the present disclosure The multivalent antibody is produced by recombinant expression of a nucleic acid encoding an antibody polypeptide chain. In some embodiments, multivalent antibodies comprise a dimerization domain and three or more antigen binding sites. In some embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites at the amino terminus of the Fc region. In some embodiments, multivalent antibodies herein comprise from about three to about eight (but preferably four) antigen binding sites. Multivalent antibodies comprise at least one polypeptide chain (and preferably two polypeptide chains), wherein one or more polypeptide chains comprise two or more variable regions. For example, one or more polypeptide chains comprise VD1- (X1) n -VD2-(X2) n -Fc, wherein VD1 is a first variable region, VD2 is a second variable region, fc is a polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. In some embodiments, one or more polypeptide chains each independently comprise: v (V) H -C H 1-Flexible Joint-V H -C H 1-Fc region chain; or V H -C H 1-V H -C H 1-Fc region chain. In some embodiments, the multivalent antibodies herein further comprise at least two (and preferably four) light chain variable region polypeptides. In some embodiments, the multivalent antibodies herein comprise from about two to about eight light chain variable region polypeptides. In some embodiments, a light chain variable region polypeptide described herein comprises a light chain variable region. In some embodiments, the light chain variable region polypeptides described herein further comprise C L A domain.
In some embodiments, the antibodies of the present disclosure are constructed to fold into multivalent form, which in some embodiments improves binding affinity, specificity, and/or increases half-life in blood. Multivalent forms of antibodies, for example, are prepared by techniques known in the art.
In some embodiments, the antibodies of the present disclosure are SMIP or binding domain immunoglobulin fusion proteins specific for a target protein. These constructs are single chain polypeptides comprising an antigen binding domain fused to an immunoglobulin domain necessary for achieving antibody effector function.
In some embodiments, the antibodies of the present disclosure comprise a single chain binding polypeptide having a heavy chain variable region and/or a light chain variable region that binds an epitope disclosed herein, and optionally having an immunoglobulin Fc region. Such molecules are single chain variable fragments (scFv), optionally with effector function or increased half-life by the presence of an immunoglobulin Fc region.
anti-CD 163 antibodies
In certain embodiments, provided herein are antibodies that specifically bind to CD163 protein. In some embodiments, the CD163 binding antibody comprises at least one heavy chain and at least one light chain. In some embodiments, the CD163 binding antibody comprises at least one polypeptide comprising a heavy chain variable domain (V H ) And at least one heavy chain comprising a light chain variable domain (V L ) Is a light chain of (c). Each V H And V L Comprising three Complementarity Determining Regions (CDRs). V (V) H And V L And the amino acid sequence of the CDRs determine the antigen binding specificity and antigen binding strength of the antibody. V (V) H And V L The domains are summarized in table 1. The amino acid sequences of the CDRs are summarized in tables 2 and 3.
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 the group consisting of intact immunoglobulins, scFv, fab, F (ab') 2 Or disulfide-linked 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 chimeric.
anti-CD 163 antibody variable domains
Table 1. Anti-CD 163 variable domain sequences.
* The underlined text in table 1 indicates CDRs, where domain boundary annotations are based on IMGT database and CDR region annotations are based on honeyger (AHo) numbering scheme.
In certain embodiments, disclosed herein are antibodies comprising a light chain variable domain (V L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 28. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 28.
In certain embodiments, disclosed herein are antibodies comprising a light chain variable domain (V L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 30. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 30.
In certain embodiments, disclosed herein are antibodies comprising a light chain variable domain (V) having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID No. 32 L ). In some embodiments, V L Has at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% of the amino acid sequence set forth in SEQ ID NO. 3291%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 32.
In certain embodiments, disclosed herein are antibodies comprising a light chain variable domain (V L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 34. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 34.
In certain embodiments, disclosed herein are antibodies comprising a light chain variable domain (V) having an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID No. 36 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 36. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 36.
In certain embodiments, disclosed herein are antibodies comprising a light chain variable domain (V L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 38. In some embodimentsIn (c), VL has an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO. 38.
In certain embodiments, disclosed herein are antibodies comprising a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 29. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 29.
In certain embodiments, disclosed herein are antibodies comprising a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 31. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 31.
In certain embodiments, disclosed herein are antibodies comprising a heavy chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 33. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 33.
In certain embodiments, disclosed herein are antibodies comprising a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 35. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 35.
In certain embodiments, disclosed herein are antibodies comprising a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 37. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 37.
In certain embodiments, disclosed herein are antibodies comprising a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 39. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 39.
In certain embodiments, disclosed herein are antibodies comprising antibodies having a specific amino acid sequence as set forth in the group consisting of seq id no A light chain variable domain (V) of an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence L ): 28, 30, 32, 34, 36 and 38; and a heavy chain variable domain (V) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in the group consisting of H ): SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37 and SEQ ID NO. 39, provided that the antibody does not comprise the light chain variable sequence of SEQ ID NO. 40 and the heavy chain variable sequence of SEQ ID NO. 41. In some embodiments, the sequence of the antibody is 100% identical at CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3.
In certain embodiments, disclosed herein are antibodies comprising a light chain variable domain (V) having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of L ): 28, 30, 32, 34, 36 and 38; and a heavy chain variable domain (V) having an amino acid sequence at least about 80% identical to an amino acid sequence set forth in the group consisting of H ): SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37 and SEQ ID NO. 39, provided that the antibody does not comprise the light chain variable sequence of SEQ ID NO. 40 and the heavy chain variable sequence of SEQ ID NO. 41. In some embodiments, the sequence of the antibody is 100% identical at CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3.
Exemplary anti-CD 163 complementarity determining regions
TABLE 2 anti-CD 163 light chain CDR sequences
TABLE 3 anti-CD 163 heavy chain CDR sequences
In certain embodiments, disclosed herein are antibodies that bind CD163, comprising a light chain CDR1 (CDR L1) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id nos: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; light chain CDR2 (CDR L2) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 (CDR L3) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
In some embodiments, an antibody that binds to CD163 comprises a light chain CDR1 having an amino acid sequence that is 100% identical to the amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; light chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
In certain embodiments, disclosed herein are antibodies that bind CD163, comprising a heavy chain CDR1 (CDR H1) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id nos: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 (CDR H2) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 (CDR H3) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, an antibody that binds to CD163 comprises a heavy chain CDR1 having an amino acid sequence that is 100% identical to the amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; heavy chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; heavy chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise a sequence at least about 100% identical to the sequence set forth in seq id no: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In certain embodiments, disclosed herein are antibodies comprising (a) a light chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id nos: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; and (b) a heavy chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, an antibody that binds CD163 comprises (a) a light chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; and (b) a heavy chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
Exemplary antibodies
In some embodiments, the antibody is V1 and comprises a light chain variable domain having the amino acid sequence set forth in SEQ ID NO. 28 (V L ) And a heavy chain variable domain having the amino acid sequence as set forth in SEQ ID NO. 29 (V H )。
In some embodiments, the antibody is V2 and comprises a light chain variable domain having the amino acid sequence set forth in SEQ ID NO. 30 (V L ) And a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO. 31 (V H )。
In some embodiments, the antibody is V3 and comprises a light chain variable domain having the amino acid sequence set forth in SEQ ID NO. 32 (V L ) And a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO. 33 (V H )。
In some embodiments, the antibody is V4 and comprises a light chain variable domain having the amino acid sequence set forth in SEQ ID NO. 34 (V L ) And a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO. 35 (V H )。
In some embodiments, the antibody is V5 and comprises a light chain variable domain having the amino acid sequence set forth in SEQ ID NO. 36 (V L ) And a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO. 37 (V H )。
In some embodiments, the antibody is V6 and comprises a light chain variable domain having the amino acid sequence set forth in SEQ ID NO. 38 (V L ) And a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO. 39 (V H )。
In some embodiments, the antibody is V1 and comprises CDR L1 having the amino acid sequence as set forth in SEQ ID NO. 7, CDR L2 having the amino acid sequence as set forth in SEQ ID NO. 2, and CDR L3 having the amino acid sequence as set forth in SEQ ID NO. 8; and CDR H1 having the amino acid sequence as set forth in SEQ ID NO. 16, CDR H2 having the amino acid sequence as set forth in SEQ ID NO. 17 and CDR H3 having the amino acid sequence as set forth in SEQ ID NO. 18.
In some embodiments, the antibody is V2 and comprises CDR L1 having the amino acid sequence as set forth in SEQ ID NO. 7, CDR L2 having the amino acid sequence as set forth in SEQ ID NO. 9 and CDR L3 having the amino acid sequence as set forth in SEQ ID NO. 10; and CDR H1 having the amino acid sequence as set forth in SEQ ID NO. 19, CDR H2 having the amino acid sequence as set forth in SEQ ID NO. 20 and CDR H3 having the amino acid sequence as set forth in SEQ ID NO. 21.
In some embodiments, the antibody is V3 and comprises CDR L1 having the amino acid sequence as set forth in SEQ ID NO. 7, CDR L2 having the amino acid sequence as set forth in SEQ ID NO. 2, and CDR L3 having the amino acid sequence as set forth in SEQ ID NO. 11; and CDR H1 having the amino acid sequence as set forth in SEQ ID NO. 22, CDR H2 having the amino acid sequence as set forth in SEQ ID NO. 23 and CDR H3 having the amino acid sequence as set forth in SEQ ID NO. 24.
In some embodiments, the antibody is V4 and comprises CDR L1 having the amino acid sequence set forth in SEQ ID No. 7, CDR L2 having the amino acid sequence set forth in SEQ ID No. 2, and CDR L3 having the amino acid sequence set forth in SEQ ID No. 8; and CDR H1 having the amino acid sequence as set forth in SEQ ID NO. 22, CDR H2 having the amino acid sequence as set forth in SEQ ID NO. 17 and CDR H3 having the amino acid sequence as set forth in SEQ ID NO. 18.
In some embodiments, the antibody is V5 and comprises CDR L1 having the amino acid sequence as set forth in SEQ ID NO. 7, CDR L2 having the amino acid sequence as set forth in SEQ ID NO. 2, and CDR L3 having the amino acid sequence as set forth in SEQ ID NO. 10; and CDR H1 having the amino acid sequence as set forth in SEQ ID NO. 16, CDR H2 having the amino acid sequence as set forth in SEQ ID NO. 17 and CDR H3 having the amino acid sequence as set forth in SEQ ID NO. 24.
In some embodiments, the antibody is V6 and comprises CDR L1 having the amino acid sequence as set forth in SEQ ID NO. 7, CDR L2 having the amino acid sequence as set forth in SEQ ID NO. 2 and CDR L3 having the amino acid sequence as set forth in SEQ ID NO. 12; and CDR H1 having the amino acid sequence as set forth in SEQ ID NO. 19, CDR H2 having the amino acid sequence as set forth in SEQ ID NO. 17 and CDR H3 having the amino acid sequence as set forth in SEQ ID NO. 18.
In some embodiments, the antibody comprises a CDR L1 having the amino acid sequence as set forth in SEQ ID NO. 13, a CDR L2 having the amino acid sequence as set forth in SEQ ID NO. 14, and a CDR L3 having the amino acid sequence as set forth in SEQ ID NO. 15; and CDR H1 having the amino acid sequence as set forth in SEQ ID NO. 25, CDR H2 having the amino acid sequence as set forth in SEQ ID NO. 26 and CDR H3 having the amino acid sequence as set forth in SEQ ID NO. 27; with the proviso that the antibody does not comprise CDR L1 having the amino acid sequence as set forth in SEQ ID NO. 1, CDR L2 having the amino acid sequence as set forth in SEQ ID NO. 2 and CDR L3 having the amino acid sequence as set forth in SEQ ID NO. 3, CDR H1 having the amino acid sequence as set forth in SEQ ID NO. 4, CDR H2 having the amino acid sequence as set forth in SEQ ID NO. 5 and CDR H3 having the amino acid sequence as set forth in SEQ ID NO. 6.
Binding affinity and immunoreactivity
The binding affinity and/or avidity of the antibody or antigen binding fragment thereof is improved by modifying the framework regions. Any suitable method for modifying the framework regions is known in the art and is contemplated herein. The choice of one or more relevant framework amino acid positions to be changed depends on a number of criteria. One criterion for selecting the relevant framework amino acid to be altered is, for example, the relative difference in amino acid framework residues between the donor molecule and the acceptor molecule. The selection of relevant framework positions to be altered using this approach has the advantage of avoiding any subjective bias in residue determination or any bias in residue contribution to CDR binding affinity.
In some embodiments, the binding interaction is manifested as intermolecular contact with one or more amino acid residues of one or more CDRs. Antigen binding involves, for example, CDRs or CDR pairs, or in some cases, V H Chain and V L Interactions of up to all six CDRs of the chain.
The binding affinity and avidity of an antibody or antigen binding fragment may be measured by Surface Plasmon Resonance (SPR) measurement, alphaLisa assay, or equilibrium dissociation constant (K D ) Is measured by flow cytometry.
K at 0.1nM to 1000nM is disclosed herein D An antibody that specifically binds to human CD 163. In some embodiments, the antibodies are below K D Specific binding to human CD 163: about 0.1nM to about 500nM, about 0.1nM to about 100nM, about 0.1nM to about 50nM, about 0.1nM to about 20nM, about 0.1nM to about 10nM, about 0.1nM to about 5nM, about 0.1nM to about 2nM, about 0.1nM to about 1nM, about 0.1nM to about 0.5nM, about 0.5nM to about 1000nM, about 0.5nM to about 500nM, about 0.5nM to about 100nM, about 0.5nM to about 50nM, about 0.5nM to about 20nM, about 0.5nM to about 10nM, about 0.5nM to about 5nM, about 0.5nM to about 2nM, about 0.5nM to about 1nM, about 1nM to about 1000nM, about 1nM to about 500nM about 1nM to about 100nM, about 1nM to about 50nM, about 1nM to about 20nM, about 1nM to about 10nM, about 1nM to about 5nM, about 1nM to about 2nM, about 2nM to about 1000nM, about 2nM to about 500nM, about 2nM to about 100nM, about 2nM to about 50nM, about 2nM to about 20nM, about 2nM to about 10nM, about 2nM to about 5nM, about 5nM to about 1000nM, about 5nM to about 500nM, about 5nM to about 100nM, about 5nM to about 50nM, about 5nM to about 20nM, about 5nM to about 10nM, about 10nM to about 1000nM, about 10nM to about 500nM, About 10nM to about 100nM, about 10nM to about 50nM, about 10nM to about 20nM, about 20nM to about 1000nM, about 20nM to about 500nM, about 20nM to about 100nM, about 20nM to about 50nM, about 50nM to about 1000nM, about 50nM to about 500nM, about 50nM to about 100nM, about 100nM to about 500nM, about 100nM to about 1000nM, about 500nM to about 1000nM. In some embodiments, the antibody is at a K of 1.8nM, 12nM, 45nM or 89nM D Specifically binds to human CD 163.
In some embodiments, the antibodies disclosed herein are raised against a K of 0.824nM D Specifically binds to human CD 163. In some embodiments, the antibodies disclosed herein are raised against a K of 0.937nM D Specifically binds to human CD 163. In some embodiments, the antibodies disclosed herein are raised against a K of 0.964nM D Specifically binds to human CD 163. In some embodiments, the antibodies disclosed herein are raised to a K of 0.991nM D Specifically binds to human CD 163. In some embodiments, the antibodies disclosed herein are raised against a K of 1.03nM D Specifically binds to human CD 163. In some embodiments, the antibodies disclosed herein are raised against a K of 1.25nM D Specifically binds to human CD 163.
The antibodies disclosed herein bind to the myeloscavenger receptor CD163, which is highly expressed on M2 macrophages. The binding affinity between the antibodies disclosed herein and IL-10 polarized M2c macrophages is measured by flow cytometry assays.
K at 0.1nM to 1000nM is disclosed herein D An antibody that specifically binds to M2c macrophages. In some embodiments, the antibodies are below K D Specific binding to M2c macrophages: about 0.1nM to about 500nM, about 0.1nM to about 100nM, about 0.1nM to about 50nM, about 0.1nM to about 20nM, about 0.1nM to about 10nM, about 0.1nM to about 5nM, about 0.1nM to about 2nM, about 0.1nM to about 1nM, about 0.1nM to about 0.5nM, about 0.5nM to about 1000nM, about 0.5nM to about 500nM, about 0.5nM to about 100nM, about 0.5nM to about 50nM, about 0.5nM to about 20nM, about 0.5nM to about 10nM, about 0.5nM to about 5nM, about 0.5nM to about 2nM, about 0.5nM to about 1nM, about 1nM to about 1000nM, about 1nM to about 500nM, about 1nM to about 100nM, about 1nM to about 50nM, about 1nM to about 20nM, about 1nM to about 10nM,About 1nM to about 5nM, about 1nM to about 2nM, about 2nM to about 1000nM, about 2nM to about 500nM, about 2nM to about 100nM, about 2nM to about 50nM, about 2nM to about 20nM, about 2nM to about 10nM, about 2nM to about 5nM, about 5nM to about 1000nM, about 5nM to about 500nM, about 5nM to about 100nM, about 5nM to about 50nM, about 5nM to about 20nM, about 5nM to about 10nM, about 10nM to about 1000nM, about 10nM to about 500nM, about 10nM to about 100nM, about 10nM to about 50nM, about 10nM to about 20nM, about 20nM to about 1000nM, about 20nM to about 500nM, about 20nM to about 100nM, about 20nM to about 50nM to about 1000nM, about 50nM to about 500nM, about 50nM to about 100nM, about 100nM to about 500nM, about 100nM to about 1000nM, about 1000nM to about 500 nM. In some embodiments, the antibody is at a K of 7.7nM D Specifically bind to M2c macrophages.
Binding epitopes
An antibody epitope may be a linear peptide sequence (i.e., "contiguous") or may be composed of a discontinuous amino acid sequence (i.e., "conformational" or "discontinuous"). In some embodiments, the antibody recognizes one or more amino acid sequences; thus, an epitope defines more than one different amino acid sequence. The epitope recognized by an antibody is determined, for example, by peptide mapping and sequence analysis techniques well known to those skilled in the art. Binding interactions are manifested as intermolecular contacts with one or more amino acid residues of the CDRs.
The human CD163 protein is a protein encoded by the CD163 gene in humans. The amino acid sequence of human CD163 is: MSKLRMVLLEDSGSADFRRHFVNLSPFTITVVLLLSACFVTSSLGGTDKELRLVDGENKCSGRVEVKVQEEWGTVCNNGWSMEAVSVICNQLGCPTAIKAPGWANSSAGSGRIWMDHVSCRGNESALWDCKHDGWGKHSNCTHQQDAGVTCSDGSNLEMRLTRGGNMCSGRIEIKFQGRWGTVCDDNFNIDHASVICRQLECGSAVSFSGSSNFGEGSGPIWFDDLICNGNESALWNCKHQGWGKHNCDHAEDAGVICSKGADLSLRLVDGVTECSGRLEVRFQGEWGTICDDGWDSYDAAVACKQLGCPTAVTAIGRVNASKGFGHIWLDSVSCQGHEPAIWQCKHHEWGKHYCNHNEDAGVTCSDGSDLELRLRGGGSRCAGTVEVEIQRLLGKVCDRGWGLKEADVVCRQLGCGSALKTSYQVYSKIQATNTWLFLSSCNGNETSLWDCKNWQWGGLTCDHYEEAKITCSAHREPRLVGGDIPCSGRVEVKHGDTWGSICDSDFSLEAASVLCRELQCGTVVSILGGAHFGEGNGQIWAEEFQCEGHESHLSLCPVAPRPEGTCSHSRDVGVVCSRYTEIRLVNGKTPCEGRVELKTLGAWGSLCNSHWDIEDAHVLCQQLKCGVALSTPGGARFGKGNGQIWRHMFHCTGTEQHMGDCPVTALGASLCPSEQVASVICSGNQSQTLSSCNSSSLGPTRPTIPEESAVACIESGQLRLVNGGGRCAGRVEIYHEGSWGTICDDSWDLSDAHVVCRQLGCGEAINATGSAHFGEGTGPIWLDEMKCNGKESRIWQCHSHGWGQQNCRHKEDAGVICSEFMSLRLTSEASREACAGRLEVFYNGAWGTVGKSSMSETTVGVVCRQLGCADKGKINPASLDKAMSIPMWVDNVQCPKGPDTLWQCPSSPWEKRLASPSEETWITCDNKIRLQEGPTSCSGRVEIWHGGSWGTVCDDSWDLDDAQVVCQQLGCGPALKAFKEAEFGQGTGPIWLNEVKCKGNESSLWDCPARRWGHSECGHKEDAAVNCTDISVQKTPQKATTGRSSRQSSFIAVGILGVVLLAIFVALFFLTKKRRQRQRLAVSSRGENLVHQIQYREMNSCLNADDLDLMNSSGGHSEPH (SEQ ID NO: 42).
Disclosed herein are antibodies that specifically bind to an epitope in human CD 163. In some embodiments, the antibodies disclosed herein bind to an epitope comprising a discontinuous amino acid sequence. In some embodiments, the antibody binds to an epitope of human CD163 comprising amino acid sequence IGRVNASKGFGHIWLDSVSCQGHEPAI (SEQ ID NO: 43). In some embodiments, the antibody binds to an epitope of human CD163 comprising amino acid sequence VVCRQLGCGSA (SEQ ID NO: 44). In some embodiments, the antibody binds to an epitope of human CD163 comprising amino acid sequence WDCKNWQWGGLTCD (SEQ ID NO: 45). In some embodiments, the antibody hybridizes to a polypeptide comprising SEQ ID NO:43-45, and an epitope of human CD163 of amino acid sequence.
Additional antibodies that specifically bind to the epitopes disclosed herein are also disclosed herein. These additional antibodies or antigen binding fragments thereof that specifically bind to the epitopes disclosed herein can be identified using techniques known in the art. For example, computational methods are used to design epitope-specific antibodies. Nimrod et al Computational Design of Epitope-Specific Functional Antibodies, cell Reports 25,2121-2131, nov.20,2018, (incorporated herein by reference). Another method may be used to identify antibodies that bind to a particular epitope from a library of antibodies that bind to an antigen, such as the following: atypical amino acids (ncAA) p-benzoyl-L-phenylalanine (pBpa) and p-azido-L-phenylalanine (pAzF) were first incorporated into target epitopes, and then antibodies crosslinked with the ncAA-incorporated epitopes after uv irradiation were selected. Since cross-linking occurs only when the distance between the antibody and the epitope is sufficiently close, the present method allows efficient selection of antibodies that specifically bind to the target epitope. Chen et al, epitope-directed antibody selection by site-specific photocrosslinking, science Advances,6 (14), eaaz7825,01Apr 2020 (incorporated herein by reference).
Modification of antibodies
In some cases, the antibodies or antigen-binding fragments thereof are modified for various purposes using techniques known in the art, such as, for example, by 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 proteolytic tolerance, reduced antigenicity and immunogenicity, improved bioavailability, reduced toxicity, improved stability, and easier formulation.
In some cases, when the antigen-binding fragment does not contain an Fc portion, the Fc portion is added (e.g., recombinantly) to the fragment, e.g., to increase the half-life of the antigen-binding fragment in the blood circulation when administered to a subject. The selection of suitable Fc regions and methods of incorporating such fragments are known in the art. For example, the Fc region of IgG is incorporated into a polypeptide of interest so as to increase its circulatory half-life without losing its biological activity, using conventional techniques known in the art. In some embodiments, the Fc portion of the antibody is further modified to increase the half-life of the antigen binding fragment in the blood circulation when administered to a subject. Modifications, for example, are determined using means conventional in the art.
Additionally, in some embodiments, antibodies and antigen binding fragments thereof are produced or expressed such that they are free of fucose on their complex N-glycosidically linked sugar chains. Removal of fucose from complex N-glycosidically linked sugar chains is known to increase effector functions of antibodies and antigen binding fragments, including, but not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Similarly, in some cases, an epitope-binding antibody or antigen-binding fragment thereof is attached at its C-terminus to all or a portion of an immunoglobulin heavy chain derived from any antibody isotype (e.g., igG, igA, igE, igD and IgM) and any isotype subclass (particularly IgG1, igG2, igG3, and IgG 4).
Additionally, in some embodiments, the antibodies or antigen binding fragments described herein are also modified such that they are capable of crossing the blood brain barrier. Such modifications of the antibodies or antigen binding fragments described herein allow for the treatment of brain diseases such as glioblastoma multiforme (GBM). Exemplary modifications that allow proteins such as antibodies or antigen binding fragments to cross the blood brain barrier are described in U.S. patent publication 2007/0082380.
Glycosylation of immunoglobulins has been shown to have a significant impact on their effector functions, structural stability, and secretion rates from antibody-producing cells. The saccharide groups responsible for these properties are typically attached to the constant (C) region of the antibody. For example, igG at C H Glycosylation at asparagine 297 in the 2 domain is essential for the full capacity of IgG to activate the classical pathway of complement dependent cell lysis (Tao and Morrison, J Immunol 143:2595 (1989)). IgM at C H Glycosylation at asparagine 402 in the 3 domain is essential for proper assembly and cell lysis activity of the antibody (Muraoka and Shulman, J Immunol 142:695 (1989)). C to IgA antibody H 1 and C H Removal of glycosylation sites in the 3 domain, such as positions 162 and 419, resulted in intracellular degradation and at least 90% inhibition of secretion (Taylor and Wall, mol Cell Biol 8:4197 (1988)). Additionally, in some embodiments, antibodies and antigen binding fragments thereof are produced or expressed such that they are free of fucose on their complex N-glycosidically linked sugar chains. Removal of fucose from complex N-glycosidically linked sugar chains is known to increase effector functions of antibodies and antigen binding fragments, including, but not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In some embodiments, these "defucosylated" antibodies and antigen binding fragments are produced by various systems utilizing molecular cloning techniques known in the art, including, but not limited to, transgenic animals, transgenic plants, or cell lines that have been genetically engineered to no longer contain the enzymes and biochemical pathways necessary to incorporate fucose into complex N-glycosidically linked sugar chains (also known as fucosyltransferase knockout animals, plants An object or a cell). Non-limiting examples of cells engineered to be fucosyltransferase knockout cells include CHO cells, SP2/0 cells, NS0 cells, and YB2/0 cells.
Glycosylation of immunoglobulins in the variable (V) region has also been observed. Sox and Hood report that about 20% of human antibodies are glycosylated in the V region (Proc Natl Acad Sci USA 66:66:975 (1970)). Glycosylation of the V domain is thought to be caused by the occasional occurrence of the N-linked glycosylation signal Asn-Xaa-Ser/Thr in the V region sequence and has not been considered in the art to play a role in immunoglobulin function.
In some cases, glycosylation at the variable domain framework residues alters the binding interaction of the antibody with the antigen. The disclosure includes criteria for selecting a limited number of amino acids in the framework or CDRs of a humanized immunoglobulin chain for mutation (e.g., by substitution, deletion, or addition of residues) to increase antibody affinity.
In some embodiments, cysteine residues are removed or introduced into the Fc region or Fc-containing polypeptide of an antibody, thereby eliminating or increasing inter-chain disulfide bond formation in that region. In some embodiments, homodimer specific binding agents or antibodies produced using such methods exhibit improved internalization ability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).
It has been shown that sequences within CDRs cause antibodies to bind to MHC class II and trigger unwanted helper T cell responses in some cases. In some embodiments, conservative substitutions allow the antibody to retain binding activity but reduce its ability to trigger an unwanted T cell response. In one embodiment, one or more of the 20 amino acids N-terminal to the heavy or light chain are removed.
In some embodiments, antibody molecules having altered carbohydrate structures are produced resulting in altered effector activity, including antibody molecules that exhibit improved ADCC activity with no or reduced fucosylation. Various methods are known in the art to accomplish this. For example, ADCC effector activity is mediated by binding of antibody molecules to fcγriii receptorThis has been shown to be dependent on C H An N-linked glycosylated saccharide structure at Asn-297 of the 2 domain. Non-fucosylated antibodies bind fcyriii receptors with increased affinity and trigger fcyriii mediated effector functions more effectively than native fucosylated antibodies. Some host cell lines, such as Lec13 or rat hybridoma YB2/0 cell lines, naturally produce antibodies with lower levels of fucosylation. An increase in the level of bisecting carbohydrate (e.g., by recombinant production of antibodies in cells that overexpress the GnTIII enzyme) has also been determined to increase ADCC activity. In some embodiments, only one of the two fucose residues is absent sufficient to increase ADCC activity.
Covalent modifications of antibodies are also included herein. In some embodiments, covalent modification of the antibody, if applicable, is performed by chemical synthesis or by enzymatic or chemical cleavage of the antibody. In some embodiments, other types of covalent modifications are introduced by reacting the targeted amino acid residue with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues.
Cysteinyl residues most often react with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to form carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with: bromotrifluoroacetone, α -bromo- β - (5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkyl maleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuric benzoate, 2-chloromercuric-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1, 3-diazole.
In some embodiments, the histidyl residue is derivatized by reaction with diethyl pyrocarbonate at a pH of 5.5-7.0 because the agent is relatively specific for the histidyl side chain. In some embodiments, p-bromobenzoyl bromide is also useful; in some embodiments, the reaction is performed at pH 6.0 in 0.1M sodium dimethyl arsenate.
In some embodiments, lysyl and amino terminal residues are reacted with succinic anhydride or other carboxylic anhydrides. Derivatization with these agents has the effect of reversing the charge of lysyl residues. Other suitable reagents for derivatizing the alpha-amino group containing residue include imidoesters such as picoline imidoesters, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2, 4-pentanedione and transaminase catalyzed reactions with glyoxylic acid.
In some embodiments, the arginyl residues are modified by reaction with one or several conventional reagents, such as phenylglyoxal, 2, 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be carried out under basic conditions due to the high pKa of the guanidine functionality. Furthermore, in some embodiments, these reagents react with lysine groups as well as arginine epsilon-amino groups.
In some embodiments, the tyrosyl residue is specifically modified by reaction with an aromatic diazonium compound or tetranitromethane due to the particular interest in introducing a spectroscopic tag to the tyrosyl residue. In some embodiments, most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Using 125 I or 131 I iodinated tyrosyl residues to prepare the marker proteins for radioimmunoassay.
The pendant carboxyl groups (aspartyl or glutamyl) are specifically modified by reaction with a carbodiimide (R-n=c=n-R '), where R and R' are different alkyl (alkyl) groups, such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azocation-4, 4-dimethylpentyl) carbodiimide. In addition, aspartyl residues and glutamyl residues are converted to asparaginyl residues and glutaminyl residues by reaction with ammonium ions.
In some embodiments, the glutaminyl residue and the asparaginyl residue are deaminated to the corresponding glutamyl residue and aspartyl residue, respectively. These residues are deamidated under neutral or basic conditions.
Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of alpha-amino groups of lysine, arginine and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification includes chemical or enzymatic coupling of the glycoside to a specific binding agent or antibody. These procedures do not require the production of polypeptides or antibodies in host cells that have the glycosylation capacity to carry out N-linked or O-linked glycosylation. In some embodiments, the saccharide is attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) amide groups of glutamine, depending on the coupling mode used.
In some embodiments, the removal of any carbohydrate moiety present on the polypeptide or antibody is accomplished chemically or enzymatically. Chemical deglycosylation involves exposing the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in cleavage of most or all of the saccharides except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine) while maintaining the antibody intact. In some embodiments, enzymatic cleavage of the carbohydrate moiety on the antibody is achieved by using various endo-and exoglycosidases.
Another type of covalent modification includes linking the antibody to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylene, or polysaccharide polymers such as dextran. Such methods are known in the art.
The affinity for binding to a predetermined polypeptide antigen is typically modulated by introducing one or more mutations into the V-region framework (typically in regions adjacent to one or more CDRs and/or in one or more framework regions). Typically, such mutations include the introduction of conservative amino acid substitutions that disrupt or create the glycosylation site sequence but do not substantially affect the hydrophilic/hydrophobic structural properties of the polypeptide. Generally, mutations introducing proline residues are avoided.
Effector function
Examples of antibody effector functions include: c1q binding and complement dependent cytotoxicity, fc receptor binding, antibody dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Typically, fc-mediated functions involve the binding of the Fc portion of an antibody to a specialized receptor molecule, "Fc receptor" or "FcR," which is expressed by the cell whose function is to be affected.
IgG is considered the most functional immunoglobulin because it performs all functions of an immunoglobulin molecule in some embodiments. IgG is the primary Ig in serum and is also the only class of Ig that passes through the placenta. IgG also fixes complement, although the IgG4 subclass does not fix complement. Macrophages, monocytes, polymorphonuclear leukocytes (PMNs) and some lymphocytes have receptors for the Fc region of IgG. Not all subclasses bind equally well: igG2 and IgG4 do not bind to Fc receptors. As a result of binding to Fc receptors on PMNs, monocytes and macrophages, cells can now better internalize antigens in some cases. IgG is an opsonin that enhances phagocytosis. Binding of IgG to Fc receptors on other types of cells results in activation of other functions.
In certain embodiments, the FcR is a native sequence human FcR. Furthermore, preferred fcrs are fcrs that bind IgG antibodies (gamma ")), and include receptors of fcγri, fcγrii and fcγriii subclasses, including allelic variants and alternatively spliced forms of these receptors. Fcyrii receptors include fcyriia ("activating receptor") and fcyriib ("inhibitory receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor fcyriia contains an immune receptor tyrosine based activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor fcyriib contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain.
"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (fcrs) present on certain cytotoxic cells, such as Natural Killer (NK) cells, neutrophils and macrophages, enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are required for such killing. Among the major cells mediating ADCC, NK cells express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. To assess ADCC activity of a molecule of interest, in some embodiments an in vitro ADCC assay is performed. Effector cells useful in such assays include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells.
Alternatively or additionally, in some embodiments, ADCC activity of the molecule of interest is assessed in vivo, e.g., in an animal model.
In some embodiments, the antibodies of the disclosure bind to a surface membrane protein of an M2-like macrophage and are internalized by the M2-like macrophage. This internalization process is thought to be involved in the observed change in the functional immunosuppressive characteristics of these cells, i.e., the differentiation of the cells from the M2 state to a weakly activated state, without killing them or inhibiting their proliferation. In some embodiments, following internalization, the antibody reduces expression of immunosuppressive soluble factors while allowing stimulation or promotion of T cells (including CD4 + Helper T cells and cytotoxic lymphocytes) or proliferated soluble factors.
For certain therapeutic applications, an internalization process is employed for the purpose of killing target cells expressing a CD163 protein or reducing the activity or proliferation of target cells expressing a CD163 protein. The amount of antibody molecules internalized will be sufficient or sufficient to kill or inhibit the growth of the cells. Depending on the potency of the antibody or antibody conjugate, in some cases, a single antibody molecule is taken up into the cell sufficient to kill the target cell to which the antibody binds. For example, certain toxins have a high killing power such that internalization of one molecule of the toxin conjugated to an antibody is sufficient to kill the target cell.
In some embodiments, the antibodies or antigen binding fragments provided herein are conjugated or linked to a therapeutic moiety, imaging or detectable moiety or affinity tag. Methods for conjugating or linking polypeptides are well known in the art. Association (binding) between the compound and the label includes any means known in the art including, but not limited to, covalent and non-covalent interactions, chemical conjugation, and recombinant techniques. In some embodiments, the antibody or antigen binding fragment thereof is conjugated to or recombinantly engineered to have an affinity tag (e.g., a purification tag). Affinity tags, such as, for example, polyhistidine (e.g., his 6) tags, are conventional in the art.
In some embodiments, the antibody or antigen binding fragment further comprises a detectable moiety. Detection is accomplished, for example, in vitro, in vivo, or ex vivo. In vitro assays that use antibodies or antigen binding fragments thereof for, e.g., detection and/or determination (quantitative, qualitative, etc.) of huCD163 protein expressed by macrophages include, but are not limited to, e.g., ELISA, RIA, and western blot. In some embodiments, in vitro detection, diagnosis, or monitoring of an antigen of an antibody occurs by obtaining a sample (e.g., a blood sample) from a subject and testing the sample in, for example, a standard ELISA assay.
Methods of treating cancer
In certain embodiments, disclosed herein are methods of treating cancer in an individual in need thereof, comprising administering to the individual an antibody disclosed herein. In some embodiments, the present disclosure provides the use of an antibody as described herein for the manufacture of a medicament for treating cancer in a human subject. In some embodiments, the antibody specifically binds to a 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.
In certain embodiments, disclosed herein are methods of modulating immune activity in a subject in need thereof, comprising administering to the subject an antibody described herein. In some embodiments, the antibody specifically binds to a 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.
In certain embodiments, disclosed herein are methods of treating a subject with a pathological or inappropriately elevated level of M2 macrophages (e.g., an inappropriately elevated relative to a level useful to promote immune-mediated tumor cell killing in a subject), the method comprising administering to the subject an antibody described herein. In some embodiments, the antibody specifically binds to a 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.
In certain embodiments, disclosed herein are methods of modulating tumor-associated macrophage activity in a tumor microenvironment, the method comprising contacting tumor-associated macrophages with an antibody disclosed herein, wherein the method results in at least one of the following effects:
(a) Reduced expression by a human macrophage of at least one marker, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15;
(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 + Proliferation of T cells, NK cells, or any combination thereof; and
(e) Promote the killing of tumor cells in the tumor microenvironment.
In certain embodiments, disclosed herein are methods of modulating tumor-associated macrophage activity in a tumor microenvironment, the method comprising contacting tumor-associated macrophages with an antibody disclosed herein, wherein the method results in at least two of the following effects:
(a) Reduced expression by a human macrophage of at least one marker, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15;
(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 + Proliferation of T cells, NK cells, or any combination thereof; and
(e) Promote the killing of tumor cells in the tumor microenvironment.
In certain embodiments, disclosed herein are methods of modulating tumor-associated macrophage activity in a tumor microenvironment, the method comprising contacting tumor-associated macrophages with an antibody disclosed herein, wherein the method results in at least three of the following effects:
(a) Reduced expression by a human macrophage of at least one marker, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15;
(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 + Proliferation of T cells, NK cells, or any combination thereof; and
(e) Promote the killing of tumor cells in the tumor microenvironment.
In certain embodiments, disclosed herein are methods of modulating tumor-associated macrophage activity in a tumor microenvironment, the method comprising contacting tumor-associated macrophages with an antibody disclosed herein, wherein the method results in at least four of the following effects:
(a) Reduced expression by a human macrophage of at least one marker, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15;
(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 + Proliferation of T cells, NK cells, or any combination thereof; and
(e) Promote the killing of tumor cells in the tumor microenvironment.
In certain embodiments, disclosed herein are methods of modulating tumor-associated macrophage activity in a tumor microenvironment, the method comprising contacting tumor-associated macrophages with an antibody disclosed herein, wherein the method results in at least five of the following effects:
(a) Reduced expression by a human macrophage of at least one marker, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15;
(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 + Proliferation of T cells, NK cells, or any combination thereof; and
(e) Promote the killing of tumor cells in the tumor microenvironment.
In certain embodiments, disclosed herein are methods of functionally redirecting tumor-associated macrophages to reduce immunosuppression in a patient suffering from cancer, the method comprising administering to the patient a therapeutic agent that modifies CD4 in the tumor microenvironment + Or CD8 + A T cell active or proliferation effective amount of a pharmaceutical composition comprising an antibody as described herein.
In certain embodiments, disclosed herein are methods of promoting lymphocyte-mediated killing of tumor cells in a human subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody as described herein.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 28 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 28. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 28.
In some embodiments, the antibody comprises a polypeptide havingA light chain variable domain (V) of an amino acid sequence which is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 30 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 30. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 30.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 32 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 32. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 32.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 34 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 34. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 34.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 36 L ). In some embodiments, V L Has an amino acid sequence at least about 75%, 80%, 81%, and a sequence set forth in SEQ ID NO. 36,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 36.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 38 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 38. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 38.
In some embodiments, the antibody comprises a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 29. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 29.
In some embodiments, the antibody comprises a heavy chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 31 H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 31. In some embodiments, V H Having a sequence as set forth in SEQ ID NO. 31Amino acid sequence 100% identical amino acid sequence.
In some embodiments, the antibody comprises a heavy chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 33 H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 33. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 33.
In some embodiments, the antibody comprises a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 35. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 35.
In some embodiments, the antibody comprises a heavy chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 37 H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 37. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 37.
In some embodiments, the antibody comprises a heavy chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO 39 H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 39. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 39.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in the group consisting of L ): 28, 30, 32, 34, 36, 38 and 40 SEQ ID NO; and a heavy chain variable domain (V) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in the group consisting of H ): SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41, provided that the antibody does not comprise the light chain variable sequence of SEQ ID NO. 40 and the heavy chain variable sequence of SEQ ID NO. 41. In some embodiments, the sequence of the antibody is 100% identical at CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of L ): 28, 30, 32, 34, 36, 38, 40 respectively; and a heavy chain variable domain (V) having an amino acid sequence at least about 80% identical to an amino acid sequence set forth in the group consisting of H )：SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33. SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41, provided that the antibody does not comprise the light chain variable sequence of SEQ ID NO. 40 and the heavy chain variable sequence of SEQ ID NO. 41. In some embodiments, the sequence of the antibody is 100% identical at CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3.
In some embodiments, the antibody comprises a light chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
In some embodiments, the antibody comprises a light chain CDR1 having an amino acid sequence that is 100% identical to an amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; light chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
In some embodiments, the antibody comprises a heavy chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, the antibody comprises a heavy chain CDR1 having an amino acid sequence that is 100% identical to an amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; heavy chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; heavy chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, the antibody comprises: (a) A light chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; and (b) a heavy chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, the antibody comprises: (a) A light chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; and (b) a heavy chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, the present disclosure provides the use of an antibody as described herein for the manufacture of a medicament for reducing immunosuppression of tumor-associated macrophages in a human subject having cancer.
In some embodiments, the present disclosure provides the use of an antibody as described herein for the manufacture of a medicament for promoting T cell mediated killing of tumor cells in a human subject having cancer.
In some embodiments, the present disclosure provides a method of treating a human subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody as described herein, thereby reducing immunosuppression of tumor-associated macrophages in the subject.
In some embodiments, the present disclosure provides a method of treating a human subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody as described herein, thereby increasing T cell-mediated tumor cell killing in the subject.
In some embodiments, the methods disclosed herein reduce the inhibition of cd8+ T cell activation and proliferation by bone marrow cells.
In some embodiments, the antibody reduces the inhibition of CAR T cell mediated killing of cancer cells by bone marrow cells.
In some embodiments, the antibody reduces suppression of NK cell mediated killing of cancer cells by ADCC by bone marrow cells.
In some embodiments, the cancer is a lung epithelial carcinoma or sarcoma. In some embodiments, the lung cancer is lung adenocarcinoma. In some embodiments, the lung cancer is non-small cell lung cancer.
In some cases, any of the methods disclosed herein further comprise administering an additional anti-cancer therapy to the subject. Anticancer therapies include, but are not limited to, surgical therapies, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy and cytokine therapy and combinations thereof. In one embodiment, the antibody or antigen-binding fragment thereof and the anti-cancer therapy are administered simultaneously or sequentially.
In some embodiments, the additional anti-cancer therapy is immunotherapy. In some embodiments, the immunotherapy is a composition comprising a checkpoint inhibitor. In some embodiments, the additional anti-cancer therapy is an immune checkpoint inhibitor.
Methods of treating fibrosis
In certain embodiments, disclosed herein are methods of treating fibrosis (such as fibrosis associated with the presence of M2-macrophages) in a subject in need thereof, the methods comprising administering to the subject an antibody disclosed herein. In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the fibrosis is a secondary fibrotic disease. In some embodiments, the present disclosure provides the use of an antibody as described herein for the manufacture of a medicament for treating fibrosis in a human subject. In some embodiments, the antibody specifically binds to a CD163 protein expressed on a human macrophage (such as a tissue resident or infiltrating macrophage) and reduces the expression of at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophage.
In certain embodiments, disclosed herein are methods of modulating immune activity in a subject in need thereof, comprising administering to the subject an antibody described herein. In some embodiments, the antibody specifically binds to a CD163 protein expressed on a human macrophage (such as a tissue resident or infiltrating macrophage) and reduces the expression of at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophage.
In certain embodiments, disclosed herein are methods of treating a subject having a pathologically or inappropriately elevated level of M2 macrophages (e.g., an inappropriately elevated level relative to a level useful for promoting proper wound healing and/or tissue regeneration in a subject), the method comprising administering to the subject an antibody described herein. In some embodiments, the antibody specifically binds to a CD163 protein expressed on a human macrophage (such as a tissue resident or infiltrating macrophage) and reduces the expression of at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophage.
In certain embodiments, disclosed herein are methods of functionally redirecting macrophages (such as tissue resident or infiltrating M2 macrophages) to reduce the pro-fibrotic function of macrophages in a patient suffering from fibrosis (such as fibrosis associated with the presence of M2-macrophages), the method comprising administering to the patient a pharmaceutical composition comprising an antibody as described herein in an amount effective to reduce activation and/or proliferation of fibroblasts in fibrotic tissue. In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the fibrosis is a secondary fibrotic disease.
In certain embodiments, disclosed herein are methods of reducing secretion of TGF-beta, PDGF, VEGF, IGF-1, galectin-3, IL-10, or a combination thereof by macrophages (such as tissue resident or infiltrating M2 macrophages) in a human subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody as described herein.
In certain embodiments, disclosed herein are methods of modulating the activity of macrophages (such as M2 macrophages) in fibrotic tissue, the method comprising contacting the macrophages with an antibody disclosed herein, wherein the method results in at least one of the following effects:
(a) Reduced expression by a human macrophage of at least one marker, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15; or (b)
(b) Antibodies are internalized by human macrophages.
In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the fibrosis is a secondary fibrotic disease.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 28 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 28. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 28.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 30 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 30. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 30.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 32 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 32. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 32.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 34 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 34. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 34.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 36 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 36. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 36.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 38 L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 38. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 38.
In some embodiments, the antibody comprises a heavy chain variable domain (V H ). In some embodiments, V H 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% identical to the amino acid sequence set forth in SEQ ID NO. 29,96%, 97%, 98% or 99% identical amino acid sequence. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 29.
In some embodiments, the antibody comprises a heavy chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 31 H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 31. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 31.
In some embodiments, the antibody comprises a heavy chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO. 33 H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 33. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 33.
In some embodiments, the antibody comprises a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 35. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 35.
In some embodiments of the present invention, in some embodiments,the antibody comprises a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 37. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 37.
In some embodiments, the antibody comprises a heavy chain variable domain (V) having an amino acid sequence at least about 70% identical to the amino acid sequence set forth in SEQ ID NO 39 H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 39. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 39.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in the group consisting of L ): 28, 30, 32, 34, 36, 38 and 40 SEQ ID NO; and a heavy chain variable domain (V) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in the group consisting of H ): SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID No. 41, provided that the antibody does not comprise the light chain variable sequence of SEQ ID No. 40 and the heavy chain variable sequence of SEQ ID No. 41. In some embodiments, the sequence of the antibody is 100% identical at CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of L ): 28, 30, 32, 34, 36, 38, 40 respectively; and a heavy chain variable domain (V) having an amino acid sequence at least about 80% identical to an amino acid sequence set forth in the group consisting of H ): SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41, provided that the antibody does not comprise the light chain variable sequence of SEQ ID NO. 40 and the heavy chain variable sequence of SEQ ID NO. 41. In some embodiments, the sequence of the antibody is 100% identical at CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3.
In some embodiments, the antibody comprises a light chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
In some embodiments, the antibody comprises a light chain CDR1 having an amino acid sequence that is 100% identical to an amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; light chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
In some embodiments, the antibody comprises a heavy chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, the antibody comprises a heavy chain CDR1 having an amino acid sequence that is 100% identical to an amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; heavy chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; heavy chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, the antibody comprises: (a) A light chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; and (b) a heavy chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, the antibody comprises: (a) A light chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; and (b) a heavy chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, the present disclosure provides the use of an antibody as described herein for the preparation of a medicament that reduces the pro-fibrotic function of macrophages (such as tissue resident or infiltrating M2 macrophages) in a human subject suffering from fibrosis (such as fibrosis associated with the presence of M2-macrophages). In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the fibrosis is a secondary fibrotic disease.
In some embodiments, the present disclosure provides the use of an antibody as described herein for the manufacture of a medicament for reducing the activation and/or proliferation of fibroblasts in a human subject suffering from fibrosis (such as fibrosis associated with the presence of M2-macrophages). In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the fibrosis is a secondary fibrotic disease.
In some embodiments, the present disclosure provides a method of treating a human subject having fibrosis (such as fibrosis associated with the presence of M2-macrophages), the method comprising administering to the subject a therapeutically effective amount of an antibody as described herein, thereby reducing the pro-fibrotic function of macrophages (such as tissue resident or infiltrating M2 macrophages) in the subject. In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the fibrosis is a secondary fibrotic disease.
In some embodiments, the present disclosure provides a method of treating a human subject having fibrosis (such as fibrosis associated with the presence of M2-macrophages), the method comprising administering to the subject a therapeutically effective amount of an antibody as described herein, thereby reducing activation and/or proliferation of fibroblasts. In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the fibrosis is a secondary fibrotic disease.
In some embodiments, the present disclosure provides a method of treating a human patient suffering from fibrosis (such as fibrosis associated with the presence of M2-macrophages), the method comprising administering to the patient an effective amount of an antibody as described herein.
In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the primary fibrotic disease is cystic fibrosis, idiopathic pulmonary fibrosis, cirrhosis, systemic sclerosis (SSc), scleroderma Graft Versus Host Disease (GVHD), nephrogenic systemic fibrosis, and radiofibrosis. Exemplary radiofibrosis includes, for example, pulmonary fibrosis resulting from tissue repair following pneumonitis (pneumonia) caused by radiotherapy. Other soft tissues that undergo radiation therapy, for example for cancer treatment, may also exhibit radioactive fibrosis, such as that of breast tissue, head and neck tissue, nerves, heart tissue, blood vessels, bone, and muscles, tendons and ligaments. "radiofibrosis syndrome" may be used to refer to a clinical manifestation of progressive fibrotic tissue sclerosis resulting from radiation therapy.
In some embodiments, the fibrosis is a secondary fibrotic disease. In some embodiments, the fibrosis is a sequelae of an acute disease or a chronic disease. In some embodiments, the disease or disorder in which fibrosis is a sequelae is cancer, a viral infection, or an autoimmune or inflammatory disease. Exemplary infections associated with fibrosis are sepsis, HIV infection, SARS-CoV-2 infection, and malaria. Exemplary autoimmune and inflammatory diseases and disorders associated with fibrosis are sickle cell disease, type 1 diabetes, type 2 diabetes, crohn's disease, celiac disease, asthma, sarcoidosis, glomerulonephritis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis, sjogren's syndrome, scleroderma, cystic Fibrosis (CF), graft-versus-host disease, allograft rejection, sarcoidosis, hemophagocytic Lymphocytosis (HLH), inflammatory arthritis, chronic Obstructive Pulmonary Disease (COPD), asthma, osteoarthritis, and multiple sclerosis.
In some embodiments, the fibrosis is pulmonary fibrosis. Pulmonary fibrosis may be associated with pulmonary diseases or disorders. One exemplary pulmonary disease is Idiopathic Pulmonary Fibrosis (IPF). Other exemplary pulmonary diseases and disorders include, but are not limited to, interstitial Lung Disease (ILD), diffuse interstitial lung disease, pulmonary sarcoidosis, acute Lung Injury (ALI), acute Respiratory Distress Syndrome (ARDS), covid-19 and allergic pneumonia.
In some embodiments, the fibrosis is cardiac fibrosis. Cardiac fibrosis may be associated with heart diseases or disorders. Exemplary heart diseases and disorders include, but are not limited to, atherosclerosis, atrial fibrillation, chronic heart failure, peripheral arterial disease, and acute coronary syndrome.
In some embodiments, the fibrosis is liver fibrosis. Liver fibrosis may be associated with liver diseases or disorders. Exemplary liver diseases and disorders include, but are not limited to, liver fibrosis, non-alcoholic fatty liver disease (NAFLD), chronic plus acute liver failure, alcoholic hepatitis, non-alcoholic steatohepatitis, cirrhosis, and viral hepatitis.
In some embodiments, the fibrosis is renal fibrosis. Renal fibrosis may be associated with diseases or disorders of the kidney. Exemplary kidney diseases and disorders include, but are not limited to, acute kidney injury, acute tubular necrosis, chronic kidney disease, and renal allograft rejection.
In some embodiments, the fibrosis is retinal fibrosis.
In some embodiments, any of the methods disclosed herein further comprise administering to the subject an additional anti-fibrotic therapy. Anti-fibrotic chemotherapies include, but are not limited to, nintedanib, pirfenidone, corticosteroids (e.g., prednisone), mycophenolate mofetil/mycophenolic acid, azathioprine, ACE inhibitors (e.g., benazepril, lisinopril, and ramipril), angiotensin II receptor blockers (ARBs), antiviral agents (e.g., hepatitis c therapy), and TGF-beta inhibitors.
In some embodiments, any of the methods disclosed herein further comprise administering to the subject an additional anti-inflammatory therapy. Anti-inflammatory therapies include, but are not limited to, corticosteroids (e.g., prednisone), non-steroidal anti-inflammatory drugs (NSAIDs, e.g., ibuprofen), pirfenidone, and other immunomodulators.
In some embodiments, other antibodies, small molecule therapeutic agents, and/or other agents are combined in separate compositions for simultaneous or sequential administration. In one embodiment, simultaneous administration includes simultaneous administration or administration of one or more compositions within 30 minutes of each other. In some embodiments, the administration occurs at the same or different sites.
In some embodiments, toxicity and therapeutic efficacy of such components are determined in cell culture or experimental animals by standard pharmaceutical procedures, e.g., for determining LD 50 (dose lethal to 50% of population) and ED 50 (50% of the dose therapeutically effective for the population). In some embodiments, the dose ratio between toxic effect and therapeutic effect is the therapeutic index, and it is expressed as LD 50 /ED 50 Ratio. In some embodiments, although using shows toxic side effects A compound, but care should be taken to design a delivery system that targets such a compound to the affected tissue site to minimize potential damage to healthy cells, thereby reducing side effects.
In some embodiments, the data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in humans. Preferably, the dosage of such compounds is such that it includes ED with little or no toxicity 50 Within a range of circulating concentrations. In some embodiments, the dosage varies within this range depending on the dosage form employed and the route of administration utilized. In some embodiments, for any compound used in the methods of the present disclosure, a therapeutically effective dose is initially estimated from a cell culture assay. In some embodiments, the dose is formulated in an animal model to achieve a composition comprising IC as determined in cell culture 50 (i.e., the concentration of test compound that achieves half-maximal inhibition). The level in plasma is measured, for example, by high performance liquid chromatography. Such information, in some cases, is used to more accurately determine the dosage useful in humans.
In some embodiments, the present disclosure provides a method of treating a patient having fibrosis (such as fibrosis associated with the presence of M2-macrophages), the method comprising administering to the patient a therapeutically effective amount of an antibody as described herein, and further comprising treating the subject with a fibrochemotherapy selected from the group consisting of surgical therapy and cytokine therapy. In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the fibrosis is a secondary fibrotic disease. In some embodiments, the antibody or antigen-binding fragment thereof and another fibrotic therapy (e.g., an anti-inflammatory agent) are administered simultaneously or sequentially.
Diagnostic products and methods
In some embodiments, disclosed herein are methods of detecting huCD163 protein or M2 macrophages in a sample or subject to assess the therapeutic status of the patient or diagnose a disease or disorder associated with or associated with the activity of M2 macrophages or tissue resident macrophages or infiltrating macrophages, such as those fibrotic diseases and disorders described herein.
In vivo detection, diagnosis or monitoring of cells or tissues for soluble huCD163 protein, expression of huCD163 protein, or presence or activity of M2 macrophages, a subject is administered an antibody or antigen binding fragment as described herein, which is conjugated to a detectable moiety. In some embodiments, the detectable moiety is visualized using art-recognized methods such as, but not limited to, the following: magnetic Resonance Imaging (MRI), fluorescence, radiological imaging, light source provided by an endoscope, laparoscope, or intravascular catheter (i.e., via detection of a photoactive agent), optical scanning, positron Emission Tomography (PET) scanning, whole-body Nuclear Magnetic Resonance (NMR), radioscintigraphy, single Photon Emission Computed Tomography (SPECT), targeted Near Infrared Region (NIR) scanning, X-rays, ultrasound. Labels for detecting compounds using such methods are also known in the art. In some embodiments, visualization of the detectable moiety allows detection, diagnosis, and/or monitoring of a condition or disease associated with M2 macrophage activity or activity of another cell expressing huCD163 protein. Additional diagnostic assays utilizing antibodies specific for the desired target protein (i.e., huCD163 protein) are known in the art and are also contemplated herein.
For in vitro detection methods, samples to be obtained from a subject include, but are not limited to, blood, tissue biopsy samples, and fluids therefrom.
Thus, the present disclosure provides antibodies and antigen binding fragments thereof that are useful for detecting or diagnosing the level of M2 macrophages, tissue resident macrophages, or infiltrating macrophages associated with a fibrotic disease or disorder, potentially indicative of a need for therapeutic treatment. In other embodiments, the antibody further comprises a second dose. In some embodiments, such agents are molecules or moieties such as, for example, reporter molecules or detectable labels. Detectable labels/moieties for such detection methods are known in the art and described in more detail below. For example, a reporter molecule is any moiety that is detected using 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, colored particles or ligands, such as biotin. In some embodiments, the detectable label comprises a compound and/or element that is detected due to its particular functional and/or chemical characteristics, their use allowing the polypeptides 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 attachment to polypeptides.
In some embodiments, the polypeptides are conjugated to a wide variety of fluorescent dyes, quenchers, and haptens such as fluorescein, R-phycoerythrin, and biotin. In some embodiments, conjugation occurs during polypeptide synthesis or after polypeptide synthesis and purification.
Alternatively, in some embodiments, the antibody, antigen binding fragment, or binding protein is conjugated to a fluorescent moiety. For example, conjugation of the polypeptide to a fluorescent moiety (e.g., R-phycoerythrin, fluorescein Isothiocyanate (FITC), etc.) is accomplished using art-recognized techniques. Many commercially available fluorescent dyes and dye conjugation kits for specific applications, such as fluorescence microscopy, flow cytometry, fluorescence Activated Cell Sorting (FACS), and the like, are commercially available.
In one non-limiting embodiment, the antibody or antigen binding fragment is associated (conjugated) with a detectable label, such as a radionuclide, dye, imaging agent, or fluorescent agent for immunodetection of binding to antigen, for visualizing binding of the antibody to M2 macrophages or soluble or bound huCD163 protein in vitro and/or in vivo.
Non-limiting examples of radiolabels include for example, 32 P、 33 P、 43 K、 52 Fe、 57 Co、 64 Cu、 67 Ga、 67 Cu、 68 Ga、 71 Ge、 75 Br、 76 Br、 77 Br、 77 As、 77 Br、 81 Rb/ 81m Kr、 87m Sr、 90 Y、 97 Ru、 99 Tc、 99m Tc、 100 Pd、 101 Rh、 103 Pb、 105 Rh、 109 Pd、 111 Ag、 111 In、 113 In、 119 Sb、 121 Sn、 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 Bi 213 Bi. In some embodiments, the radiolabel is attached to the compound using conventional chemistry known in the antibody imaging arts. Radiolabeled compounds are useful in vitro diagnostic techniques and in vivo radiological imaging techniques and radioimmunotherapy.
In some embodiments, the compositions of antibodies and antigen binding fragments described herein are also useful as non-therapeutic agents (e.g., as affinity purification agents).
Pharmaceutical composition and administration
In certain embodiments, disclosed herein are pharmaceutical compositions comprising an antibody disclosed herein and a pharmaceutically acceptable excipient.
In some embodiments, the antibody comprises a light chain variable domain (V L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 28. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 28.
In some embodiments of the present invention, in some embodiments,the antibody comprises a light chain variable domain (V L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 30. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 30.
In some embodiments, the antibody comprises a light chain variable domain (V L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 32. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 32.
In some embodiments, the antibody comprises a light chain variable domain (V L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 34. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 34.
In some embodiments, the antibody comprises a light chain variable domain (V L ). In some embodiments, V L Has a nucleotide sequence as set forth in SEQ ID NO 36At 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. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 36.
In some embodiments, the antibody comprises a light chain variable domain (V L ). In some embodiments, V L Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 38. In some embodiments, V L Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 38.
In some embodiments, the antibody comprises a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 29. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 29.
In some embodiments, the antibody comprises a heavy chain variable domain (V H ). In some embodiments, V H Has at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid to the amino acid sequence set forth in SEQ ID NO. 31Sequence. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 31.
In some embodiments, the antibody comprises a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 33. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 33.
In some embodiments, the antibody comprises a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 35. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 35.
In some embodiments, the antibody comprises a heavy chain variable domain (V H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 37. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 37.
In some embodiments, the antibody comprises a polypeptide havingA heavy chain variable domain (V) of an amino acid sequence which is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO 39 H ). In some embodiments, V H Has an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 39. In some embodiments, V H Has an amino acid sequence which is 100% identical to the amino acid sequence set forth in SEQ ID NO. 39.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in the group consisting of L ): 28, 30, 32, 34, 36, 38 and 40 SEQ ID NO; and a heavy chain variable domain (V) having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in the group consisting of H ): SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41, provided that the antibody does not comprise the light chain variable sequence of SEQ ID NO. 40 and the heavy chain variable sequence of SEQ ID NO. 41. In some embodiments, the sequence of the antibody is 100% identical at CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3.
In some embodiments, the antibody comprises a light chain variable domain (V) having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of L ): 28, 30, 32, 34, 36, 38, 40 respectively; and having an amino acid sequence at least about as set forth in the group consisting ofHeavy chain variable domain of 80% identical amino acid sequence (V H ): SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41, provided that the antibody does not comprise the light chain variable sequence of SEQ ID NO. 40 and the heavy chain variable sequence of SEQ ID NO. 41. In some embodiments, the sequence of the antibody is 100% identical at CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3.
In some embodiments, the antibody comprises a light chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
In some embodiments, the antibody comprises a light chain CDR1 having an amino acid sequence that is 100% identical to an amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; light chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
In some embodiments, the antibody comprises a heavy chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, the antibody comprises a heavy chain CDR1 having an amino acid sequence that is 100% identical to an amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; heavy chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence listed in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; heavy chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, the antibody comprises: (a) A light chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; and (b) a heavy chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In some embodiments, the antibody comprises: (a) A light chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; and (b) a heavy chain CDR1 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; a heavy chain CDR3 having an amino acid sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27; provided that the antibody does not comprise the sequences listed in: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
Such compositions can be used 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 antibodies.
In some embodiments, the excipient is a carrier, buffer, stabilizer, or other suitable material known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance will depend on the route of administration.
In some embodiments, pharmaceutical formulations comprising an antibody or antigen-binding fragment identified by the methods described herein are prepared for storage in the form of a lyophilized formulation or an aqueous solution by mixing a protein of the desired purity with an optional physiologically acceptable carrier, excipient, or stabilizer (see, e.g., remington's Pharmaceutical Sciences, 16 th edition, osol, a. Editions (1980)). Acceptable carriers or stabilizers are those which are non-toxic to the recipient at the dosage and concentration employed and include: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethyldiammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides A peptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salts form counterions, such as sodium; metal complexes (e.g., zn-protein complexes); and/or nonionic surfactants, such asOr polyethylene glycol (PEG). In certain embodiments, the pharmaceutical composition comprises an antibody at a concentration between 5mg/mL and 200 mg/mL; preferably between 10mg/mL and 100 mg/mL.
The acceptable carrier is physiologically acceptable to the subject to which it is administered and retains the therapeutic properties of the compound with which it is administered/in which it is administered. Acceptable carriers and formulations thereof are generally described, for example, in the following: remington' sPharmaceutical Sciences, supra. One exemplary carrier is physiological saline. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that participates in carrying or transporting the subject compound from the site of administration of one organ or portion of the body to another organ or portion of the body or in an in vitro assay system. Each carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject to which it is to be administered. The acceptable carrier should also not alter the specific activity of the subject compound.
In another embodiment, the pharmaceutical compositions disclosed herein further comprise acceptable additives to improve the stability of the compounds in the composition and/or to control the release rate of the composition. Acceptable additives do not alter the specific activity of the subject compounds. Exemplary acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. In some embodiments, the acceptable additives are combined with an acceptable carrier and/or excipient, such as dextrose. Alternatively, exemplary acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 to increase the stability of the peptide 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 in storage.
In one embodiment, the pharmaceutical compositions disclosed herein contain an isotonic buffer, such as phosphate, acetate, or TRIS buffer, in combination with a tonicity agent, such as a polyol, sorbitol, sucrose, or sodium chloride, which increases tonicity and stabilizes. In one embodiment, the tonicity agent is present in the composition in an amount of about 5%.
In another embodiment, the pharmaceutical composition disclosed herein comprises 0.01% to 0.02% wt/vol surfactant in order to prevent aggregation and for stabilization.
In another embodiment, the pharmaceutical composition disclosed herein has a pH in the range of 4.5-6.5 or 4.5-5.5.
In some embodiments, the pharmaceutical compositions disclosed herein also contain more than one active compound necessary for the indication being treated, such as those having complementary activities that do not adversely affect each other. For example, the methods of treatment also provide immunosuppressants. Such molecules are suitably present in combination in an amount effective for the intended purpose.
In some embodiments, the active ingredient is embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, in hydroxymethyl cellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. Such techniques are disclosed in the following: remington's Pharmaceutical Sciences, supra.
Suspensions and crystalline forms of antibodies are also contemplated herein; methods for preparing suspensions and crystalline forms are known to those skilled in the art.
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 accomplished by filtration through sterile filtration membranes. In some embodiments, the resulting solution is packaged for use or filtered under sterile conditions and lyophilized, and the lyophilized product is combined with a sterile solution prior to administration.
In some embodiments, lyophilization is employed to stabilize the polypeptide for long-term storage, such as when the polypeptide is relatively unstable in a liquid composition.
In some embodiments, some excipients, such as, for example, polyols (including mannitol, sorbitol, and glycerol); sugar (including glucose and sucrose); and amino acids (including alanine, glycine, and glutamic acid) as stabilizers for freeze-dried products. In some embodiments, polyols and sugars are also used to protect polypeptides from freeze and dry induced damage and to enhance stability during storage in the dry state. In some embodiments, the sugar is effective during both the lyophilization process and during storage. Other classes of molecules, including mono-and disaccharides and polymers such as PVP, have also been reported as stabilizers for lyophilized products.
For injection, in some embodiments, the pharmaceutical compositions disclosed herein are powders suitable for reconstitution with the appropriate solutions described above. Examples of these include, but are not limited to, freeze-dried powder, spin-dried powder or spray-dried powder, amorphous powder, granules, precipitates or microparticles. For injection, the composition optionally contains stabilizers, pH modifiers, surfactants, bioavailability modifiers, and combinations of these.
In some embodiments, sustained release articles are prepared. Suitable examples of sustained-release articles include semipermeable matrices of solid hydrophobic polymers containing the antibody, wherein the matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides (see, e.g., U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron depot. Tm. (including injectable microspheres 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 enable molecular release for more than 100 days, while certain hydrogels enable protein release for shorter periods of time. In some embodiments, while the encapsulated antibodies remain in the body for a long period of time, they denature or aggregate as a result of exposure to 37 ℃ moisture, resulting in loss of biological activity and possible changes in immunogenicity. In some cases, the rational strategy designed for stabilization depends on the mechanisms involved. For example, if the aggregation mechanism is found to be the formation of intermolecular S-S bonds through thio-disulfide interchange, stabilization is achieved in some cases by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
In some embodiments, the pharmaceutical compositions disclosed herein are designed for short-acting, rapid release, long-acting, or sustained release as described herein. In one embodiment, the pharmaceutical compositions disclosed herein are formulated for controlled release or for slow release.
The pharmaceutical composition is administered, for example, by injection, including, but not limited to, subcutaneous, intravitreal, intradermal, intravenous, intraarterial, intraperitoneal, intracerebral spinal or intramuscular injection. Excipients and carriers for formulating the compositions for each injection type are contemplated herein. The following description is merely exemplary in nature and is not intended to limit the scope of the compositions. Compositions for injection include, but are not limited to, aqueous solutions (in the case of water solubility) or dispersions, sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, cremophor el.tm. (BASF, parippany, n.j.), 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 glycols, and the like), and suitable mixtures thereof. Fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In some embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride are included in the composition. In some embodiments, the resulting solution is packaged for use as is or lyophilized; in some embodiments, the lyophilized preparation is later combined with a sterile solution prior to administration. For intravenous injection or injection at the lesion site, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen free and has suitable pH, isotonicity and stability. Those skilled in the art will be well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, ringer's injection or lactated ringer's injection. In some embodiments, preservatives, stabilizers, buffers, antioxidants, and/or other additives are included as desired. In some embodiments, the sterile injectable solution is prepared by: the desired amount of active ingredient is incorporated into an appropriate solvent, as desired, together with one or a combination of the ingredients listed above, and then filter sterilized. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
In some embodiments, the composition is administered intravenously conventionally, such as, for example, by injection of a unit dose. For injection, in some embodiments, the active ingredient is in the form of a parenterally acceptable aqueous solution that is substantially pyrogen free and has suitable pH, isotonicity and stability. In some embodiments, one prepares a suitable solution using, for example, an isotonic vehicle (such as sodium chloride injection, ringer's injection, lactated ringer's injection). In some embodiments, preservatives, stabilizers, buffers, antioxidants, and/or other additives are included as desired. Additionally, in some embodiments, the composition is administered via aerosolization. (Lahn et al Int Arch Allergy Immunol 134:49-55 (2004)).
For parenteral administration, the antibodies are formulated in unit dose injectable forms (solutions, suspensions, emulsions) along with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as non-volatile oils and ethyl oleate are also used. Liposomes are used as carriers. The vehicle contains small amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. Antibodies are typically formulated in such vehicles at a concentration of about 1mg/mL to 10 mg/mL.
In one embodiment, the pharmaceutical compositions disclosed herein are lyophilized, for example, to increase shelf life in storage. When considering the use of the compositions in the medicaments or any methods provided herein, in some embodiments, it is contemplated that the compositions are substantially free of pyrogens such that the compositions do not cause an inflammatory or unsafe allergic reaction when administered to a human subject. The pyrogens of the test compositions and the preparation of compositions that are substantially free of pyrogens are well known to those of ordinary skill in the art and are accomplished in some embodiments using commercially available kits.
In some embodiments, the acceptable carrier contains a compound that stabilizes, increases, or delays absorption or clearance. Such compounds include, for example, sugars such as glucose, sucrose or dextran; low molecular weight proteins; a composition that reduces clearance or hydrolysis of peptides; or excipients or other stabilizers and/or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. In some embodiments, the detergent is also used to stabilize or increase or decrease the absorption of the pharmaceutical composition, including the liposomal carrier. To prevent digestion, in some embodiments, the compound is complexed with the composition to render it resistant to acid hydrolysis and enzymatic hydrolysis, or in some embodiments, the compound is complexed in a suitable resistant carrier such as a liposome. Methods for protecting compounds from digestion are known in the art.
In some embodiments, the composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be administered depends on the subject to be treated, the ability of the subject's immune system to utilize the active ingredient, and the extent of binding capacity desired. The exact amount of active ingredient required to be administered depends on the judgment of the practitioner and is unique to each individual. Suitable regimens for initial administration and booster injections are also variable, but are typically repeated at intervals of one or more hours following initial administration by subsequent injections or other administrations. Alternatively, continuous intravenous infusion sufficient to maintain concentration in blood is contemplated.
In some embodiments, the present disclosure provides for the use of a composition described herein for the manufacture of a medicament for the treatment of a condition, disease or disorder described herein. In some embodiments, the medicament is formulated based on physical characteristics of the subject in need of treatment and is formulated as a single or more than one formulation based on the stage of the condition, disease or disorder. In some embodiments, the medicament is packaged in a suitable package with an appropriate label for distribution to hospitals and clinics, wherein the label is used to indicate treatment of a subject having a disease described herein. In some embodiments, the medicament is packaged as a single or more than one unit. In some embodiments, the dosage of the composition and instructions for administration are contained with the package as described below. The disclosure also relates to a medicament comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.
In some embodiments, the varying factors include the amount of active ingredient in the composition, the formulation of the composition, and the mode of administration, to provide an amount of active ingredient that is effective to achieve the desired therapeutic response for each subject without undue toxicity to the subject. The selected dosage level will depend on a variety of factors including the activity of the particular compound employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound employed, the duration of the treatment, other drugs, compounds and/or substances used in combination with the particular composition employed, the age, sex, weight, condition, general health, diet, and the like of the subject being treated as well as similar factors well known in the medical arts.
In some embodiments, the antibodies and antigen-binding fragments described herein are administered to a subject at different doses and within different time frames. Non-limiting dosages include about 0.01mg/kg, about 0.05mg/kg, about 0.1mg/kg, about 0.5mg/kg, about 1mg/kg, about 5mg/kg, about 10mg/kg, about 20mg/kg, about 30mg/kg, about 40mg/kg, about 50mg/kg, about 60mg/kg, about 70mg/kg, about 80mg/kg, about 90mg/kg, about 100mg/kg, about 125mg/kg, about 150mg/kg, about 175mg/kg, about 200mg/kg, or any integer therebetween. Additionally, in some embodiments, one or more doses of the antibody or antigen binding fragment are administered twice a week, once every two weeks, once every three weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks, once every 12 weeks, or a combination of any of several weeks therein. Administration cycles are also contemplated, such as, for example, once or twice a week for 4 weeks, followed by two weeks without treatment. Additional dosing cycles are also contemplated within the present disclosure, including, for example, different combinations of dosages and weekly cycles described herein.
In some embodiments, the therapeutically effective amount of the composition varies and depends on the severity of the disease and the weight and general state of the subject being treated, but generally ranges from about 1.0 μg/kg to about 100mg/kg body weight, or from about 10 μg/kg to about 30mg/kg, or from about 0.1mg/kg to about 10mg/kg, or from about 1mg/kg to about 10mg/kg per application. Depending on the response to the disorder or condition and the subject's tolerance to treatment, administration may be once daily, once a few days, once a week, twice a month, once a month or more or less frequent as desired. In some embodiments, it is desirable to maintain the dose for a longer period of time, such as 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, or 12 weeks or more, until the desired inhibition of the symptoms of the disorder occurs, and adjust the dose as needed. The progress of the therapy is readily monitored by conventional techniques and assays.
In some embodiments, the antibodies of the present disclosure are administered intravenously in physiological solution at a dose ranging from 0.01mg/kg to 100mg/kg, at a frequency ranging from once daily to once weekly to once monthly (e.g., once daily, once every two days, once every three days, or 2, 3, 4, 5, or 6 times weekly), preferably at a dose ranging from 0.1mg/kg to 45mg/kg, 0.1mg/kg to 15mg/kg, or 0.1mg/kg to 10mg/kg, at a frequency of 2 or 3 times weekly, or at most 45mg/kg once monthly.
Responses are achieved when a subject experiences a partial or complete alleviation or alleviation of the signs or symptoms of a disease and particularly includes, but is not limited to, an extension of survival. Depending on prognostic factors including the number of relapses, disease stage and other factors, the expected progression-free survival time is measured, for example, in months to years. Extended survival includes, but is not limited to, a period of at least 1 month (mo), about at least 2 months (mo), about at least 3 months, about at least 4 months, about at least 6 months, about at least 1 year, about at least 2 years, about at least 3 years, or longer. In some embodiments, total survival is also measured in months to years. In some embodiments, the symptoms of the subject remain static or decrease.
In some cases, a physician or veterinarian of ordinary skill in the art will readily determine and prescribe the effective amount of the composition required (ED 50 ). For example, a physician or veterinarian may begin the dosage of the compound employed in the composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Alternatively, in some embodiments the dosage is kept constant.
In some embodiments, the composition (an antibody or antigen binding fragment described herein) is administered alone or in combination with a second composition, either simultaneously or sequentially, depending on the condition to be treated. In one embodiment, the second therapeutic treatment is a fibrosis therapy (fibrosis therapy) or a fibrosis treatment (fibrosis therapeutic). In one embodiment, the second therapeutic treatment is the treatment of a disease or disorder associated with fibrosis, such as an infection, an autoimmune disease or disorder, an inflammatory disease or disorder, or mechanical injury. When two or more compositions are administered, the compositions are administered, for example, in combination (sequentially or simultaneously). In some embodiments, the composition is administered in a single dose or more than one dose.
In some embodiments, the composition is formulated to be pyrogen-free when formulated for administration to a human subject. The pyrogens of the test compositions and the preparation of pyrogen-free pharmaceutical compositions are well known to those of ordinary skill in the art.
In some embodiments, the antibody or antigen binding fragment thereof is formulated for administration to a subject by any suitable route, including, but not limited to, injection. Injections include, for example, subcutaneous injections, peritoneal injections, intravenous injections, intramuscular injections, or spinal injections into the cerebrospinal fluid (CSF). In some embodiments, administration is at one, two, three, four, five, six, seven or more injection sites. In one embodiment, administration is via six injection sites.
For in vivo applications, the contacting occurs, for example, via administration of a composition (such as described herein) to the subject in any suitable manner. In some embodiments, the antibodies described herein are administered systemically or locally by any suitable means, including via parenteral, subcutaneous, intraperitoneal, intraspinal, intrapulmonary, and intranasal administration, and if local treatment is desired, intralesional administration. Parenteral routes include, for example, intravenous, intra-arterial, intraperitoneal, epidural, intramuscular, and intrathecal administration. In some embodiments, such administration is, for example, bolus infusion (bolus), continuous infusion, or pulsed infusion. In some embodiments, the composition is administered by injection, depending in part on whether the administration is brief or chronic. Other modes of administration are contemplated, including topical administration, particularly transdermal, transmucosal, rectal, oral or topical administration, for example, through a catheter placed near the desired site.
Antibody technology
As will be appreciated by those skilled in the art, the general description of antibodies and methods of making and using the same herein also applies to individual antibody polypeptide components and antibody fragments.
The antibodies of the present disclosure are polyclonal or monoclonal antibodies. However, in a preferred embodiment, they are monoclonal. In a particular embodiment, the antibody of the present disclosure is a human antibody. Methods for producing polyclonal and monoclonal antibodies are known in the art.
In some embodiments, antibodies, antigen binding fragments, and other proteins that bind huCD163 expressed by M2 macrophages produced using such methods are tested for one or more of binding affinity, avidity, and regulatory capacity.
In some embodiments, antibodies or antigen-binding fragments thereof that bind to the huCD163 protein are identified using conventional methods. In some embodiments, the antibodies and antigen binding fragments are evaluated for one or more of binding affinity, binding rate, dissociation rate, and avidity. Measurement of such parameters is accomplished, for example, using assays including, but not limited to: enzyme-linked immunosorbent assay (ELISA), ELISPot assay, scatchard assay, surface plasmon resonance (e.g., BIACORE) assay, etc., competitive binding assay, and the like. In one non-limiting embodiment, an ELISA assay is used to measure the binding capacity of a particular antibody or antigen binding fragment that binds to the huCD163 protein. Liljeblad et al Glyco J17:323-9 (2000) describe a surface plasmon resonance technique.
In some embodiments, antibodies according to the present disclosure are recombinantly produced using vectors and methods available in the art, as described further below. In some embodiments, the human antibodies are also produced by in vitro activated B cells (see U.S. Pat. nos. 5,567,610 and 5,229,275).
In some embodiments, the human antibody is capable of being administeredIn transgenic animals (e.g., mice) that produce a complete human antibody repertoire in the presence of endogenous immunoglobulin production. For example, it has been described that the heavy chain linking region of antibodies in chimeric and germ-line mutant mice (J H ) Homozygous deletion of the gene results in complete inhibition of endogenous antibody production. The transfer of an array of human germline immunoglobulin genes into such germline mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., jakobovits et al, proc Natl Acad Sci USA,90:2551 (1993); jakobovits et al Nature 362:255-58 (1993); bruggemann et al, year in immunol.,7:33 (1993); U.S. Pat. nos. 5,545,806, 5,569,825, 5,591,669; U.S. patent No. 5,545,807; and WO 97/17852. In some embodiments, such animals are genetically engineered to produce human antibodies comprising the polypeptides of the disclosure.
For example, antibodies are isolated and purified from culture supernatants or ascites fluid (if antibodies are produced in animals) using methods known in the art, such as by saturated ammonium sulfate precipitation, euglobulin precipitation methods, caproic acid methods, caprylic acid methods, ion exchange chromatography (DEAE or DE 52), or affinity chromatography using an anti-Ig column or protein A, G or L column.
As described above, the present disclosure also provides antibody fragments. In some cases, it may be advantageous to use antibody fragments rather than whole antibodies. For example, in some embodiments, smaller sized fragments allow for rapid clearance and result in improved access to certain tissues such as organs (e.g., lung, kidney, liver, or heart). Examples of antibody fragments include: fab, F (ab') 2 And Fv fragments; a diabody; a linear antibody; single chain antibodies and multispecific antibodies formed from antibody fragments.
Various techniques have been developed to generate antibody fragments. Traditionally, these fragments were obtained via proteolytic digestion of the intact antibody (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 the recombinant host cell. In some cases In embodiments, fab, fv and ScFv antibody fragments are all expressed in and secreted from e.coli (e.coli), allowing for the easy production of large amounts of these fragments. In some embodiments, the F (ab ') -SH fragment is recovered directly from E.coli and chemically coupled to form F (ab') 2 Fragments (Carter et al, bio/Technology 10:163-167 (1992)). According to another method, in some embodiments, F (ab') 2 Fragments are isolated directly from recombinant host cell culture. C comprising Fc region derived from IgG H Fab and F (ab') with increased in vivo half-life of the two loops of the 2 domain salvage receptor binding epitope 2 Fragments are described in U.S. Pat. nos. 5,869,046 and 6,121,022. Other techniques for generating antibody fragments will be apparent to the skilled artisan.
In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. patent No. 5,571,894; and No. 5,587,458. Fv and sFv are the only species with complete binding sites (binding sites) that do not contain constant regions. They are therefore suitable for reducing non-specific binding during in vivo use. In some embodiments, the sFv fusion protein is constructed to produce fusion of effector proteins at the amino-terminus or carboxy-terminus of the sFv. See Antibody Engineering, incorporated by reference, borrebaeck, supra. In some embodiments, the antibody fragment is a "linear antibody," e.g., as described, for example, in U.S. Pat. No. 5,641,870. In some embodiments, such linear antibody fragments are monospecific or bispecific.
Methods for preparing 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 dimer (Gruber et al, J Immunol 152:5368 (1994)), linear antibody (Zapata et al, protein Eng 8:1057-62 (1995)); and chelating recombinant antibodies (Neri et al, JMol Biol 246:367-73 (1995)).
The traditional generation of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al Nature 305:537-9 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is accomplished, for example, by affinity chromatography. A similar procedure is disclosed in WO 93/08829 and Traunecker et al, EMBO J10:3655-9 (1991).
According to various methods, an antibody variable region having a desired binding specificity (antibody-antigen binding site, anti-antigen combining sites) is fused to an immunoglobulin constant domain sequence. Preferably, the fusion is with an Ig heavy chain constant domain comprising at least part of a hinge, C H Region 2 and C H Zone 3. Preferably, the first heavy chain constant region (C H 1) Comprising the sites required for light chain bonding, present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host cell. This provides greater flexibility in adjusting the mutual proportions of the four polypeptide fragments in embodiments when unequal ratios of the four polypeptide chains used in the construction provide for optimal yields of the desired bispecific antibody. However, when expression of at least two polypeptide chains at an equal ratio results in high yield or when the ratio has no significant effect on the yield of the desired chain combination, the coding sequences of two or all four polypeptide chains may be inserted into a single expression vector.
Bispecific antibodies consist of, for example, a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of immunoglobulin light chains in only half of the bispecific molecule provides an easy way of separation. This method is disclosed in WO 94/04690. For further details on the production of bispecific antibodies, see, for example, suresh et al, methods enzymes 121:210 (1986).
In some embodiments, the interface between a pair of antibody molecules is engineered to maximize the percentage of heterodimers recovered from recombinant cell cultures according to another approach described in U.S. patent No. 5,731,168. Preferred interfaces comprise C H 3 at least a portion of the domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine), a compensating "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers rather than other unwanted end products such as homodimers.
Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one of the antibodies in the heterologous conjugate is coupled to avidin and the other is coupled to biotin. For example, such antibodies have been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/20373 and EP 03089). In some embodiments, the heteroconjugate antibody is prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with some crosslinking techniques. Another approach is designed to prepare tetramers by adding a streptavidin coding sequence at the C-terminus of the scFv. Streptavidin consists of four subunits, so when scFv-streptavidin folds, the four subunits associate to form a tetramer (Kipriyanov et al Hum Antibodies Hybridomas 6 (3): 93-101 (1995)).
In some embodiments, according to another method for preparing bispecific antibodies, the interface between a pair of antibody molecules is engineered so as to maximize the percentage of heterodimers recovered from recombinant cell culture. C with interface containing antibody constant structural domain H 3 at least a portion of the domain. In the method, toOne or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine), a compensating "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers rather than other unwanted end products such as homodimers. See WO 96/27011.
Techniques for producing bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies are prepared using chemical ligation. Brennan et al, science 229:81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to yield F (ab') 2 Fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the ortho dithiol and prevent intermolecular disulfide formation. The resulting F (ab') fragment is then converted to a Thionitrobenzoate (TNB) derivative. One of the F (ab ') -TNB derivatives is then reconverted to F (ab ') -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other F (ab ') -TNB derivative to form a bispecific antibody. In some embodiments, the bispecific antibodies produced are used as agents for selective immobilization of enzymes.
Recent developments have facilitated the direct recovery of F (ab') -SH fragments from E.coli, which are, for example, chemically coupled to form bispecific antibodies. SHalaby et al, J Exp Med 175:217-25 (1992) describe a humanized bispecific antibody F (ab') 2 Production of molecules. Each F (ab') fragment was secreted by E.coli alone and subjected to in vitro directed chemical coupling to form bispecific antibodies. The bispecific antibody thus formed is capable of binding to cells overexpressing ErbB2 receptor and normal human T cells, as well as triggering the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell cultures are also described. For example, leucine zippers have been used to generate bispecific antibodies. Kostelny et al, J Immunol 148 (5): 1547-53 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the F (ab') portion of two different antibodies by gene fusion. The antibody homodimers are reduced at the hinge region to form monomers, and then oxidized to form antibody heterodimers. In some embodiments, the method is used to produce antibody homodimers.
Identification and preparation of antibodies
In some embodiments, the polynucleotide sequence encoding the antibody, variable region thereof, or antigen binding fragment thereof is determined using conventional sequencing techniques and subcloned into an expression vector for recombinant production of the antibody. This is accomplished by: obtaining mononuclear cells from the blood of a subject; generating B cell clones from the mononuclear cells; inducing B cells into antibody-producing plasma cells; and screening the supernatant produced by the plasma cells to determine if it contains antibodies. In some embodiments, the identification of other antibodies specific for antibodies of the disclosure is accomplished using similar methods. For example, after antibody-producing B cell clones are identified, reverse transcription polymerase chain reaction (RT-PCR) is performed to clone DNA encoding the antibody variable regions or portions thereof. These sequences are then subcloned into expression vectors suitable for recombinant production of human antibodies. In some embodiments, binding specificity is confirmed by determining the ability of an antibody to bind to an M2 cell or other cell expressing a human CD163 polypeptide expressed by an M2 cell.
In particular embodiments of the methods described herein, B cells isolated from peripheral blood or lymph nodes are sorted, e.g., based on they being CD19 positive, and plated specifically, e.g., as low as each well Shan Xibao, in, e.g., 96-well, 384-well, or 1536-well configurations. Cells are induced to differentiate into antibody-producing cells, such as plasma cells, and culture supernatants are harvested and tested for binding to cells expressing the target polypeptide on their surface using, for example, FMAT or FACS analysis. Positive Kong Jingli Quan Kong RT-PCR was then performed to amplify the heavy and light chain variable regions of the IgG molecules expressed by the clone progeny plasma cells. The resulting PCR products encoding the heavy and light chain variable regions or portions thereof are subcloned into a human antibody expression vector for recombinant expression. The resulting recombinant antibodies are then tested to confirm their original binding specificity, and in some embodiments are further tested for cross-reactivity against other cells or proteins.
Thus, in one embodiment, the method of identifying antibodies is performed as follows. First, full length or about full length CD163cDNA is transfected into a cell line for expression of the CD163 polypeptide. Next, antibodies in a human plasma or serum sample of the individual that bind to the polypeptides expressed by the cells are tested. And finally, the binding of MAbs derived from plasma or seropositive individuals to CD163 polypeptides expressed by the same cells is characterized. In this case the fine specificity of the MAb is further determined in some embodiments.
In some embodiments, methods useful in accordance with the art and described herein include isolating polynucleotides encoding antibodies or portions thereof of the present disclosure from cells expressing the antibodies by polymerase chain reaction amplification using primers specific for conserved regions of human antibody polypeptides. For example, light and heavy chain variable regions are cloned from B cells according to molecular biology techniques described below: WO 92/02551; U.S. Pat. nos. 5,627,052; or Babcook et al Proc Natl Acad Sci USA 93:7843-48 (1996). In certain embodiments, polynucleotides encoding all or a portion of both the heavy chain variable region and the light chain variable region of an IgG molecule expressed by clone progeny plasma cells expressing the antibody are subcloned and sequenced. In some embodiments, the sequence of the encoded polypeptide is readily determined from the polynucleotide sequence.
The isolated polynucleotides encoding the polypeptides of the present disclosure are subcloned into expression vectors to recombinantly produce the antibodies and polypeptides of the present disclosure using procedures known in the art and described herein.
In some embodiments, the binding characteristics of an antibody (or fragment thereof) to a CD163 polypeptide or M2 cell are generally determined and assessed using immunodetection methods, including, for example, immunofluorescence-based assays, such as Immunohistochemistry (IHC) and/or Fluorescence Activated Cell Sorting (FACS). In some embodiments, the immunoassay method comprises a control and procedure to determine whether the antibody specifically binds to a CD163 polypeptide or M2 macrophage and does not recognize a control cell, e.g., an M1 cell, or a host cell transfected to express a control protein or cross-react therewith.
After serum pre-screening to identify patients producing antibodies to CD163 polypeptides or to M2 macrophages, the methods of the present disclosure generally include isolating or purifying B cells from a biological sample previously obtained from the patient or subject. In some embodiments, the patient or subject has been currently or previously diagnosed as having or suspected of having fibrosis, or the patient or subject is deemed to be not fibrotic. Typically, the patient or subject is a mammal, and in particular embodiments is a human. In some embodiments, the biological sample is any sample comprising B cells, including, but not limited to, fibrotic tissue, lymph node or lymph node tissue, pleural effusion, peripheral blood, ascites, or cerebrospinal fluid (CSF). In some embodiments, B cells are isolated from different types of biological samples, such as biopsies of fibrotic tissue or other biological samples affected by fibrosis. However, in some embodiments, it is understood that any biological sample comprising B cells is used in any embodiment of the present disclosure.
After isolation, the B cells are induced to produce antibodies, for example, by culturing the B cells under conditions that support proliferation or development of the B cells into plasma cells (plasmacytes), plasmablasts, or plasmablasts. Antibodies are then screened, typically using high throughput techniques, to identify antibodies that specifically bind to a target antigen, such as a particular tissue, cell, or polypeptide. In certain embodiments, the specific antigen, e.g., cell surface polypeptide to which the antibody binds, is unknown, while in other embodiments, the antigen to which the antibody specifically binds is known.
In some embodiments, according to the present disclosure, B cells are isolated from a biological sample, e.g., a tissue, peripheral blood or lymph node sample, or fibrotic tissue, by any means known and available in the art. B cells are typically sorted by FACS based on the presence of B cell specific markers (e.g., CD19, CD138, and/or surface IgG) on their surface. However, in some embodiments other methods known in the art are employed, such as, for example, column purification using CD19 magnetic beads or IgG-specific magnetic beads, followed by elution from the column. However, in some embodiments, magnetic separation of B cells with any marker results in the loss of certain B cells. Thus, in certain embodiments, rather than sorting isolated cells, ficoll purified mononuclear cells isolated from fibrotic tissue are plated directly to the appropriate or desired number of specificities per well.
To identify antibody-producing B cells, the B cells are typically plated at low density (e.g., single cell specificity per well, 1-10 cells per well, 10-100 cells per well, 1-100 cells per well, less than 10 cells per well, or less than 100 cells per well) in a multi-well plate or microtiter plate, e.g., in a 96-well, 384-well, or 1536-well configuration. In some embodiments, when B cells are initially plated at a density of more than one cell per well, then the methods of the present disclosure include the step of subsequently diluting the cells in the well identified as producing antigen-specific antibodies until single cell specificity per well is reached, thereby facilitating identification of B cells producing antigen-specific antibodies. In some embodiments, the cell supernatant or a portion thereof and/or the cells are frozen and stored for future testing and subsequent recovery of the antibody polynucleotide.
In certain embodiments, the B cells are cultured under conditions conducive to antibody production by the B cells. For example, B cells are cultured under conditions conducive to proliferation and differentiation of the B cells to yield antibody-producing plasmablasts, plasma cells (plasmacytes) or plasma cells (plasmacytes). In certain embodiments, the B cells are cultured in the presence of a B cell mitogen such as Lipopolysaccharide (LPS) or CD40 ligand. In a particular embodiment, B cells are differentiated into antibody-producing cells by culturing the B cells with feeder cells and/or other B cell activators such as CD40 ligands.
In some embodiments, cell culture supernatants are tested or isolated from cells using conventional methods available in the art, including those described hereinThe ability of the antibodies obtained to bind to the target antigen. In certain embodiments, culture supernatants are tested for the presence of antibodies that bind to target antigens using a high throughput method. For example, B cells are cultured in a multi-well microtiter dish such that a robotic plate processor is used to sample more than one cell supernatant simultaneously and test for the presence of antibodies that bind to the target antigen. In particular embodiments, the antigen is bound to beads (e.g., paramagnetic beads or latex beads) to facilitate capture of the antibody/antigen complex. In other embodiments, the antigen and antibody are fluorescently labeled (with different labels) and FACS analysis is performed to identify the presence of antibodies that bind to the target antigen. In one embodiment, FMAT is used TM Analysis and instrumentation (Applied Biosystems, foster City, calif.) determined antibody binding. FMAT is a fluorescent macroscopic confocal platform for high throughput screening that enables mixing and reading, non-radioactive assays using living cells or beads.
In comparing the binding of an antibody to a particular target antigen (e.g., a biological sample such as a diseased tissue or cell, a fibrotic tissue or cell, or an infectious agent) and the binding of an antibody to a control sample (e.g., a biological sample such as a normal cell, a comparison cell from another species, a different fibrotic tissue or cell, a different tissue or cell, or a different infectious agent), in some embodiments, an antibody is considered to preferentially bind to a particular target antigen if it binds at least twice, at least three times, at least five times, or at least ten times more than the amount of the control sample.
In some embodiments, polynucleotides encoding antibody chains, variable regions thereof, or fragments thereof, are isolated from cells using any means available in the art. In one embodiment, the polynucleotides are isolated using Polymerase Chain Reaction (PCR), such as reverse transcription PCR (RT-PCR), using conventional procedures available in the art using oligonucleotide primers that specifically bind to heavy or light chain encoding polynucleotide sequences or complements thereof. In one embodiment, kong Jingli Quan Kong RT-PCR is positive to amplify the heavy and light chain variable regions of the IgG molecule expressed by the clone progeny plasma cells. In some embodiments, these PCR products are sequenced according to procedures conventional in the art, and then the products encoding the heavy and light chain variable regions or portions thereof are subcloned into a human antibody expression vector and re-expressed (see, e.g., U.S. patent No. 7,112,439). In some embodiments, the nucleic acid molecules encoding the M2 macrophage specific antibodies or fragments thereof as described herein are propagated and expressed according to any of a variety of well-known procedures for nucleic acid excision, ligation, transformation, and transfection. Thus, in certain embodiments, expression of the antibody fragment is preferred in a prokaryotic host cell such as E.coli (see, e.g., pluckaphun et al, methods Enzymol 178:497-515 (1989)). In certain other embodiments, expression of the antibody or antigen binding fragment thereof is preferably in a eukaryotic host cell such as yeast (e.g., saccharomyces cerevisiae (Saccharomyces cerevisiae), schizosaccharomyces pombe (s.pombe), pichia pastoris); animal cells (including mammalian cells) or plant cells. Examples of suitable animal cells include, but are not limited to, myeloma, COS, CHO, or hybridoma cells. Examples of plant cells include tobacco cells, maize cells, soybean cells and rice cells. In some embodiments, nucleic acid vectors for expressing exogenous sequences in a particular host system are designed by methods known to those of ordinary skill in the art and based on the present disclosure, and then polynucleotide sequences encoding M2 macrophage specific antibodies (or fragments thereof) are inserted. The regulatory elements will vary depending on the particular host.
In some embodiments, one or more replicable expression vectors comprising polynucleotides encoding variable and/or constant regions are prepared and used to transform an appropriate cell line, e.g., a non-producing myeloma cell line such as a mouse NSO line, or a bacterium such as e.coli, in which production of antibodies will occur. In order to obtain efficient transcription and translation, the polynucleotide sequences in each vector should comprise appropriate regulatory sequences, in particular promoter and leader sequences operably linked to the variable region sequences.
Specific methods of producing antibodies in this manner are generally well known and routinely used. For example, sambrook et al, molecular Cloning, A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory, new York,1989 describe molecular biology procedures; see also Sambrook et al, 3 rd edition, cold Spring Harbor Laboratory, new York, (2001). Although not required, in certain embodiments, the polynucleotide region encoding the recombinant antibody is sequenced. DNA sequencing is performed, for example, in any manner known in the art or using any system known in the art. Basic sequencing techniques are described in, for example, sanger et al Proc Natl Acad Sci USA 74:5463 (1977)) and Amersham International plc sequencing handbooks and include modifications thereof.
In certain embodiments, the resulting recombinant antibodies or fragments thereof are then tested to confirm their original specificity, and in some embodiments are further tested for cross-reactivity, e.g., with related polypeptides. In certain embodiments, antibodies identified or produced according to the methods described herein are tested for internalization ability or other effector function using conventional methods.
Packaging, kit and prefilled container
Also provided herein are kits comprising one or more of the compounds described above. In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof as described herein in a suitable container means.
In some embodiments, there is provided a container device comprising a composition described herein. In some embodiments, the container means is any suitable container containing, for example, a liquid or lyophilized composition, including, but not limited to, a vial, syringe, bottle, intravenous (IV) bag, or ampoule. In some embodiments, the syringe contains any volume of liquid suitable for injection into a subject, including but not limited to 0.5cc, 1cc, 2cc, 5cc, 10cc, or more.
Provided herein are kits comprising one or more compositions described herein. In some embodiments, provided herein are kits for treating a subject with fibrosis, the kits comprising an antibody as described herein and a fibrosis therapy.
In some embodiments, provided herein are kits for treating fibrosis, the kits comprising an antibody as described herein and a label attached to or packaged with a container, the label describing use of the antibody in combination with a fibrochemotherapeutic method.
In some embodiments, provided herein are kits for treating fibrosis, the kits comprising a fibrotic therapy and a label attached to or packaged with a container that describes the use of a fibrotic therapy (e.g., an anti-inflammatory agent) with an antibody as described herein.
In some embodiments, the container means of the kit will generally comprise at least one vial, test tube, flask, bottle, ampoule, syringe, intravenous (IV) bag, and/or other container means in which to place and/or preferably suitably aliquot at least one polypeptide. Provided herein are container devices comprising the compositions described herein.
In some embodiments, the kit comprises a tightly closed device for holding at least one fusion protein, detectable moiety, reporter molecule, and/or other reagent container for commercial sale. In some embodiments, such containers include injection and/or blow molded plastic containers in which the desired vials are stored. In some embodiments, the kit further comprises printed materials for use of the materials in the kit.
In some embodiments, the packages and kits further comprise buffers, preservatives, and/or stabilizers in the pharmaceutical formulation. In some embodiments, each component of the kit is packaged in a separate container and all of the different containers may be in a single package. In some embodiments, the kits of the disclosure are designed for refrigerated or room temperature storage.
Additionally, in some embodiments, the formulation contains a stabilizer to increase the shelf life of the kit and includes, for example, bovine Serum Albumin (BSA). In some embodiments, where the composition is lyophilized, the kit contains additional solution preparations to reconstitute the lyophilized formulation. Acceptable reconstitution solutions are well known in the art and include, for example, pharmaceutically acceptable Phosphate Buffered Saline (PBS).
In some embodiments, the packages and kits further comprise one or more components for assaying, such as, for example, ELISA assays. Samples to be tested in the present application include, for example, blood, plasma, tissue sections and secretions, urine, lymph and products thereof. In some embodiments, the packages and kits further comprise one or more components (e.g., syringe, cup, swab, etc.) for collecting the sample.
In some embodiments, the packages and kits further comprise a label specifying information required by the U.S. FDA or similar regulatory agency, for example, product description, amount and mode of administration, and/or therapeutic indication. The packages provided herein may comprise any composition as described herein.
The term "packaging material" refers to the physical structure that contains the components of the kit. In some embodiments, the packaging material maintains sterility of the components and is made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoule, etc.). In some embodiments, the label or package insert contains the appropriate written instructions. Thus, in some embodiments, the kit further comprises a label or instructions for using the kit components in any of the methods of the present disclosure. In some embodiments, the kit comprises the compound in a package or dispenser and instructions for administering the compound in the methods described herein.
In still further embodiments, the kit further comprises a container means for fibrotic therapy.
In some embodiments, the instructions comprise instructions for performing any of the methods described herein, including methods of treatment. In some embodiments, the instructions additionally comprise an indication of a satisfactory clinical endpoint or any adverse symptoms occurring, or additional information required by regulatory authorities, such as the food and drug administration (Food and Drug Administration), for the human subject.
In some embodiments, the instructions are on "printed matter", for example, within or affixed to the paper or paperboard of the kit, or on a label or packaging material affixed to the kit, or affixed to a vial or tube containing the components of the kit. In some embodiments, the instructions are additionally contained on a computer-readable medium, such as, for example, CD-ROM, DVD, flash memory devices, solid state memory, magnetic disks and disk devices, magnetic tape, cloud computing systems, servers, and the like. In some cases, the program and instructions are encoded on a medium permanently, substantially permanently, semi-permanently, or non-temporarily.
Provided herein are container devices comprising the compositions described herein. In some embodiments, the container means is any suitable container containing a liquid or lyophilized composition, including but not limited to a vial, syringe, bottle, intravenous (IV) bag, or ampoule. In some embodiments, the syringe contains any volume of liquid suitable for injection into a subject, including but not limited to 0.5cc, 1cc, 2cc, 5cc, 10cc, or more.
Provided herein are kits comprising the compositions described herein. In some embodiments, provided herein are kits for treating fibrosis, the kits comprising an antibody as described herein in combination with a fibrotic therapeutic agent.
In some embodiments, provided herein are kits for treating fibrosis (such as fibrosis associated with the presence of M2-macrophages), the kits comprising an antibody as described herein and a label attached to or packaged with a container describing use of the antibody or antigen binding fragment thereof with additional anti-fibrotic therapy or anti-inflammatory therapy. In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the fibrosis is a secondary fibrotic disease.
In some embodiments, provided herein are kits for treating fibrosis (such as fibrosis associated with the presence of M2-macrophages), the kits comprising an additional anti-fibrotic or anti-inflammatory therapy and a label attached to or packaged with the container that describes the use of the additional anti-fibrotic or anti-inflammatory therapy with an antibody as described herein. In some embodiments, the fibrosis is a primary fibrotic disease. In some embodiments, the fibrosis is a secondary fibrotic disease.
Examples
The present disclosure will be further illustrated in the following examples, which are provided for illustrative purposes only and are not intended to limit the present disclosure in any way.
EXAMPLE 1 affinity maturation
The results of phage ELISA showed that AB101-scFv-HL could be displayed at the N-terminus of the pIII protein at the phage tail by the E.coli TG1/pCantab 5E phage display system. EC of binding of AB101-E and AB101-C to huCD163 50 The values were 4.34nM and 5.44nM, respectively.
Phage display scFv libraries of VH and VL mutations (labeled scFv-AB101VHmu-VLwt library and VHwt-VLmu library) were designed and constructed via error-prone PCR.
scFv-AB101VHmu-VLwt library
scFv-AB101 VHwt-VLmu library
Based on sequence alignment analysis based on antibody databases and structural modeling analysis of Discovery Studio, a hotspot mutant phage display scFv library (labeled scFv-AB101 hotspot mutant library) was designed and constructed via NNK primers.
scFv-AB101 hotspot mutation library
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The capacity of the 3 mutation libraries was 1.13×10, respectively 10 (VHmu-VLwt library), 1.03X10 10 (VHwt-VLmu library) and 1.0X10 9 (library of hot spot mutations). The quality of each library was tested by sequencing with the S1 primer. The correct rate for each mutation library was 77.8% (VHmu-VLmu library), 70% (VHwt-VLmu library) and 75% (hot spot mutation library), respectively. These results indicate that three high quality libraries were constructed.
Library screening was performed on three mutant libraries labeled scFv-Ab101 VHmu-VLwt library, VHwt-VLmu library, and hot spot mutant library.
SDS-PAGE analysis was performed on the target antigen human CD 163. The molecular weight of human CD163 is about 140KD, with purity higher than 90%, without any degradation. Biotin-labeled human CD163 (labeled b-huCD 163) was produced and QC tested. In detail, biotin labeling efficiency was evaluated using SDS-PAGE associated with streptavidin, and the results showed an efficiency of more than 95%. All the results indicate that the quality of human CD163 and b-huCD163 has high levels.
Four rounds of library screening were performed based on three mutant libraries against b-huCD 163. Enrichment of all three libraries, namely scFv-AB101 VHmu-VLwt library, VHwt-VLmu library and hot spot mutation library, was found.
VHmu-VLwt library
138 clones were randomly selected from round 4 output of VHmu-VLwt library screening and placed in 96-well plates (labeled 1-46, 137-182 and 273-318) and QC monoclonal phage ELISA was performed using the AB101 parental phage as a control. Based on phage ELISA results, 40 highly positive clones were selected from batches 1-3 for DNA sequencing (clones 178 and 181 failed to sequence).
VHwt-VLmu library
138 clones were randomly selected from round 4 output of VHwt-VLmu library screening and placed in 96-well plates (labeled 47-92, 183-228 and 319-364) and QC monoclonal phage ELISA was performed using the AB101 parental phage as a control. Based on phage ELISA results, 42 highly positive clones were selected from batches 1-3 for DNA sequencing.
Library of hot spot mutations
138 clones were randomly selected from round 4 output of hotspot mutation library screening and placed in 96-well plates (labeled 93-136, 229-272, 365-408) and QC-monoclonal phage ELISA was performed using AB101 parental phage as a control. Based on phage ELISA results, 17 highly positive clones were selected from batches 1-3 for DNA sequencing. In total 97 highly positive clones were selected for DNA sequencing.
Sub-library construction and screening
VHm (containing mutations in VH) sequences from round 4 output pool of VHm-VLwt library and VLm (containing mutations in VL) sequences from round 4 output pool of VHwt-VLm library were amplified separately and randomly assembled into scFvs via overlap PCR to construct a sub-library. An endpoint sub-library was constructed and the library capacity was 3.8X10 9 . Two rounds of library screening were performed based on the sub-library against the antigen. After this, 180 clones (labeled clone 409-588) were randomly selected from the 2 nd round output of the sub-library for monoclonal phage ELISA. Based on the monoclonal phage ELISA results, 60 clones with the highest positive signals were selected for sequencing. Based on the DNA sequencing results, 56 clones were successfully sequenced, and 4 clones (clones 411, 462, 520 and 556) failed to obtain the correct sequence. Based on the sequence characteristics, 5 sequences were selected for further IgG antibody eukaryotic expression and affinity evaluation.
Example 2 determination of binding affinity of antibodies
Using AB101 parent AB as a control, BLI affinity detection was performed on 6 clones with one AB concentration. All clones had lower K D Values, indicating that they have enhanced affinity compared to the AB101 parent AB.
Material
Antigen: human CD163 protein (biotinylation)
The following antibodies were tested: variant 1 (V1) -variant 6 (V6). Molecular Weight (MW) and concentration are as described in table 4:
TABLE 4 Table 4
Buffer solution: PBS, PBST
The device comprises: forteBio Octet RED96 (Pall), SA Biosensor
Scheme for the production of a semiconductor device
The samples were prepared as follows: antigen b-huCD163 was diluted to 2 μg/mL with PBS buffer; the AB101 parent Ab was diluted to a range of concentrations (100 nM/150nM/200nM/250 nM) with PBST buffer. The results are described in table 5:
TABLE 5
Clone name K D (M) Ka(M -1 ·s -1 ) Kd(s -1 ) Ab concentration (nM)
AB101 parent Ab 8.78×10 -7 3.37×10 4 2.96×10 -2 50.0nM
V1 1.25×10 -9 8.95×10 5 1.12×10 -3 50.0nM
V2 9.91×10 -10 8.78×10 5 8.70×10 -4 50.0nM
V3 9.37×10 -10 9.87×10 5 9.25×10 -4 50.0nM
V4 8.24×10 -10 1.06×10 6 8.74×10 -4 50.0nM
V5 1.03×10 -9 9.51×10 5 9.78×10 -4 50.0nM
V6 9.64×10 -10 9.21×10 5 8.87×10 -4 50.0nM
V4 was selected for BLI affinity measurement at 4 Ab concentrations. The V4 antibody was diluted to a range of concentrations (6.25 nM/12.5nM/25nM/50 nM) with PBST buffer. The results are described in table 6:
TABLE 6
Affinity was determined as follows:
the SA sensor was placed in PBS buffer for 15 min. Different solutions were added to the 96-well plates, 200 μl per well, according to the following sequence.
PBS buffer 2μg/mL b-huCD163 PBST buffer Ab solution
PBS buffer 2μg/mL b-huCD163 PBST buffer Ab solution
PBS buffer 2μg/mL b-huCD163 PBST buffer Ab solution
PBS buffer 2μg/mL b-huCD163 PBST buffer Ab solution
PBS buffer 2μg/mL b-huCD163 PBST buffer PBST buffer
Notably, clone 4 had a size of 1.76X10 -10 K of (2) D When combined with a magnetic resonance system having a magnetic resonance frequency of 6.61×10 -7 K of (2) D Clone 4 showed about 3755 fold enhancement in affinity when compared to the AB101 parent AB of the value.
EXAMPLE 3 treatment of lung epithelial cancer
Administering one or more therapeutically effective doses of an antibody disclosed herein to a subject diagnosed with or suspected of having lung epithelial cancer. Lung epithelial cancer is ameliorated or eliminated after treatment.
EXAMPLE 4 treatment of pulmonary sarcoma
One or more therapeutically effective doses of an antibody disclosed herein are administered to a subject diagnosed with or suspected of having pulmonary sarcoma. Pulmonary sarcomas are ameliorated or eliminated after treatment.
Example 5-alleviation of fibrotic pathology in a subject suffering from primary fibrotic disease
One or more therapeutically effective doses of an antibody disclosed herein are administered to a subject diagnosed with or suspected of having a primary fibrotic disease, such as Interstitial Lung Disease (ILD), idiopathic Pulmonary Fibrosis (IPF), diffuse interstitial lung disease, liver fibrosis, and liver cirrhosis. Fibrotic pathology is ameliorated or eliminated after treatment.
Example 6-alleviation of fibrotic pathology in a subject suffering from fibrosis as a secondary disease
Administering one or more therapeutically effective doses of an antibody disclosed herein to a subject diagnosed with or suspected of having fibrosis as a complication of a non-fibrotic disease (e.g., an infection, autoimmune disease or disorder, cancer, or inflammatory disease or disorder) and determined or suspected of having M2 macrophages present in fibrotic tissue. Fibrotic pathology is ameliorated or eliminated after treatment.
Example 7-alleviation of fibrotic pathology in a subject suffering from fibrosis due to tissue injury
One or more therapeutically effective doses of an antibody disclosed herein are administered to a subject diagnosed with or suspected of having fibrosis as a complication of tissue injury (e.g., radiation-induced injury or mechanical injury) and determined to have M2 macrophages present in the injured tissue. Fibrotic pathology is ameliorated or eliminated after treatment.
Example 8-high affinity variant of AB101 shows stronger binding to CD163 on SU-DHL-1WT cells
DHL-1 (ATCC; accession No. CRL-2955) is a phagocytic human anaplastic large cell lymphoma cell line that constitutively expresses CD163, but lacks Fc receptor (FcR) expression on the cell surface. These cells were cultured in suspension in RPMI-1640 medium (Hyclone; no. SH 30027.02) supplemented with 10% fetal bovine serum (Hyclone; no. SH 30396.03).
To evaluate the role of FcR in binding to AB101 and its high affinity variants, CD 163-expressing SU-DHL-1 cells were treated with nucleic acids encoding human CD64 or FcgammaRI (origin; accession number RC207487L 3V), CD32a or FcgammaRIIa (G)&PBiosciences; number LTV 2124P) and CD16 or fcyriii (origin; lentiviral transduction numbered RC206429L 3V). SU-DHL-1 cells (5X 10) 4 Individual) with lentiviral particles encoding the respective FCRs (CD 16, CD32a and CD 64) in the presence of 8 μg/mL polybrene (Millipore; no. TR-1003-G) was treated with 20-50 particles per cell to generate SU-DHL-1 cell line with stable FcR expression. Cells were infected at Room Temperature (RT) by centrifugation at 800 Xg for 1 hour and subsequently transferred to an incubator (37 ℃, 5% CO) 2 ) Lasting for 4 hoursWhen (1). The virus-containing medium was then removed and the cells were allowed to recover for 3 days in fresh medium, then selected in medium containing 0.3. Mu.g/mL puromycin (ThermoFisher; accession number A1113803). Lentiviral particles deliver the respective FcR genes into the target cells at the time of infection and then integrate the contents into the host genome with high efficiency. By selecting stable FcR expression using puromycin as a selectable marker, populations of cells expressing CD16, CD32 and CD64 are isolated and cultured. FcR expression on puromycin resistant cell populations was confirmed by flow cytometry using a BD FACSymphony cytometer and FcR specific antibodies.
Binding of AB101, representative variant V3 and hIgG1 isotype controls to SU-DHL-1 wild-type (WT) cells or fcγr expressing cells was assessed by flow cytometry. Isotype control is a proprietary human IgG1 mAb with known specificity and the same IgG1 Fc region as AB101 and V3. SU-DHL-1 cells were washed once with PBS and resuspended in Zombie UV live/dead stain (BioLegend, accession number 423107) (1:500) at RT for 20 minutes. The cells were then washed with FACS buffer (pbs+1% fbs+1mM EDTA (Fisher Scientific, nos. 15575-038)) and resuspended in FACS blocking solution (FACS buffer containing 10% FBS and 0.5mg/mL igg 1) at RT for 20 min. The cells in blocking buffer were incubated at 2.5X10 4 Individual cells/well were transferred into 384-well plates and titrated antibodies (AB 101, V3 and hIgG1 isotype control) were added directly to each well at a 2 x final assay concentration. Cells were incubated with antibody for 20 min at RT. The cells were washed three times with FACS buffer and then resuspended in FACS buffer for use in FACSymphony TM Collected on a cytometer (BD Biosciences).
Although SU-DHL-1 wild-type cells constitutively expressed CD163, no appreciable AB101 binding was observed (fig. 1A). Optimal binding of AB101 to SU-DHL-1 cells in this assay requires co-expression of an Fc receptor, e.g., fcgammaRIII (FIG. 1B), fcgammaRI (FIG. 1C), or FcgammaRII (FIG. 1D). In contrast, V3 bound well to CD163 expressed on wild-type cells even in the absence of expressed Fc receptors (fig. 1A), and this binding was not significantly altered by co-expression of fcγr (fig. 1B-1D). In these assays, the hIgG1 control antibody showed negligible or no binding, indicating that Fc receptor binding alone was insufficient to show measurable binding of the IgG1 antibody in the assay.
Example 9-high affinity variants of AB101 showed stronger binding to recombinant CD163 protein (according to ELISA)
Binding of the CD163 variant antibodies to His-tagged recombinant human CD163 (R & D Systems, numbered 1607-CD-050) proteins was determined using ELISA. Recombinant proteins were diluted to 5 μg/mL in PBS and added at 25 μl per well to 384-well high binding ELISA plates (Greiner Bio-One, accession No. 781061) and incubated overnight at 4 ℃. Plates were washed three times with PBS using a BioTek ELx405 Select microplate washer (wash program elisa_384_pbs_3×_wash) and then blocked with 90 μl/well of blocking buffer (2% nonfat milk powder/pbs+0.05% Tween 20) for 1 hour at room temperature.
After blocking, 25 μl of primary antibody per well was added to the plate and incubated for 1 hour at RT. Isotype control is a proprietary mAb in the human IgG1 framework with known specificity. After primary antibody binding, plates were washed three times with PBS using EL405x (washing program elisa_384_pbs_3×_wash). The secondary antibody is goat anti-human IgG F (ab') 2 HRP (Jackson Immunoresearch, numbered 109-035-097). The secondary antibody was diluted to 1:2500 in 2% nonfat milk powder/PBS and added to the wells at 25 μl per well and incubated for 1 hour at RT. Plates were washed four times with PBS using EL405× (wash program elisa_384_pbs_4×_wash). After removal of the last wash, 25. Mu.L/well of pure 1-Step was added TM Ultra TMB-ELISA substrate solution (ThermoFisher, accession number 34028) and plates were incubated at room temperature for 10-15 min, protected from light. After development, the reaction was stopped by adding 25 μl of 0.3M HCl per well and the plates were read at 450nm using a SpectraMax M5e instrument.
As shown in fig. 2A-2F, variants of AB101, i.e., V1 (fig. 2A), V2 (fig. 2B), V3 (fig. 2C), V4 (fig. 2D), V5 (fig. 2E) and V6 (fig. 2F), and AB101 parent antibody bound to huCD163 in this assay, while isotype controls did not show appreciable binding. Binding of AB101 parent antibody to huCD163EC 50 The value is 84.39pM, while the EC of the variant of AB101 50 Values ranged from 1.87pM to 3.05pM, showing 27-to 45-fold stronger binding than the AB101 parent antibody (table 7).
Table 7: EC of antibodies to huCD163 50 Value of
Antibodies to EC 50 (nM)
AB101 84.39
V1 1.87
V2 2.23
V3 2.79
V4 3.05
V5 2.83
V6 2.68
Example 10: preparation of isolated monocytes from donor samples
Apheresis products can be collected from donors (apheresis products) and used in the artAutologous monocytes and T cells are isolated using techniques commonly used in the art. Here, human monocytes and T cells are isolated from White Blood Cells (WBCs) according to standard techniques. During the collection process, WBC is captured in an integrated leukoreduction system (LRS) chamber (Trima, numbered 2490-08) or LeukoPaks (numbered 4510-01,Full LeukoPak,BloodWorks Northwest,Seattle,WA). Through standard density gradient centrifugation Premium 1.073,GE Healthcare, accession number 17-5449-52) Peripheral Blood Mononuclear Cells (PBMC) were purified from the LRS chamber or LeukoPaks. The supernatant was discarded and the pellet (pellet) was resuspended in 20mL EasySep TM Buffer (STEMCELL Technologies, no. 20144) for PBMC counting and for further isolation of monocytes and T cells.
Using EasySep TM Human monocyte isolation kit (STEMCELL Technologies, no. 19359) isolated monocytes according to manufacturer's instructions.
EasySep can be used TM Human cd3+ T cells, cd4+ T cells, and cd8+ T cell isolation kits (STEMCELL Technologies, numbers 19051, 17952, and 17953) isolated total cd3+ T cells, cd4+ T cells, and cd8+ T cells according to the manufacturer's instructions. These negative selection kits use antibodies to label unwanted cell types for removal, allowing the desired target cells to be isolated from the sample in an untouched manner.
Macrophages can be produced from PBMC-derived monocytes using techniques commonly used in the art, such as those exemplified below.
Production of M0 macrophages: on day 0, monocytes from individual donors were isolated in M0 medium (90% X-VIVO TM 15 25,000-50,000 cells/100. Mu.L/well of 25,000-50,000 cells/well of (Lonza accession No. O4-418Q) +10% heat-inactivated FBS (Hyclone, accession No. SH 30396.03) +100ng/mL human M-CSF (PeproTech, accession No. 300-25)) were plated in 96-well culture plates (ThermoFisher (Costar), accession No. 09-761-145). The cells were incubated at 37℃with 5% CO 2 Incubation was performed for 5 to 6 days to generate M0 macrophages.
M2c giantProduction of phagocytes: on days 5-6 of culture, M0 macrophages were polarized to M2c macrophages by gentle aspiration of medium from each plate and replacement with 100. Mu.L/well of M2c medium (M0 medium with 20ng/mL human IL-10 (PeproTech, accession number 200-10)). The cells were incubated at 37℃with 5% CO 2 Incubate for two days. On days 7-8 of culture, M2c macrophages were ready for assay setup.
Example 11-high affinity variants of AB101 showed stronger binding to M2c macrophages (according to FACS)
To assess the binding kinetics of the variant antibodies to CD163 expressed on cells, binding studies were performed with immunosuppressive M2c macrophages. Macrophages are isolated and prepared according to standard methods such as, for example, the method described in example 10 above.
M2C cells were incubated in macrophage dissociation solution DXF (Promocell, accession number C-41330) for 15 min at RT and removed from the flask to X-VIVO TM 15 in serum-free medium (Lonza, accession No. O4-418Q).
After centrifugation, the cells were washed once with PBS and resuspended in Zombie UV live/dead stain (BioLegend, accession number 423107) (1:500) at RT for 20 minutes. The cells were then washed with FACS buffer (pbs+1% fbs+1mM EDTA (Fisher Scientific, nos. 15575-038)) and resuspended in FACS blocking solution (FACS buffer containing 10% FBS and 0.5mg/mL human IgG 1) at RT for 20 min. The cells in blocking buffer were incubated at 2.5X10 4 Individual cells/well were transferred to 384 well plates and titrated antibodies were added directly to each well at 2 x final assay concentration. Cells were incubated with antibody for 20 min at RT. The cells were washed three times with FACS buffer and then resuspended in FACS buffer for use in FACSymphony TM Collected on a cytometer (BD Biosciences).
As shown in fig. 3, these representative variants showed improved binding to M2c macrophages compared to the parent AB101 antibody. Human IgG1 (hIgG 1) isotype control showed no binding to M2c macrophages.
Example 12 high affinity variants of AB101 retain specificity and demonstrate improved binding to CD163+ cells in whole blood
Binding of antibodies to a subset of human immune cell lineages was assessed by flow cytometry. Briefly, whole blood with heparin as an anticoagulant was obtained (Bloodworks Northwest). Fc receptor blocking reagent was added directly to whole blood to final concentrations of 10% FBS, 0.5mg/mL human IgG1 and 0.05% sodium azide in a final sample volume of 400 μl. After 20 minutes of RT blocking, APC-labeled anti-CD 163 antibodies AB101, V3, human IgG1 isotype control, anti-CD 163 clone R20 and murine IgG1 isotype control were added. After 30 minutes, red Blood Cells (RBCs) were lysed with RBC lysis buffer followed by Zombie UV viability staining. The cells were then buffered with FACS buffer (containing 1% FBS, 2mM EDTA and 0.05% NaN 3 Is included in the liquid) and resuspended in FACS buffer containing 10% FBS and 10% FcX blocking liquid. Then, the antibody phenotyping mixture was added (table 8 a) and incubated at room temperature in the dark for 30 minutes. Cells were washed with FACS buffer and resuspended in FACS buffer for use with acsympany TM The data were collected by a cytometer (BD BioSciences). Analysis was performed using FlowJo software. Populations were identified according to the gating parameters as described in table 8 b.
Table 8a: antibody phenotyping mixtures
Table 8b: immune cell lineages
Cell type Gating control
CD4 + T cell CD3 + CD4 +
CD8 + T cell CD3 + CD8 +
B cell CD19 + HLA-DR +
NK cells CD3 - CD56 +
Neutrophils SSC High height CD15 +
Classical monocytes CD14 + HLA-DR + CD16 -
Intermediate monocytes CD14 + HLA-DR + CD16 +
Non-classical monocytes CD14 - HLA-DR + CD16 +
Bone marrow dendritic cells CD14 - HLA-DR + CD11c +
CD163 expression was detected on classical monocytes, intermediate monocytes and a portion of bone marrow dendritic cells (mDC) using commercially available anti-CD 163 clone R20 (R & D Systems, no. FAB16072R 10) as shown in fig. 4A-4C. Comparison of staining across monocytes and DCs using anti-CD 163 clone R20 showed that CD163 expression was different across cell types (classical monocytes > intermediate monocytes > mDC). No appreciable binding was observed with cd4+ T cells and cd8+ T cells, B cells, NK cells, neutrophils, non-classical monocytes and neutrophils (data not shown).
Consistent with CD163 expression, AB101 and V3 bound specifically to monocytes, while no appreciable binding was found to human neutrophils, B cells, NK cells, cd4+ T cells or cd8+ T cells. Human IgG1 isotype control (IgG 1) did not show detectable binding to any of the immune cell lineages evaluated. V3 shows improved binding to cd163+ cells in whole blood. The gMFI of V3 binding to DC, classical, intermediate and non-classical monocytes was increased 4.3-fold, 9.8-fold, 9.2-fold and 3-fold compared to the geometric MFI of AB101 (fig. 4A-4C).
Example 13A-CD163 antibodies alleviate M2 c-mediated immunosuppression in M2c/CD8+ T cell co-cultures as measured by T cell proliferation
Inhibition of T cells in tumor microenvironments by tumor-associated macrophages contributes to the immunosuppressive tumor microenvironment. This activity can be modeled by co-cultures of autologous monocyte-derived M2 macrophages with activated cd8+ T cells or cd4+ T cells, and T cell proliferation and cytokine expression levels can be used as alternatives to T cell activation.
After polarization of M0 macrophages to M2c macrophages, the supernatant was removed from the M2c macrophage 96-well plate and assayed with 100. Mu.L of assay medium (X-VIVO) containing 0.625. Mu.g/mL OKT3 (thermo Fisher, accession No. 14-0037-82) +/-CD163 antibodies TM 15 medium+10% FBS) and 5% CO at 37℃ 2 Incubation was continued for 1-2 hours. When M2c macrophages were contacted with antibody, autologous CD8+ T cells isolated as described above were subjected to 5. Mu.M CellTrace TM The violet proliferation dye (ThermoFisher, code C34571) was added at 37deg.C with 5% CO 2 Marking for 20 minutes. Excess CellTrace TM Washed off with ice-cold assay medium, labeled cd8+ T cells were resuspended in assay medium, and then added to the M2 c/antibody preparation at a ratio of 1:1 of M2c to cd8+ T cells. However, the method is thatThe cells were then incubated at 37℃with 5% CO 2 Incubate for 72 hours.
The supernatant containing the T cells was transferred to a V-bottom 96-well plate, centrifuged to pellet the T cells, and the culture supernatant was collected and frozen at-20℃for human IFNγ cytokine analysis by ELISA (R & D Systems; duoset ELISA, accession number DY 285B). T cell pellet was blocked with True Stain Fc blocking solution (BioLegend) and stained with anti-human CD8-BUV395 (BD Biosciences) for 20 min at 4 ℃. Cells were washed and stained with e780 viability dye for 15 min in the dark at RT, washed with FACS buffer and resuspended in 200 μl of FACS buffer for collection on a BD Symphony flow cytometer (BD Biosciences). The percentage of proliferating cd8+ T cells was analyzed using FlowJO software and reported as the percentage of cd8+ CellTrace negative dividing cells. In the M2c/T cell co-culture assay, CD163 antibodies alleviated M2 c-macrophage mediated immunosuppression by restoring CD8+ T cell proliferation (FIG. 5A). V3 was about 40-fold more potent than the parent AB101 antibody in alleviating M2 c-mediated inhibition of cd8+ T cell proliferation (table 9).
Table 9: EC of antibody-induced T cell proliferation 50 Value of
Antibodies to EC 50 (nM)
AB101 10.10
V3 0.27
Example 13B-CD163 antibodies alleviate M2 c-mediated immunosuppression in M2c/CD8+ T cell co-cultures as measured by perforin secretion
Antibody treatment during M2c/T cell co-culture alleviates M2 c-mediated immunosuppression and induces an effective cytotoxic response by anti-CD 3 activated cd8+ T cells. Cd8+ T cells isolated from 2 to 3 study subjects were activated with anti-CD 3 antibody (OKT 3,0.25 μg/mL) in the presence of M2c macrophages. M2c/T cell co-cultures were treated with 20. Mu.g/mL anti-CD 163 antibody, human IgG1 isotype control or medium alone (M2 c). Supernatants were removed 72 hours after anti-CD 3 stimulation and perforin secretion was quantified by a magnetic bead-based immunoassay. In this assay, the CD163 antibody rescued the perforin response of depleted T cells from M2c macrophage-mediated immunosuppression (fig. 5B), whereas the control IgG1 antibody did not show activity. V3 antibodies were about 100-fold more potent than the parent AB101 antibodies in alleviating M2 c-mediated inhibition of perforin secretion by cd8+ T cells (table 10).
Table 10: antibody-initiated EC of perforin secretion 50 Value of
Antibodies to EC 50 (nM)
AB101 36.45
V3 0.33
Example 13C-CD163 antibody alleviating M2C-mediated immunosuppression in M2C/T cell blast co-cultures as measured by IFN gamma secretion
Most T cells in the tumor microenvironment are depleted, showing reduced cytokine expression and effector function, which contributes to cancer immune evasion. Reversing T cell depletion and restoring antitumor potential represents a promising strategy for treating cancer. We used a co-culture of depleted T cells and M2c (a system that mimics macrophage-mediated immunosuppression) to evaluate the ability of CD163 antibodies to rescue the functional activity of depleted T cells from immunosuppression. Depleted T cells with a blast-like morphology can be generated from human PBMCs by repeating (3×) Phytohemagglutinin (PHA) stimulation.
Frozen PBMC or freshly isolated PBMC were used to reconstitute human IL-2 (R) in medium (IMDM (Thermo Fisher, accession number 12440053) +10% human AB serum (Sigma Aldrich, accession number H4522) +2. Mu.g/mL PHA-L (Sigma Aldrich, accession number 11249738001) +4 ng/mL)&D Systems, no. 202-IL)), 1X 10 in 6 Individual cells/mL at 37℃with 5% CO 2 Incubation was carried out for 10 days, with cell passaging and medium exchange taking place on days 4 and 7. On day 10, T cell blast cells were collected using standard methods.
To measure the ability of CD163 antibodies to rescue the functional activity of T cell blast cells from M2 c-mediated immunosuppression, old medium was removed from cultured M2c macrophages and incubated with 50 μl of X-VIVO containing 4X final concentration of CD163 or isotype control antibody TM 15 medium+10% FBS replacement, and at 37 ℃, 5% CO 2 Incubate for 2 hours. OKT3 antibody was diluted to 0.25. Mu.g/mL and added to M2 c/antibody samples and incubated at 37℃with 5% CO 2 Incubate for 30 minutes. Finally, T cell blast was added to the sample at a ratio of 1:1T cell blast to M2c, and at 37 ℃, 5% CO 2 Incubate for 24 hours. The supernatant was collected and analyzed for human gamma interferon (ifnγ) cytokines by ELISA. Each treatment condition was set in triplicate.
In this assay, the CD163 antibody rescued ifnγ responses of depleted T cells from M2C macrophage-mediated immunosuppression (fig. 5C), whereas the control IgG1 antibody showed no activity. In alleviating M2 c-mediated inhibition of ifnγ release by CD 8T cells, V3 was shown to be more than 10-fold stronger than AB101 antibody (table 11).
Table 11: antibodies toPrimed ifnγ secreted EC 50 Value of
Antibodies to EC 50 (nM)
AB101 6.84
V3 0.47
。
Sequence listing
<110> Oncordones Pang Si Co
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<151> 2021-04-01
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<221> VARIANT
<222> (7)..(7)
<223 >/substitution= "Q" or "T" or "S" or "N" or "a" or "G"
<220>
<221> VARIANT
<222> (8)..(8)
<223 >/substitution= "G" or "a" or "S"
<220>
<221> VARIANT
<222> (10)..(10)
<223 >/substitution= "S" or "a" or "G" or "N"
<220>
<221> SITE
<222> (1)..(10)
<223 >/note = "variant residues given in sequence have no preference for those residues in the annotation of variant positions" for "a variant residue"
<400> 15
Gln Gln Ser Tyr Ser Thr Pro Arg Gly Thr
1 5 10
<210> 16
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<400> 16
Ser Tyr Asp Met His
1 5
<210> 17
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<400> 17
Val Ile Ser Glu Asp Gly Ser Asn Lys Tyr Asn Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 18
<211> 24
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<400> 18
Glu Asn Val Arg Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Ser Ser Glu
1 5 10 15
Tyr Tyr Tyr Tyr Gly Leu Asp Val
20
<210> 19
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<400> 19
Ser Glu Thr Met His
1 5
<210> 20
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<400> 20
Val Ile Ser Glu Asp Gly Ser Asn Lys Tyr His Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 21
<211> 24
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<400> 21
Glu Asn Val Arg Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Asn Ser Glu
1 5 10 15
Tyr Tyr Tyr Tyr Gly Met Asp Val
20
<210> 22
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<400> 22
Ser Tyr Val Met His
1 5
<210> 23
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<400> 23
Val Ile Ser Glu Asp Gly Ser Asn Lys Tyr Glu Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 24
<211> 24
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<400> 24
Glu Asn Val Arg Pro Tyr Tyr Asp Phe Trp Arg Gly Tyr Asn Ser Glu
1 5 10 15
Tyr Tyr Tyr Tyr Gly Leu Asp Val
20
<210> 25
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<220>
<221> VARIANT
<222> (2)..(2)
<223 >/substitution= "E" or "Q" or "D"
<220>
<221> VARIANT
<222> (3)..(3)
<223> "D" or "T" or "V" or "S" or "G" or "E"
<220>
<221> SITE
<222> (1)..(5)
<223 >/note = "variant residues given in sequence have no preference for those residues in the annotation of variant positions" for "a variant residue"
<400> 25
Ser Tyr Ala Met His
1 5
<210> 26
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<220>
<221> VARIANT
<222> (4)..(4)
<223 >/substitution= "E" or "Q" or "D"
<220>
<221> VARIANT
<222> (11)..(11)
<223 >/substitution= "N" or "H" or "E" or "D" or "K" or "Q" or "R"
<220>
<221> SITE
<222> (1)..(17)
<223 >/note = "variant residues given in sequence have no preference for those residues in the annotation of variant positions" for "a variant residue"
<400> 26
Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 27
<211> 24
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic peptides'
<220>
<221> VARIANT
<222> (11)..(11)
<223 >/substitution= "R" or "K" or "H"
<220>
<221> VARIANT
<222> (14)..(14)
<223 >/substitution= "S" or "N" or "T" or "a" or "Q"
<220>
<221> VARIANT
<222> (22)..(22)
<223 >/substitution= "L" or "I" or "V"
<220>
<221> SITE
<222> (1)..(24)
<223 >/note = "variant residues given in sequence have no preference for those residues in the annotation of variant positions" for "a variant residue"
<400> 27
Glu Asn Val Arg Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Tyr Ser Glu
1 5 10 15
Tyr Tyr Tyr Tyr Gly Met Asp Val
20
<210> 28
<211> 109
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 28
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Gln Arg
85 90 95
Gly Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 29
<211> 132
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 29
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Glu Asp Gly Ser Asn Lys Tyr Asn Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Asn Val Arg Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Ser
100 105 110
Ser Glu Tyr Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr
115 120 125
Val Thr Val Ser
130
<210> 30
<211> 109
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 30
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Asn Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Thr Arg
85 90 95
Gly Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 31
<211> 133
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 31
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Glu
20 25 30
Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Glu Asp Gly Ser Asn Lys Tyr His Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Asn Val Arg Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Asn
100 105 110
Ser Glu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
115 120 125
Val Thr Val Ser Ser
130
<210> 32
<211> 109
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 32
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Gln Arg
85 90 95
Gly Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 33
<211> 133
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 33
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Glu Asp Gly Ser Asn Lys Tyr Glu Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Asn Val Arg Pro Tyr Tyr Asp Phe Trp Arg Gly Tyr Asn
100 105 110
Ser Glu Tyr Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr
115 120 125
Val Thr Val Ser Ser
130
<210> 34
<211> 109
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 34
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Gln Arg
85 90 95
Gly Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 35
<211> 133
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 35
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Glu Asp Gly Ser Asn Lys Tyr Asn Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Asn Val Arg Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Ser
100 105 110
Ser Glu Tyr Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr
115 120 125
Val Thr Val Ser Ser
130
<210> 36
<211> 109
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 36
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Thr Arg
85 90 95
Gly Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 37
<211> 133
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 37
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Glu Asp Gly Ser Asn Lys Tyr Asn Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Asn Val Arg Pro Tyr Tyr Asp Phe Trp Arg Gly Tyr Asn
100 105 110
Ser Glu Tyr Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr
115 120 125
Val Thr Val Ser Ser
130
<210> 38
<211> 109
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 38
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Thr Gly
85 90 95
Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 39
<211> 133
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 39
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Glu
20 25 30
Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Glu Asp Gly Ser Asn Lys Tyr Asn Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Asn Val Arg Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Ser
100 105 110
Ser Glu Tyr Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr
115 120 125
Val Thr Val Ser Ser
130
<210> 40
<211> 109
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 40
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Arg
85 90 95
Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 41
<211> 166
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthetic polypeptide'
<400> 41
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Asn Val Arg Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Tyr
100 105 110
Ser Glu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
115 120 125
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
130 135 140
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
145 150 155 160
Leu Val Lys Asp Tyr Phe
165
<210> 42
<211> 1121
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 42
Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp
1 5 10 15
Phe Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Thr Val Val
20 25 30
Leu Leu Leu Ser Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Asp
35 40 45
Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val
50 55 60
Glu Val Lys Val Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp
65 70 75 80
Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr
85 90 95
Ala Ile Lys Ala Pro Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg
100 105 110
Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp
115 120 125
Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln
130 135 140
Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asn Leu Glu Met Arg
145 150 155 160
Leu Thr Arg Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe
165 170 175
Gln Gly Arg Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asp His
180 185 190
Ala Ser Val Ile Cys Arg Gln Leu Glu Cys Gly Ser Ala Val Ser Phe
195 200 205
Ser Gly Ser Ser Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp
210 215 220
Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His
225 230 235 240
Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val
245 250 255
Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val
260 265 270
Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly
275 280 285
Thr Ile Cys Asp Asp Gly Trp Asp Ser Tyr Asp Ala Ala Val Ala Cys
290 295 300
Lys Gln Leu Gly Cys Pro Thr Ala Val Thr Ala Ile Gly Arg Val Asn
305 310 315 320
Ala Ser Lys Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln
325 330 335
Gly His Glu Pro Ala Ile Trp Gln Cys Lys His His Glu Trp Gly Lys
340 345 350
His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly
355 360 365
Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly
370 375 380
Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg
385 390 395 400
Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys
405 410 415
Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala
420 425 430
Thr Asn Thr Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser
435 440 445
Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His
450 455 460
Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu
465 470 475 480
Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly
485 490 495
Asp Thr Trp Gly Ser Ile Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala
500 505 510
Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu
515 520 525
Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu
530 535 540
Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala
545 550 555 560
Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val
565 570 575
Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys
580 585 590
Glu Gly Arg Val Glu Leu Lys Thr Leu Gly Ala Trp Gly Ser Leu Cys
595 600 605
Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu
610 615 620
Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala Arg Phe Gly Lys
625 630 635 640
Gly Asn Gly Gln Ile Trp Arg His Met Phe His Cys Thr Gly Thr Glu
645 650 655
Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys
660 665 670
Pro Ser Glu Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln
675 680 685
Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr
690 695 700
Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg
705 710 715 720
Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His
725 730 735
Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp
740 745 750
Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala
755 760 765
Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp
770 775 780
Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser
785 790 795 800
His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val
805 810 815
Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg
820 825 830
Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly
835 840 845
Thr Val Gly Lys Ser Ser Met Ser Glu Thr Thr Val Gly Val Val Cys
850 855 860
Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Pro Ala Ser Leu
865 870 875 880
Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro
885 890 895
Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys
900 905 910
Arg Leu Ala Ser Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys
915 920 925
Ile Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile
930 935 940
Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu
945 950 955 960
Asp Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu
965 970 975
Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp
980 985 990
Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys
995 1000 1005
Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp
1010 1015 1020
Ala Ala Val Asn Cys Thr Asp Ile Ser Val Gln Lys Thr Pro Gln
1025 1030 1035
Lys Ala Thr Thr Gly Arg Ser Ser Arg Gln Ser Ser Phe Ile Ala
1040 1045 1050
Val Gly Ile Leu Gly Val Val Leu Leu Ala Ile Phe Val Ala Leu
1055 1060 1065
Phe Phe Leu Thr Lys Lys Arg Arg Gln Arg Gln Arg Leu Ala Val
1070 1075 1080
Ser Ser Arg Gly Glu Asn Leu Val His Gln Ile Gln Tyr Arg Glu
1085 1090 1095
Met Asn Ser Cys Leu Asn Ala Asp Asp Leu Asp Leu Met Asn Ser
1100 1105 1110
Ser Gly Gly His Ser Glu Pro His
1115 1120
<210> 43
<211> 27
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 43
Ile Gly Arg Val Asn Ala Ser Lys Gly Phe Gly His Ile Trp Leu Asp
1 5 10 15
Ser Val Ser Cys Gln Gly His Glu Pro Ala Ile
20 25
<210> 44
<211> 11
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 44
Val Val Cys Arg Gln Leu Gly Cys Gly Ser Ala
1 5 10
<210> 45
<211> 14
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 45
Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp
1 5 10
<210> 46
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> source
<223 >/note= "description of artificial sequence: synthesis of 6xHis tag'
<400> 46
His His His His His His
1 5

Claims (156)

1. An antibody comprising:
(a) A heavy chain variable region (VH) having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and
(b) A light chain variable region (VL) having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of: 28, 30, 32, 34, 36, 38 and 40 SEQ ID NO;
with the proviso that the antibody does not comprise a light chain variable region (VL) having the sequence set forth in SEQ ID NO. 40 and a heavy chain variable region (VH) having the sequence set forth in SEQ ID NO. 41.
2. The antibody of claim 1, wherein the light chain variable region (VL) has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
3. The antibody of claim 1, wherein the light chain variable region (VL) has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
4. The antibody of claim 1, wherein the light chain variable region (VL) has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
5. The antibody of claim 1, wherein the light chain variable region (VL) has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
6. The antibody of claim 1, wherein the light chain variable region (VL) has a sequence 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
7. The antibody of claim 1, wherein the heavy chain variable region (VH) has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41.
8. The antibody of claim 1, wherein the heavy chain variable region (VH) has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41.
9. The antibody of claim 1, wherein the heavy chain variable region (VH) has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41.
10. The antibody of claim 1, wherein the heavy chain variable region (VH) has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41.
11. The antibody of claim 1, wherein the heavy chain variable region (VH) has a sequence 100% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41.
12. The antibody of any one of claims 1-11, wherein the antibody is 100% identical at CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3.
13. The antibody of any one of claims 1-12, wherein CDR H1 has a sequence listed in the amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25.
14. The antibody of any one of claims 1-13, wherein CDR H2 has a sequence listed in the amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26.
15. The antibody of any one of claims 1-14, wherein CDR H3 has a sequence listed in the amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27.
16. The antibody of any one of claims 1-15, wherein CDR L1 has a sequence listed in the amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13.
17. The antibody of any one of claims 1-16, wherein CDR L2 has a sequence listed in the amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14.
18. The antibody of any one of claims 1-17, wherein CDR L3 has a sequence listed in the amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15.
19. An antibody comprising:
(a) Light chain CDR1 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; a light chain CDR2 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and a light chain CDR3 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id nos: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15; and
(b) Heavy chain CDR1 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; a heavy chain CDR2 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; heavy chain CDR3 having an amino acid sequence that is at least about 80% identical to an amino acid sequence set forth in the group consisting of seq id no: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27;
Provided that the antibody does not comprise at least the sequences listed in seq id no: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
20. The antibody of claim 19, wherein the CDR L1 has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; the CDR L2 has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and the CDR L3 has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15.
21. The antibody of claim 19, wherein the CDR L1 has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; the CDR L2 has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and the CDR L3 has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15.
22. The antibody of claim 19, wherein the CDR L1 has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; the CDR L2 has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and the CDR L3 has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15.
23. The antibody of claim 19, wherein the CDR L1 has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; the CDR L2 has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and the CDR L3 has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15.
24. The antibody of claim 19, wherein the CDR L1 has a sequence 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 13; the CDR L2 has a sequence 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 9 and SEQ ID NO. 14; and the CDR L3 has a sequence at least 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 15.
25. The antibody of claim 19, wherein the CDR H1 has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; the CDR H2 has a sequence at least 85% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; and the CDR H3 has a sequence at least 85% identical to an amino acid selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27.
26. The antibody of claim 19, wherein the CDR H1 has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; the CDR H2 has a sequence at least 90% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; and the CDR H3 has a sequence at least 90% identical to an amino acid selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27.
27. The antibody of claim 19, wherein the CDR H1 has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; the CDR H2 has a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; and the CDR H3 has a sequence at least 95% identical to an amino acid selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27.
28. The antibody of claim 19, wherein the CDR H1 has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; the CDR H2 has a sequence at least 99% identical to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; and the CDR H3 has a sequence at least 99% identical to an amino acid selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27.
29. The antibody of claim 19, wherein the CDR H1 has a sequence at least 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 4, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22 and SEQ ID NO. 25; the CDR H2 has a sequence at least 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 5, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 26; and the CDR H3 has a sequence at least 100% identical to an amino acid selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27.
30. The antibody of claim 19, comprising:
(a) A heavy chain variable region (VH) having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and
(b) A light chain variable region (VL) having a sequence at least 80% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
31. The antibody of claim 19, comprising:
(a) A heavy chain variable region (VH) having a sequence at least 85% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and
(b) A light chain variable region (VL) having a sequence at least 85% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
32. The antibody of claim 19, comprising:
(a) A heavy chain variable region (VH) having a sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and
(b) A light chain variable region (VL) having a sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
33. The antibody of claim 19, comprising:
(a) A heavy chain variable region (VH) having a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and
(b) A light chain variable region (VL) having a sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
34. The antibody of claim 19, comprising:
(a) A heavy chain variable region (VH) having a sequence at least 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and
(b) A light chain variable region (VL) having a sequence at least 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
35. The antibody of claim 19, comprising:
(a) A heavy chain variable region (VH) having a sequence 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39 and SEQ ID NO. 41; and
(b) A light chain variable region (VL) having a sequence 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 and SEQ ID NO. 40.
36. The antibody of any one of claims 1-35, wherein the antibody further comprises a human heavy chain constant region or a human light chain constant region.
37. The antibody of claim 36, wherein the human heavy chain constant region is IgG1 or IgG4 or a fragment thereof.
38. The antibody of any one of claims 1-37, wherein the antibody binds to an Fc receptor.
39. The antibody of claim 38, wherein the Fc receptor is expressed on macrophages.
40. The antibody of any one of claims 1-39, wherein the antibody is an antibody fragment comprising: single heavy chain, single light chain, fab, F (ab') 2 Fd, scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, bispecific Fab 2 Trispecific Fab 3 A single chain binding polypeptide, a dAb fragment or a diabody.
41. The antibody of any one of claims 1-40, wherein the antibody binds to a dendritic cell.
42. The antibody of any one of claims 1-41, wherein the antibody binds to classical monocytes.
43. The antibody of any one of claims 1-42, wherein the antibody binds to intermediate monocytes.
44. The antibody of any one of claims 1-43, wherein the antibody binds to non-classical monocytes.
45. The antibody of any one of claims 1-44, wherein the antibody binds to immunosuppressive bone marrow cells.
46. The antibody of claim 45, wherein the immunosuppressive bone marrow cells are located in a tumor microenvironment.
47. The antibody of claim 45 or 46, wherein the immunosuppressive bone marrow cells are macrophages or bone marrow derived suppressor cells.
48. The antibody of claim 47, wherein the human macrophage is an M2 macrophage or an M2-like macrophage.
49. The antibody of claim 47 or 48, wherein the human macrophage is an M2a, M2b, M2c, or M2d macrophage.
50. The antibody of any one of claims 47-49, wherein the macrophage is a tumor-associated macrophage.
51. The antibody of any one of claims 1-50, wherein the antibody binds to a CD163 protein.
52. The antibody of claim 51, wherein the CD163 protein is a glycoform of CD 163.
53. The antibody of claim 52, wherein the CD163 protein is a 150kDa glycoform of CD 163.
54. The antibody of any one of claims 51-53, wherein the antibody does not specifically bind to the 130kDa glycoform of CD163 expressed by the human macrophages.
55. The antibody of any one of claims 51-54, wherein the CD163 protein is a component of a cell surface complex comprising at least one other protein expressed by the macrophage.
56. The antibody of claim 55, wherein the at least one other protein is a galectin-1 protein, a LILRB2 protein, a casein kinase II protein, or any combination thereof.
57. The antibody of any one of claims 1-56, wherein the antibody specifically binds to a CD163 epitope comprising amino acid sequence SEQ ID No. 42.
58. The antibody of any one of claims 1-56, wherein the antibody specifically binds to a CD163 epitope comprising amino acid sequence SEQ ID No. 43.
59. The antibody of any one of claims 1-56, wherein the antibody specifically binds to a CD163 epitope comprising amino acid sequence SEQ ID No. 44.
60. The antibody of any one of claims 1-56, wherein the antibody specifically binds to a CD163 epitope comprising each of the amino acid sequences SEQ ID No. 42, SEQ ID No. 43, and SEQ ID No. 44.
61. The antibody of any one of claims 51-60, wherein the antibody alters the expression of at least one marker on the macrophage.
62. The antibody of claim 61, wherein the at least one marker on human macrophages is CD16, CD64, TLR2, or Siglec-15.
63. The antibody of any one of claims 1-62, wherein the antibody is at a K of 0.5nM to 100nM D Specifically binds to CD 163.
64. The antibody of claim 63, wherein the antibody is at a K of 0.5nM to 50nM D Specifically binds to CD 163.
65. The antibody of claim 64, wherein the antibody is at a K of 0.5nM to 10nM D Specifically binds to CD 163.
66. The antibody of claim 65, wherein the antibody is at a K of 0.5nM to 1.5nM D Specifically binds to CD 163.
67. The antibody according to claim 66,wherein the antibody is at a K of 0.5nM to 1.0nM D Specifically binds to CD 163.
68. The antibody of any one of claims 1-67, wherein the antibody is at a K of 0.5nM to 100nM D Specifically binds to human M2c macrophages.
69. The antibody of claim 68, wherein the antibody is at a K of 0.5nM to 50nM D Specifically binds to human M2c macrophages.
70. The antibody of claim 69, wherein the antibody is at a K of 0.5nM to 10nM D Specifically binds to human M2c macrophages.
71. The antibody of claim 70, wherein the antibody is at a K of 0.5nM to 1.5nM D Specifically binds to human M2c macrophages.
72. The antibody of claim 71, wherein the antibody is at a K of 0.5nM to 1.0nM D Specifically binds to human M2c macrophages.
73. A method of treating a cancer or fibrotic disease or disorder associated with the presence of M2-macrophages in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody of any one of claims 1-72.
74. The method of claim 73, wherein binding of the antibody to macrophages promotes immune cell function as measured by one or both of the following parameters:
(a) Activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof; and
(b) Proliferation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof.
75. The method of claim 74, wherein the activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof is measured as an enhanced level of IFN- γ, TNF- α, or perforin, or any combination thereof.
76. The method of any one of claims 73-75, wherein binding of the antibody to a macrophage is non-cytotoxic to the macrophage.
77. The method of any one of claims 73-76, wherein binding of the antibody to macrophages results in at least one of:
(a) The macrophage has reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15;
(b) Internalizing said antibody by said macrophage;
(c) Secretion of IFN-gamma, TNF-alpha and perforin;
(d) Activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof;
(e) Proliferation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof; and
(f) Promote the killing of tumor cells in the tumor microenvironment.
78. The method of claim 77, wherein said combining results in: two or more of (a) to (e); three or more of (a) to (e); four or more of (a) to (e); or all of (a) to (e).
79. The method of any one of claims 73-78, wherein binding of the antibody to macrophages increases immunostimulatory activity in the tumor microenvironment.
80. The method of any one of claims 73-79, wherein binding of the antibody to a macrophage reduces immunosuppressive activity of the macrophage.
81. The method of any one of claims 73-80, wherein binding of the antibody to a macrophage reduces the tumor promoting activity of the macrophage.
82. The method of any one of claims 73-81, wherein binding of the antibody promotes cd4+ T cell activation, cd4+ T cell proliferation, or both cd4+ T cell activation and cd4+ T cell proliferation.
83. The method of any one of claims 73-82, wherein binding promotes expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by cd4+ T cells.
84. The method of any one of claims 73-83, wherein binding to the antibody promotes cd8+ T cell activation, cd8+ T cell proliferation, or both cd8+ T cell activation and cd8+ T cell proliferation.
85. The method of any one of claims 73-84, wherein binding of the antibody promotes expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by cd8+ T cells.
86. The method of any one of claims 73-85, wherein binding of the antibody protein promotes cytotoxic lymphocyte-mediated killing of cancer cells.
87. The method of any one of claims 73-86, wherein binding of the antibody promotes NK cell-mediated killing of tumor cells.
88. The method of any one of claims 73-87, wherein binding of the antibody promotes expression of IL-2 by T cells.
89. The method of any one of claims 73-88, wherein binding of the antibody increases cd4+ T cells, CD196-T cells, cxcr3+ T cells, CCR4-T cells, or any combination thereof.
90. The method of any one of claims 73-89, wherein binding of the antibody to macrophages reduces inhibition of cytotoxic T cell mediated killing of tumor cells in the tumor microenvironment.
91. The method of any one of claims 73-90, wherein the cancer is lung cancer.
92. The method of any one of claims 73-91, wherein the cancer is lung epithelial cancer or lung sarcoma.
93. The method of any one of claims 73-92, wherein the cancer is lung adenocarcinoma.
94. The method of any one of claims 73-93, further comprising administering an anti-cancer therapeutic to the subject.
95. The method of any one of claims 73-78, wherein binding of the antibody to a macrophage reduces the pro-fibrotic function of the macrophage.
96. The method of any one of claims 73-78, wherein the fibrotic disease or disorder is pulmonary fibrosis.
97. The method of any one of claims 73-78, wherein the fibrotic disease or disorder is cardiac fibrosis.
98. The method of any one of claims 73-78, wherein the fibrotic disease or disorder is liver fibrosis.
99. The method of any one of claims 73-78, wherein the fibrotic disease or disorder is renal fibrosis.
100. The method of any one of claims 73-78, wherein the fibrotic disease or disorder is retinal fibrosis.
101. The method of any one of claims 73-78, wherein the fibrosis is a primary fibrotic disease or disorder.
102. The method of claim 101, wherein the primary fibrotic disease or disorder is Idiopathic Pulmonary Fibrosis (IPF).
103. The method of claim 102, wherein the primary fibrotic disease or disorder is cirrhosis.
104. The method of claim 103, wherein the primary fibrotic disease or disorder is systemic sclerosis.
105. The method of claim 103, wherein the primary fibrotic disease or disorder is radiofibrosis.
106. The method of claim 103, wherein the primary fibrotic disease or disorder is scarring associated with mechanical injury.
107. The method of any one of claims 73-78, wherein the fibrosis is a secondary fibrotic disease.
108. The method of claim 107, wherein the secondary fibrotic disease is associated with a disease or disorder selected from the group consisting of: infection, autoimmune diseases or disorders, cancer, and inflammatory diseases or disorders.
109. The method of claim 107, wherein the secondary fibrotic disease is associated with a disease or disorder selected from the group consisting of: atherosclerosis, atrial fibrillation, chronic heart failure, peripheral arterial disease, acute coronary syndrome, nonalcoholic fatty liver disease (NAFLD), chronic acute liver failure, acute kidney injury, acute tubular necrosis, and chronic kidney disease.
110. The method of claim 108, wherein the infection is selected from the group consisting of: sepsis, HIV infection, SARS-CoV-2 infection, acute viral hepatitis, chronic viral hepatitis and malaria.
111. The method of claim 108, wherein the autoimmune or inflammatory disease or disorder is selected from the group consisting of: acute Lung Injury (ALI), acute Respiratory Distress Syndrome (ARDS), allergic pneumonia, alcoholic hepatitis, non-alcoholic steatohepatitis, viral hepatitis, sickle cell disease, type 1 diabetes, type 2 diabetes, crohn's disease, celiac disease, asthma, sarcoidosis, glomerulonephritis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis, sjogren's syndrome, scleroderma, cystic Fibrosis (CF), graft versus host disease, allograft rejection, renal allograft rejection, sarcoidosis, pulmonary sarcoidosis, hemophagocytic Lymphocytosis (HLH), inflammatory arthritis, chronic Obstructive Pulmonary Disease (COPD), asthma, osteoarthritis, fibromas and multiple sclerosis.
112. The method of any one of claims 73 and 95-111, further comprising administering to the subject an anti-inflammatory therapy.
113. A pharmaceutical composition comprising (a) the antibody of any one of claims 1-72 and (b) a pharmaceutically acceptable excipient.
114. The pharmaceutical composition of claim 113, wherein the pharmaceutically acceptable excipient is selected from the group consisting of: stabilizers, buffers, surfactants, fillers, solvents, tonicity or osmotic pressure modifiers and antioxidants.
115. The pharmaceutical composition of claim 114, comprising two or more pharmaceutically acceptable excipients independently selected from the group consisting of: stabilizers, buffers, surfactants, fillers, solvents, tonicity agents and antioxidants.
116. An antibody according to any one of claims 1-72 for use as a medicament.
117. The antibody of any one of claims 1-72 for use in treating a cancer or fibrotic disease or disorder associated with the presence of M2-macrophages in a subject in need thereof.
118. The antibody for use according to claim 116 or 117, wherein binding of the antibody to macrophages promotes immune cell function as measured by one or both of the following parameters:
(a) Activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof; and
(b) Proliferation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof.
119. The antibody for use according to claim 118, wherein the activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof is measured as an enhanced level of IFN- γ, TNF- α, or perforin, or any combination thereof.
120. The antibody for use according to any one of claims 116-119, wherein binding of the antibody to a macrophage is non-cytotoxic to the macrophage.
121. The antibody for use according to any one of claims 116-120, wherein binding of the antibody to macrophages results in at least one of the following effects:
(a) The macrophage has reduced expression of at least one marker, wherein the at least one marker is CD16, CD64, TLR2, or Siglec-15;
(b) Internalizing said antibody by said macrophage;
(c) Secretion of IFN-gamma, TNF-alpha and perforin;
(d) Activation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof;
(e) Proliferation of cd4+ T cells, cd8+ T cells, NK cells, or any combination thereof; and
(f) Promote the killing of tumor cells in the tumor microenvironment.
122. The antibody for use according to claim 121, wherein the binding results in: two or more of (a) to (e); three or more of (a) to (e); four or more of (a) to (e); or all of (a) to (e).
123. The antibody for use according to any one of claims 116-122, wherein binding of the antibody to macrophages increases immunostimulatory activity in the tumor microenvironment.
124. The antibody for use according to any one of claims 116-123, wherein binding of the antibody to a macrophage reduces the immunosuppressive activity of the macrophage.
125. The antibody for use according to any one of claims 116-124, wherein binding of the antibody to a macrophage reduces the tumor promoting activity of the macrophage.
126. The antibody for use according to any one of claims 116-125, wherein binding of the antibody promotes cd4+ T cell activation, cd4+ T cell proliferation or both cd4+ T cell activation and cd4+ T cell proliferation.
127. The antibody for use of any one of claims 116-126, wherein binding promotes expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by cd4+ T cells.
128. The antibody for use according to any one of claims 116-127, wherein binding to the antibody promotes cd8+ T cell activation, cd8+ T cell proliferation, or both cd8+ T cell activation and cd8+ T cell proliferation.
129. The antibody for use of any one of claims 116-128, wherein binding of the antibody promotes expression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by cd8+ T cells.
130. The antibody for use according to any one of claims 116-129, wherein binding of the antibody protein promotes cytotoxic lymphocyte-mediated killing of cancer cells.
131. The antibody for use of any one of claims 116-130, wherein binding of the antibody promotes NK cell-mediated killing of tumor cells.
132. The antibody for use according to any one of claims 116-131, wherein binding of the antibody promotes expression of IL-2 by T cells.
133. The antibody for use of any one of claims 116-132, wherein binding of the antibody increases cd4+ T cells, CD196-T cells, cxcr3+ T cells, CCR4-T cells, or any combination thereof.
134. The antibody for use of any one of claims 116-133, wherein binding of the antibody to macrophages reduces inhibition of cytotoxic T cell mediated tumor cell killing in the tumor microenvironment.
135. The antibody for use according to any one of claims 117-134, wherein the cancer is lung cancer.
136. The antibody for use of any one of claims 117-135, wherein the cancer is a lung epithelial cancer or a lung sarcoma.
137. The antibody for use according to any one of claims 117-136, wherein the cancer is lung adenocarcinoma.
138. The antibody for use of any one of claims 117-137, wherein the antibody is formulated for administration to the subject in combination with an anti-cancer therapeutic.
139. The antibody for use according to any one of claims 117-119, wherein binding of the antibody to a macrophage reduces the pro-fibrotic function of the macrophage.
140. The antibody for use according to any one of claims 117-120, wherein the fibrotic disease or disorder is pulmonary fibrosis.
141. The antibody for use according to any one of claims 117-120, wherein the fibrotic disease or disorder is cardiac fibrosis.
142. The antibody for use according to any one of claims 117-120, wherein the fibrotic disease or disorder is liver fibrosis.
143. The antibody for use according to any one of claims 117-120, wherein the fibrotic disease or disorder is renal fibrosis.
144. The antibody for use according to any one of claims 117-120, wherein the fibrotic disease or disorder is retinal fibrosis.
145. The antibody for use according to any one of claims 117-120, wherein the fibrosis is a primary fibrotic disease or disorder.
146. The antibody for use according to claim 145, wherein the primary fibrotic disease or disorder is Idiopathic Pulmonary Fibrosis (IPF).
147. The antibody for use according to claim 145, wherein the primary fibrotic disease or disorder is cirrhosis.
148. The antibody for use according to claim 145, wherein the primary fibrotic disease or disorder is systemic sclerosis.
149. The antibody for use of claim 145, wherein the primary fibrotic disease or disorder is radiofibrosis.
150. The antibody for use according to claim 145, wherein the primary fibrotic disease or disorder is scarring associated with mechanical injury.
151. The antibody for use according to any one of claims 117-120, wherein the fibrosis is a secondary fibrotic disease.
152. The antibody for use according to claim 151, wherein the secondary fibrotic disease is associated with a disease or disorder selected from the group consisting of: infection, autoimmune diseases or disorders, cancer, and inflammatory diseases or disorders.
153. The antibody for use according to claim 151, wherein the secondary fibrotic disease is associated with a disease or disorder selected from the group consisting of: atherosclerosis, atrial fibrillation, chronic heart failure, peripheral arterial disease, acute coronary syndrome, nonalcoholic fatty liver disease (NAFLD), chronic acute liver failure, acute kidney injury, acute tubular necrosis, and chronic kidney disease.
154. The antibody for use of claim 152, wherein the infection is selected from the group consisting of: sepsis, HIV infection, SARS-CoV-2 infection, acute viral hepatitis, chronic viral hepatitis and malaria.
155. The antibody for use of claim 152, wherein the autoimmune or inflammatory disease or disorder is selected from the group consisting of: acute Lung Injury (ALI), acute Respiratory Distress Syndrome (ARDS), allergic pneumonia, alcoholic hepatitis, non-alcoholic steatohepatitis, viral hepatitis, sickle cell disease, type 1 diabetes, type 2 diabetes, crohn's disease, celiac disease, asthma, sarcoidosis, glomerulonephritis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis, sjogren's syndrome, scleroderma, cystic Fibrosis (CF), graft versus host disease, allograft rejection, renal allograft rejection, sarcoidosis, pulmonary sarcoidosis, hemophagocytic Lymphocytosis (HLH), inflammatory arthritis, chronic Obstructive Pulmonary Disease (COPD), asthma, osteoarthritis, fibromas and multiple sclerosis.
156. The method of any one of claims 116 and 139-155, wherein the antibody is formulated for administration to the subject in combination with an anti-inflammatory therapy.
CN202280022616.1A 2021-01-20 2022-01-14 Immunomodulatory antibodies and uses thereof Pending CN117062835A (en)

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US202163200897P 2021-04-01 2021-04-01
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