Anti-CD47 antibodies and methods of use

Abstract

The present disclosure relates to antibodies and antibody derivatives that bind to CD47 (also known as IAP, MER6, and OA3) and methods of use thereof. In some embodiments, the anti-CD47 antibodies or antibody derivatives disclosed herein exhibit reduced binding to red blood cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2020 / 118320, filed September 28, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to antibodies and antibody derivatives that bind to CD47 and methods of use thereof. [Background technology]

[0003] CD47 (also known as IAP, MER6, and OA3) is a membrane receptor with an extracellular N-terminal domain, five transmembrane domains, and a C-terminal intracellular tail. It binds to various membrane integrins and two soluble ligands, thrombospondin-1 (TSP-1) and signal-regulatory protein α (SIRPα). CD47 participates in various cellular processes, including cell apoptosis, proliferation, adhesion, and migration. It also plays an important role in immune and angiogenic responses. Specifically, CD47 functions as a "don't eat me" signal to macrophages and contributes to the maintenance of immune tolerance of non-malignant cells under physiological conditions. A wide range of tumor cells overexpress these immunosuppressive signaling molecules, contributing to their survival. Given the important role of CD47 in immune regulation, there is a need in the art to develop therapeutic molecules and methods targeting CD47 for immunotherapy and cancer treatment. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides isolated monoclonal antibodies and antibody derivatives that specifically bind to CD47 with high affinity, including monospecific anti-CD47 antibodies and multispecific antibodies that bind to CD47 and one or more additional targets. In some embodiments, the antibodies or antibody derivatives disclosed herein comprise full-length antibodies that bind to CD47. In some embodiments, the antibodies or antibody derivatives disclosed herein comprise scFvs that bind to CD47. The present disclosure also provides methods of making and using the antibodies and antibody derivatives disclosed herein and pharmaceutical compositions comprising the same, e.g., methods for treating diseases and conditions, such as cancer. The present invention is based, in part, on the discovery of novel antibodies that bind to CD47, which can target tumor cells and / or enhance the immune response against tumor cells, while exhibiting reduced binding and / or toxicity to normal cells (e.g., red blood cells).

[0005] The present disclosure provides an antibody that binds to CD47, the antibody comprising: a) a heavy chain variable region; and b) a light chain variable region, wherein the heavy chain variable region comprises: (1) a heavy chain variable region CDR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, and 61, or a variant thereof comprising up to about three amino acid substitutions; (2) a heavy chain variable region CDR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, and 62, or a variant thereof comprising up to about three amino acid substitutions; and (3) an amino acid sequence of any one of SEQ ID NOs: 3, 13, 23, 33, 43, 53, and 63, or a variant thereof comprising up to about three amino acid substitutions. and a heavy chain variable region CDR-H3 comprising a variant thereof, wherein the light chain variable region comprises: (1) a light chain variable region CDR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 14, 24, 34, 44, 54, and 64, or a variant thereof comprising up to about three amino acid substitutions; (2) a light chain variable region CDR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 5, 15, 25, 35, 45, 55, and 65, or a variant thereof comprising up to about three amino acid substitutions; and (3) a light chain variable region CDR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, and 66, or a variant thereof comprising up to about three amino acid substitutions.

[0006] In some embodiments, the antibody is present in a concentration of 1×10 -8 In some embodiments, the antibody binds to CD47 with a KD of 5×10 -9 In some embodiments, the antibody binds to CD47 with a KD of about 1 x 10 -11 M to approximately 1 x 10 -8 In some embodiments, the antibody binds to CD47 with a KD of about 1 x 10 -10 M to approximately 1 x 10 -8 Binds to CD47 at the KD of M.

[0007] In some embodiments, the antibody cross-competes with a reference anti-CD47 antibody, the reference anti-CD47 antibody comprising: a) a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 1; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and (4) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 4; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 6. a) a light chain variable domain (VL) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 11; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 12; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 13; and b) a heavy chain variable domain (VH) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 14; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 15; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16. c) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 21, (2) a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 22, and (3) a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 24, (2) a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 25, and (3) a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 26; d) a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 31, a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 32, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 33; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 34, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 36; e) (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 41,(2) a heavy chain variable domain (VH) sequence comprising: (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 42; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 43; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 44; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 45; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 46; f) a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 51; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 53; g) a light chain variable domain (VL) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 61, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 62, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 63, and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 65, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 66.

[0008] In some embodiments, the antibody comprises a) a heavy chain variable region; and b) a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain comprise the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain, respectively, contained in a reference heavy chain variable region, and the reference heavy chain variable region is selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, and 67. wherein the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain respectively comprise the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain contained in a reference light chain variable region, and the reference light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, 58, and 68.

[0009] In some embodiments, the antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 1; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 4; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 11; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 12; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 13; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 14; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 15; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 21; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 22; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 23; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 24; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 25; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 31; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 32; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 33; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 34; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 35; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 36.In some embodiments, the antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 41; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 42; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 43; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 44; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 45; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 51; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 53; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 54; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 55; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 61; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 62; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 63; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 65; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 66.

[0010] In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:18. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:27 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:37 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:38. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:47 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:48. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:57 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:58. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:67 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:68.

[0011] In some embodiments, the antibody comprises a human framework. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

[0012] In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region comprises a human Fc region. In some embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions.

[0013] In some embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In some embodiments, the Fc region comprises an IgG1 Fc region. In some embodiments, the Fc region comprises an IgG4 Fc region. In some embodiments, the IgG4 Fc region comprises an S228P mutation. In some embodiments, the Fc region comprises a C-terminal lysine. In some embodiments, the Fc region comprises a deletion of a C-terminal lysine.

[0014] In some embodiments, the antibody is comprised in a multispecific antibody (e.g., a bispecific antibody), wherein the multispecific antibody comprises a second antibody portion that specifically binds to a second antigen. In some embodiments, the second antigen is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is selected from the group consisting of Her-2, EGFR, PDL1, MSLN, c-Met, B-cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial glycoprotein (EGF), and the like. Interleukin-2 (EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine kinase erb-B2, 3, 4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis A (CA1.9.9), Lewis Y (LeY), phosphatidylinositol proteoglycan-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), inclusion protein 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), nephroblastoma protein (WT-1), tyrosine kinase transmembrane receptor type 1 (ROR1), PVR, PVRL2, and any combination thereof. In some embodiments, the second antigen is an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is selected from the group consisting of TIGIT, PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof. In some embodiments, the second antigen is an immune co-stimulatory molecule or a subunit of the T cell receptor / CD3 complex.In some embodiments, the immune costimulatory molecule is selected from the group consisting of CD28, ICOS, CD27, 4-1BB, OX40, and CD40, and any combination thereof. In some embodiments, the subunit of the T cell receptor / CD3 complex is selected from the group consisting of CD3γ, CD3δ, CD3ε, and any combination thereof.

[0015] The present disclosure provides immunoconjugates comprising any of the antibodies disclosed herein linked to a therapeutic agent or label. In some embodiments, the therapeutic agent is a cytotoxin or a radioisotope. In some embodiments, the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

[0016] The present disclosure provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising an antibody disclosed herein. In some embodiments, the antibody is an scFv.

[0017] The present disclosure provides an immune response cell comprising a CAR disclosed herein. In some embodiments, the immune response cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, and a myeloid cell. In some embodiments, the immune response cell is a T cell.

[0018] The present disclosure also provides pharmaceutical compositions, in some embodiments, comprising: a) an antibody, immunoconjugate, or immune response cell disclosed herein; and b) a pharmaceutically acceptable carrier agent.

[0019] The present disclosure further provides nucleic acids encoding any of the antibodies disclosed herein, vectors comprising any of the nucleic acids disclosed herein, and host cells comprising the nucleic acids or vectors disclosed herein.

[0020] The present disclosure provides methods of producing the antibodies disclosed herein, in some embodiments, the methods comprising expressing the antibody in a host cell disclosed herein and isolating the antibody from the host cell.

[0021] The present disclosure further provides methods of reducing tumor burden in a subject, in some embodiments, comprising administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition disclosed herein.

[0022] In some embodiments, the method reduces the number of tumor cells. In some embodiments, the method reduces the size of a tumor. In some embodiments, the method eradicates a tumor in a subject. In some embodiments, the tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof.

[0023] The present disclosure further provides methods of treating and / or preventing a neoplasm in a subject, hi some embodiments, the methods comprise administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition disclosed herein.

[0024] The present disclosure further provides methods for extending survival of a subject having a neoplasm, in some embodiments, the methods comprising administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition disclosed herein.

[0025] In some embodiments, the neoplasm is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

[0026] The present disclosure provides any of the antibodies disclosed herein for use as a medicament. The present disclosure further provides any of the antibodies disclosed herein for use in treating cancer. The present disclosure further provides a pharmaceutical composition disclosed herein for use as a medicament. The present disclosure further provides a pharmaceutical composition disclosed herein for use in treating cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof.

[0027] The present disclosure provides kits comprising the antibodies, immunoconjugates, pharmaceutical compositions, nucleic acids, vectors, or immune response cells disclosed herein. In some embodiments, the kits include instructions for treating and / or preventing neoplasms. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 shows the CD47 binding ability of anti-CD47 antibodies. Antibodies selected from a naive phage library are tested for binding to Raji cells (B-cell lymphoma) by flow cytometry. An anti-CD47 reference antibody (magrolimab analog) serves as a positive control. An IgG isotype control (anti-PD1 antibody) serves as a negative control. [Figure 2A] Figures 2A and 2B show the CD47 binding ability of clonal M1 mutants to tumor and normal cells. Figure 2A shows the whole cell binding of M1 mutants to Jurkat (a human leukemia cell line). Figure 2B shows the whole cell binding of M1 mutants to red blood cells (RBCs). An anti-CD47 reference antibody (magrolimab analog) serves as a positive control. Bevacizumab (an anti-VEGF-A antibody) serves as a negative control. [Figure 2B] Same as above. [Figure 3A]Figures 3A-3B show the whole cell binding capacity of M1 constant region variants to tumor and normal cells. Binding of M1 variants with constant region modifications to Jurkat cells (3A) and RBCs (3B) is tested. An anti-CD47 reference antibody (magrolimab analog) serves as a positive control. Trastuzumab (anti-HER2 antibody) serves as a negative control. [Figure 3B] Same as above. [Figure 4] Figure 4 shows the inhibition of human SIRPα binding to CD47 by the clone M1 mutant. Anti-CD47 antibodies are tested for their ability to inhibit the binding of human SIRPα to CD47-expressing Raji cells by flow cytometry. An anti-CD47 reference antibody (magrolimab analog) serves as a positive control. An IgG isotype control (anti-PD1 antibody) serves as a negative control. Anti-CD47 monoclonal antibodies block the binding of human SIRPα to CD47-expressing Jurkat cells, as measured by the mean fluorescence intensity (MFI) of staining. [Figure 5] Figure 5 shows the coagulation activity of selected antibody clones. The red blood cell (RBC) agglutination induced by serially diluted clone M1 and its mutants is shown. An anti-CD47 reference antibody (magrolimab analog) serves as a positive control. An IgG isotype control (anti-PD1 antibody) serves as a negative control. [Figure 6A]Figures 6A-6D show the macrophage-mediated phagocytosis of M1 mutants on Jurkat cells and red blood cells (RBCs). Jurkat cells or RBCs were labeled with CFSE and incubated with serially diluted M1 mutants, anti-CD47 reference antibodies, or isotype control antibodies (anti-PD1 antibodies). The antibody-Jurkat mixture was incubated with Raw264.7 (6A) or PBMC-derived macrophages (6C). The antibody-RBC mixture was incubated with Raw264.7 (6B) or PBMC-derived macrophages (6D). After 2 hours, macrophages were analyzed by flow cytometry to determine phagocytic activity. Phagocytic activity is expressed as the percentage of CFSE+F4 / 80+ cells among total F4 / 80+ cells or the percentage of CFSE+CD14+ cells among total CD14+ cells. The M1 mutant induces phagocytosis in Jurkats (as shown in (6A) and (6C)), but not in RBCs (as shown in (6B) and (6D)). [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 7] Figure 7 shows the antitumor activity of the M1 mutant in a WiDr (human colon carcinoma) xenograft tumor model. WiDr was implanted subcutaneously into NOD-SCID mice (n = 8 per group). Seven days after tumor inoculation, the first agent of each antibody was administered intraperitoneally. After implantation, mice were treated intraperitoneally with the antibody twice weekly for 3 weeks. All data points are means ± SEM. [Figure 8] Figure 8 shows the antitumor activity of the M1 mutant in an NCI-H82 (human small cell lung cancer) xenograft tumor model. NCI-H82 cells were implanted subcutaneously into NOD-SCID mice (n = 8 per group). Seven days after tumor inoculation, the first dose of each antibody was administered. After implantation, mice were treated intraperitoneally with the antibody at the indicated time points. All data points in the tumor growth curves are means ± SEM. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present disclosure provides isolated monoclonal antibodies and antibody derivatives that specifically bind to CD47 with high affinity, including monospecific anti-CD47 antibodies and multispecific antibodies that bind to CD47 and one or more additional targets. In some embodiments, the antibodies or antibody derivatives disclosed herein comprise full-length antibodies that bind to CD47. In some embodiments, the antibodies or antibody derivatives disclosed herein comprise scFvs that bind to CD47. The present disclosure also provides methods of making and using the antibodies and antibody derivatives disclosed herein and pharmaceutical compositions comprising the same, e.g., methods for treating diseases and conditions, such as cancer. The present invention is based, in part, on the discovery of novel antibodies that bind to CD47, which can target tumor cells and / or enhance immune responses against tumor cells, while exhibiting reduced binding to normal cells (e.g., red blood cells) and / or off-target effects.

[0030] For clarity, and not by way of limitation, specific embodiments of the presently disclosed subject matter are divided into: 1. Definition, 2. Antibodies and antibody derivatives, 3.How to use, 4. Drug formulations, and 5.Product.

[0031] 1.Definition As used herein, the term "antibody" includes full-length antibodies and any antigen-binding fragment thereof (i.e., antibody fragments). An "antibody" may be a separate molecule or part of an antibody derivative. Exemplary antibody derivatives include, but are not limited to, multifunctional antibodies (e.g., multispecific antibodies (e.g., bispecific antibodies)), antigen-recognizing receptors (e.g., chimeric antigen receptors), antibody conjugates comprising additional protein or non-protein moieties (e.g., antibody-drug conjugates or polymer-coated antibodies), and other multifunctional molecules comprising antibodies.

[0032] "Full-length antibody," "intact antibody," or "whole antibody" refers to an antibody having a heavy chain that is substantially identical in structure to a native antibody or that includes an Fc region as defined herein. In some embodiments, a full-length antibody comprises two heavy chains and two light chains. In some embodiments, the variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the heavy and light chains can be referred to as "VH" and "VL," respectively. The variable regions of both the heavy and light chains generally contain three highly variable loops, referred to as complementarity-determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by well-known conventions, such as those of Kabat, Chothia, MacCallum, IMGT, and AHo, as described below. The three CDRs of a heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit multiple effector functions. Antibodies are classified based on the amino acid sequence of the antibody heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, each characterized by the presence of α, δ, ε, γ, and μ heavy chains. Some of the major antibody classes are divided into subclasses, e.g., IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgG1 (α1 heavy chain), or IgG2 (α2 heavy chain). In some embodiments, a full-length antibody is glycosylated. In some embodiments, a full-length antibody comprises glycans linked to its Fc region. In some embodiments, a full-length antibody comprises bisecting glycans. As used herein, the terms "antigen-binding portion," "antibody fragment," and "antibody portion" of an antibody refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFv-Fc), single-domain antibodies, VHH, VHH-Fc, nanobodies, domain antibodies, bivalent domain antibodies, or any other fragment of an antibody that binds to an antigen, or a combination thereof. "VHH" refers to a single-domain antibody isolated from a camelid. In some embodiments, the VHH comprises the heavy chain variable region of a camelid heavy chain antibody. In some embodiments, the VHH has a size not exceeding about 25 kDa. In some embodiments, the VHH has a size not exceeding about 20 kDa. In some embodiments, the VHH has a size not exceeding about 15 kDa.

[0033] An antibody that "cross-competes for binding" with a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).

[0034] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. The fragment consists of a dimer of one heavy-chain variable region and one light-chain variable region in tight, non-covalent association. The folding of the two domains releases six highly variable loops (three loops on each of the heavy and light chains), which provide the amino acid residues for antigen binding and confer the antibody's binding specificity for the antigen. However, even a single variable domain (or half an Fv containing only three CDRs specific for an antigen) can recognize and bind to an antigen, albeit with lower affinity than the complete binding site.

[0035] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is a VFv that is linked to a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide is an antibody fragment comprising an antibody domain. H and V L The scFv further comprises a polypeptide linker between the domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0036] For purposes herein, a "recipient human framework" or "human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human shared framework. A recipient human framework that is "derived" from a human immunoglobulin framework or a human shared framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the sequence of the VL recipient human framework and the VL human immunoglobulin framework sequence or the human shared framework sequence are the same.

[0037] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, the term "binding affinity" as used herein refers to the binding affinity within a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y may typically be expressed as a dissociation constant (KD). Affinity may be measured by conventional methods known in the art, including those described herein. Specific descriptions and exemplary embodiments for measuring binding affinity are provided below.

[0038] An "affinity matured" antibody refers to an antibody that has one or more modifications in one or more CDRs or hypervariable regions (HVRs) relative to a parent antibody that does not possess such modifications, which modifications improve the affinity of the antibody for antigen.

[0039] As used herein, "CD47," "CD47 protein," or "CD47 polypeptide" refers to any CD47 polypeptide from any vertebrate origin (including mammals, e.g., primates (e.g., humans and cynomolgus monkeys)), or any fragment thereof, and may optionally contain at most one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions, additions, and / or deletions. The term covers full-length, unprocessed CD47 and any form of CD47 produced by processing in cells. The term further covers naturally occurring variants of CD47, e.g., splice variants or allelic variants. In some embodiments, the CD47 polypeptide comprises or has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homology or identity to a sequence having NCBI reference numbers: NP_001768.1, NP_942088.1, or NP_001369235.1 (homology herein can be measured using standard software, such as BLAST or FASTA). In some embodiments, the CD47 polypeptide comprises or has an amino acid sequence that is all or a continuous portion of SEQ ID NO: 103.

[0040] The term "ECD of CD47" refers to the extracellular domain of CD47. In some embodiments, the extracellular domain of CD47 is the N-terminal extracellular domain of CD47. In some embodiments, the N-terminal ECD of an exemplary CD47 polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 104.

[0041] The terms "anti-CD47 antibody" and "antibody that binds to CD47" refer to an antibody that can bind to CD47 with sufficient affinity so that it can be used as a diagnostic and / or therapeutic agent that targets CD47. In one embodiment, the binding of an anti-CD47 antibody to an unrelated, non-CD47 protein is less than about 10% of the binding of the antibody to CD47, e.g., as measured by BIACORE. (登録商標) In some embodiments, antibodies that bind to CD47 have a binding affinity of < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -12 M, e.g., 10 -9 M to 10 -10 In some embodiments, the anti-CD47 antibody binds to a CD47 epitope that is conserved in CD47 from different species. In some embodiments, the anti-CD47 antibody binds to an epitope on CD47 in the ECD of the protein.

[0042] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In some embodiments, the chimeric antibodies disclosed herein comprise a camelid heavy chain variable region and a human Fc region.

[0043] As used herein, the term "CDR" or "complementarity determining region" refers to discontinuous antigen-binding sites within the variable regions of heavy and / or light chains. These specific regions have been described by Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol., 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc The CDRs defined in the above references are described in MP et al., Dev. Comp. Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlapping amino acid residues or subsets of amino acid residues when compared with each other. However, the application of any one definition to refer to an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues covering the CDRs defined in the above references are listed in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art and include, for example, Abhinandan and Martin, Mol. Immunol. [Molecular Immunology], 45:3832-3839 (2008), Ehrenmann F. et al., Nucleic Acids Res. [Nucleic Acids Research], 38:D301-D307 (2010), and Adolf-Bryfogle J. et al., Nucleic Acids Res. [Nucleic Acids Research], 43:D432-D438 (2015).The contents of the references referred to in this section are incorporated herein by reference in their entirety, may be used in this application, and may be included in one or more claims herein.

[0044] [Table 1]

[0045] The phrases "variable domain residue numbering, e.g., according to Kabat" or "amino acid position numbering, e.g., according to Kabat" and variations thereof refer to the numbering system used for heavy or light chain variable domains in the antibody assembler of Kabat et al. (see above). Using this numbering system, the actual linear amino acid sequence may have fewer or more amino acids corresponding to shortening or insertion of variable domain FRs or CDRs. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (based on Kabat residue 52a) and inserted residues after heavy chain FR residue 82 (e.g., based on Kabat residues 82a, 82b, and 82c, etc.). The Kabat residue numbering for a given antibody can be determined by aligning the antibody sequence with the "standard" Kabat numbered sequence at the regions of homology.

[0046] In some embodiments, the amino acid residues covering the CDRs of a single domain antibody are defined according to the IMGT nomenclature of Lefranc et al. (see above). In some embodiments, the amino acid residues covering the CDRs of a full-length antibody or scFv are defined according to the Kabat nomenclature of Kabat et al. (see above). In some embodiments, the residue numbering in an immunoglobulin heavy chain (e.g., an Fc region) is that of the EU index as set forth in Kabat et al. (see above). The "EU index as set forth in Kabat" is the residue numbering of the human IgG1 EU antibody.

[0047] "Framework" or "FR" refers to those variable domain residues other than the CDR residues as herein defined.

[0048] A "humanized" antibody refers to a chimeric antibody with amino acid residues from non-human CDRs / HVRs or human FRs. In some embodiments, a humanized antibody comprises at least one, and typically two, substantially all variable domains, in which all or substantially all HVRs / CDRs correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0049] A "human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced by any of the techniques disclosed herein for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies may be produced using a variety of techniques known in the art, including phage display libraries. See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also usable for producing monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. [News in Immunology], 5:368-74 (2001). Human antibodies can be produced by administering antigen to transgenic animals, e.g., immunized xenomouses, in which the endogenous gene locus has been disabled, but which have been modified to produce such antibodies in response to antigen challenge (e.g., XENOMOUSE (商標)(For technology, see U.S. Patent Nos. 6,075,181 and 6,150,584.) For human antibodies produced by human B cell hybridoma technology, see also, e.g., Li et al., Proc. Natl. Acad. Sci. USA [Publication of the National Academy of Sciences of the United States of America] 103:3557-3562 (2006).

[0050] "Percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the compared polypeptide, after alignment of the sequences (taking into account any conservative substitutions as part of sequence identity). For purposes of determining percent amino acid sequence identity, alignment can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters to use for measured alignment, including any algorithm that achieves maximum alignment within the full length of the sequences being compared. However, for purposes herein, the sequence comparison computer program MUSCLE is used to generate percent amino acid sequence identity values ​​(Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0051] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, when a position in each of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions in the two sequences divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Comparisons are usually performed when two sequences are aligned to maximize homology.

[0052] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of the immunoglobulin molecule, the variable domain, which contains the antigen-binding site. The constant domain is the C H 1. C H 2 and C H 3 Domains (C H ) and light chain C L domain.

[0053] The "light chains" of any antibody (e.g., immunoglobulin) from any mammalian species can be designated as one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), depending on the amino acid sequence of their constant domains.

[0054] The "CH1 domain" (also called "C1" for "H1" domain) is typically from about amino acid 118 to about amino acid 215 (EU numbering system).

[0055] The "hinge region" is usually defined as the region of IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the S—S bonds between the heavy chains in the same positions.

[0056] The "CH2 domain" (also called the "C2" domain) of the human IgG Fc region typically spans from about amino acid 231 to about amino acid 340. The CH2 domain is relatively unique because it is not tightly paired with another domain. Conversely, two N-linked sugar branches are inserted between the two CH2 domains in intact, native IgG molecules. Speculation suggests that the sugar chains may provide an alternative domain-domain pairing and contribute to the stabilization of the CH2 domain. Burton, Molec Immunol., 22:161-206 (1985).

[0057] The "CH3 domain" (also called the "C2" domain) comprises the residues between the CH2 domain and the C-terminus of the Fc region (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence), usually at amino acid residue 446 or 447 of an IgG.

[0058] As used herein, the term "Fc region" or "fragment crystallizable region" is intended to define the C-terminal region of an immunoglobulin heavy chain and includes native-sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is typically defined to extend from the amino acid residue at position Cys226, or from Pro230, to its carboxy-terminus. For example, the C-terminal lysine (residue 447 in the EU numbering system) of the Fc region can be removed during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition may include antibodies with all K447 residues removed, antibodies with K447 residues intact, and a mixture of antibodies with or without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0059] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native human FcR. Furthermore, a preferred FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), which have similar amino acid sequences and are primarily distinguished by their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein covers other FcRs and includes FcRs identified in the future.

[0060] As used herein, the term "epitope" refers to the specific atom or amino acid group on an antigen to which an antibody or antibody derivative binds. Two antibodies or antigen-binding portions can bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0061] As used herein, the terms "specifically bind," "specifically recognize," and "having specificity for" refer to a measurable and reproducible interaction, e.g., binding between a target and an antibody or antibody portion, which determines the presence of the target in the presence of heterologous molecules (including biomolecules). For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to that target with greater affinity or avidity, greater readiness, and / or longer duration than binding to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is about 10% less than the extent of binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a binding affinity of ≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≦10 -12 Dissociation constant of M (K D ) In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding may include, but is not limited to, exclusive binding. The binding specificity of an antibody or antigen-binding domain may be determined experimentally by methods known in the art. Such methods include Western blot, ELISA test, RIA test, ECL test, IRMA test, EIA test, BIACORE test, and the like. TM These include, but are not limited to, peptide scanning and peptide sequencing.

[0062] An "isolated" antibody (or construct) is one that has been identified, isolated, and / or recovered from a component (e.g., natural or recombinant) of its production environment. In some embodiments, the isolated polypeptide is free from or substantially free from binding to all other components from the production environment.

[0063] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminant nucleic acid molecule normally associated with it in its production environment. In some embodiments, an isolated nucleic acid is free from or substantially free from all components associated with the production environment. The form of the isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is different from its naturally occurring form or background. Thus, an isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptides and antibodies described herein that is naturally present in a cell. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0064] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to DNA for a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Typically, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0065] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0066] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid, and includes the primary target cell and its progeny.

[0067] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of transfers. The nucleic acid content of the progeny may not be completely identical to that of the parent cell and may contain mutations. As used herein, mutant progeny that have the same function or biological activity as cells screened or selected from naturally transformed cells are included.

[0068] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the subject is a human.

[0069] An "effective amount" of a drug refers to an amount that effectively achieves a desired therapeutic or prophylactic effect at a desired dosage and for a desired period of time. The particular dosage may vary depending on one or more of the particular drug selected, the administration regimen that needs to be followed, whether the drug is administered in combination with other compounds, the time of administration, the tissue being imaged, and the physical delivery system with which the drug is associated.

[0070] A "therapeutically effective amount" of a substance / molecule, agonist, or antagonist of the present application may vary depending on factors such as the disease state, age, sex, and weight of the individual and the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the substance / molecule, agonist, or antagonist are counteracted by the therapeutically beneficial effects. A therapeutically effective amount can be delivered in one or more administrations.

[0071] As used herein, "treatment" or "treating" refers to an approach for obtaining beneficial or desired results (including clinical results). For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the rate of disease progression, ameliorating the disease state, providing partial or complete relief of the disease, reducing the dosage of one or more other drugs required to treat the disease, delaying disease progression, improving or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also covers reducing the pathological consequences of cancer (e.g., tumor volume). The methods of the present application contemplate any one or more of these aspects of treatment. "Treatment" does not necessarily mean that the disease being treated is cured.

[0072] It is to be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0073] As used herein, "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., is limited by the measurement system. In some embodiments, "about" may mean within three or more standard differences, in accordance with practice in the art. In some embodiments, "about" can refer to a range of at most 20% (e.g., at most 10%, at most 5%, or at most 1%) of a given value. In some embodiments, particularly with respect to biological systems and methods, the term may mean within an order of magnitude of a value, e.g., within 5-fold or 2-fold.

[0074] As used herein, the term "modulation" refers to a change in a positive or negative direction. Exemplary modulations include about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100% modification.

[0075] As used herein, the term "increase" refers to a modification in a positive direction of at least about 5%. The modification may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0076] As used herein, the term "reduction" refers to a negative alteration of at least about 5%. The alteration may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0077] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0078] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0079] "Effector functions" refer to those biological activities of the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0080] An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules (including, but not limited to, cytotoxic agents).

[0081] The term "drug formulation" is a formulation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain other ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0082] As used herein, a "pharmaceutically acceptable carrier agent" refers to an ingredient in a drug formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carrier agents include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0083] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is responsible for binding the antibody to an antigen. In some embodiments, the heavy and light chain variable domains (VH and VL, respectively) of a natural antibody typically have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs. (See, e.g., Kindt et al., Kuby Immunology, 61st ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain is sufficient to confer antigen-binding specificity. Furthermore, VH or VL domains can be used to isolate antibodies that bind to a specific antigen from among those that do, and to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0084] As used herein, the term "antigen-recognizing receptor" refers to a receptor that can activate an immune response cell (e.g., a T cell) in response to its binding to an antigen. Non-limiting examples of antigen-recognizing receptors include natural and modified T cell receptors ("TCRs") and chimeric antigen receptors ("CARs").

[0085] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen-binding domain fused to an intracellular signaling domain capable of activating or stimulating immune response cells, and a transmembrane domain. In some embodiments, the extracellular antigen-binding domain of the CAR comprises an antibody or antibody fragment, e.g., a VHH or scFv. In some embodiments, the antibody (e.g., a VHH or scFv) is fused to a transmembrane domain, which is fused to an intracellular signaling domain. In some embodiments, the CAR is selected to have high binding affinity or avidity for the antigen.

[0086] "Immune response cell" refers to a cell or its precursor or progeny that acts in the immune response.

[0087] 2. Antibodies and antibody derivatives The present disclosure provides antibodies and antibody derivatives. In some embodiments, the present disclosure is based in part on the discovery of monoclonal antibodies that bind to CD47, which can be used in anti-tumor therapy, where the antibodies selectively target tumor cells and / or inhibit CD47-mediated signaling pathways, thereby inducing beneficial anti-tumor effects on tumor cells. In some embodiments, the antibodies disclosed herein are antagonistic antibodies that inhibit CD47 receptor function. In some embodiments, the anti-CD47 antibodies inhibit the interaction of the CD47 receptor with one or more of its ligands. In some embodiments, the anti-CD47 antibodies block or reduce immune inhibitory signals mediated by the CD47 receptor. In some embodiments, the anti-CD47 antibodies exhibit reduced or reduced binding and / or toxicity to normal cells (e.g., erythrocytes) compared to a reference antibody (e.g., a magrolimab analog). In some embodiments, the anti-CD47 antibodies exhibit superior therapeutic efficacy compared to a reference antibody (e.g., a magrolimab analog).

[0088] In some embodiments, the antibodies of the present disclosure may be or comprise monoclonal antibodies (including chimeric, humanized, or human antibodies). In some embodiments, the antibodies disclosed herein comprise humanized antibodies. In some embodiments, the antibodies comprise a cognate human framework, e.g., a human immunoglobulin framework or a human shared framework. In some embodiments, the antibodies disclosed herein comprise a human antibody.

[0089] In some embodiments, an antibody of the present disclosure may be an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In some embodiments, the antibody is a full-length antibody (e.g., a complete IgG4 antibody) or other antibody class or isotype as defined herein. In some embodiments, an antibody or antibody derivative of the present disclosure may incorporate any of the features described herein (e.g., in Sections 2.1-2.12, detailed herein), either singly or in combination.

[0090] The antibodies and antibody derivatives of the present disclosure can be used, for example, to diagnose or treat neoplasia or cancer. In some embodiments, tumorigenesis and cancers whose growth can be inhibited using the antibodies of the present disclosure include tumorigenesis and cancers that typically respond to immunotherapy. In some embodiments, tumorigenesis and cancers include breast cancer (e.g., breast cell carcinoma), ovarian cancer (e.g., ovarian cell carcinoma), and renal cell carcinoma (RCC). Other examples of cancers that can be treated with the methods of the present disclosure include melanoma (e.g., metastatic malignant melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma), nasopharyngeal cancer, head or neck cancer, skin cancer or intraocular malignant melanoma, uterine cancer, rectal cancer, anal region cancer, stomach cancer, and the like. tumors, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, external vaginal cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, breast cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureter cancer, breast cancer, pelvic cancer, central nervous system (CNS) neoplasms, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including asbestos-induced cancers (e.g., mesothelioma)), and combinations of the above cancers.

[0091] 2.1.1 Exemplary Anti-CD47 Antibodies The present disclosure provides isolated antibodies that bind to the CD47 protein. In some embodiments, the anti-CD47 antibodies of the present disclosure bind to the ECD of CD47. In some embodiments, the anti-CD47 antibodies bind to the N-terminal ECD of CD47 comprising the amino acid sequence set forth in SEQ ID NO: 104. In some embodiments, the anti-CD47 antibodies bind to the same epitope as the anti-CD47 antibodies described herein (e.g., clone M1 and its variants (e.g., M1#21 and M1#55)).

[0092] In some embodiments, the anti-CD47 antibodies disclosed herein may be used as antagonists of CD47-based signaling pathways. In some embodiments, the anti-CD47 antibodies can block or reduce the interaction between the CD47 receptor and one or more of its ligands. In some embodiments, the anti-CD47 antibodies can reduce the interaction between the CD47 receptor and its ligand by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or about 99.9%. In some embodiments, the anti-CD47 antibodies can block immune inhibitory signaling downstream of the CD47 receptor. In some embodiments, treatment with an anti-CD47 antibody exerts an anti-tumor effect in a subject, thereby reducing tumor growth and / or prolonging the subject's survival. In some embodiments, the anti-CD47 antibodies increase the immune response and / or anti-tumor activity of immune cells (e.g., T cells and / or NK cells). In some embodiments, the anti-CD47 antibody exhibits reduced anti-targeting effect by exhibiting reduced binding to normal cells (e.g., erythrocytes) and / or reduced toxicity compared to a reference anti-CD47 antibody (e.g., a magrolimab analog). In some embodiments, the anti-CD47 antibody exhibits superior anti-tumor efficacy compared to a reference anti-CD47 antibody (e.g., a magrolimab analog). Magrolimab, also known as Hu5F9-G4, is a clinical-stage anti-CD47 antibody published in Liu et al. (2015) "Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential," PLOS ONE [Public Science Library · Integrated] 10(9):e0137345.

[0093] In some embodiments, the antibody is present in an amount of about 1 x 10 -7 In some embodiments, the antibody binds to CD47 with a KD of about 1 x 10 -8In some embodiments, the antibody binds to CD47 with a KD of about 5×10 -9 In some embodiments, the antibody binds to CD47 with a KD of about 1 x 10 -9 In some embodiments, the antibody binds to CD47 with a KD of about 1 x 10 -10 In some embodiments, the antibody binds to CD47 with a KD of about 1 x 10 -11 M to approximately 1 x 10 -7 In some embodiments, the antibody binds to CD47 with a KD of about 1 x 10 -10 M to approximately 1 x 10 -8 In some embodiments, the antibody binds to CD47 with a KD of about 1 x 10 -10 M to about 5 x 10 -8 In some embodiments, the antibody binds to CD47 with a KD of about 1 x 10 -10 M to approximately 1 x 10 -9 In some embodiments, the antibody binds to CD47 with a KD of about 2 x 10 -10 M to about 5 x 10 -9 In some embodiments, the antibody binds to CD47 with a KD of about 1 x 10 -9 M to about 5 x 10 -8 In some embodiments, the antibody binds to CD47 with a KD of about 1 x 10 -10 M to about 5 x 10 -9 Binds to CD47 at the KD of M.

[0094] In some embodiments, the anti-CD47 antibody comprises a) a heavy chain variable region; and b) a light chain variable region, wherein the heavy chain variable region comprises: (1) a heavy chain variable region CDR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, and 61, or a variant thereof comprising up to about three amino acid substitutions; (2) a heavy chain variable region CDR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, and 62, or a variant thereof comprising up to about three amino acid substitutions; and (3) an amino acid sequence of any one of SEQ ID NOs: 3, 13, 23, 33, 43, 53, and 63, or a variant thereof comprising up to about three amino acid substitutions. and a heavy chain variable region CDR-H3 comprising a variant thereof, wherein the light chain variable region comprises: (1) a light chain variable region CDR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 14, 24, 34, 44, 54, and 64, or a variant thereof comprising up to about three amino acid substitutions; (2) a light chain variable region CDR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 5, 15, 25, 35, 45, 55, and 65, or a variant thereof comprising up to about three amino acid substitutions; and (3) a light chain variable region CDR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, and 66, or a variant thereof comprising up to about three amino acid substitutions.

[0095] In some embodiments, the anti-CD47 antibody cross-competes with a reference anti-CD47 antibody, the reference anti-CD47 antibody comprising: a) a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 1; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 4; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 6. a) a light chain variable domain (VL) sequence comprising (1) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (2) CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and (3) CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 13; and a light chain variable domain (VH) sequence comprising (1) CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (2) CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (3) CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16. c) a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 21, (2) a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 22, and (3) a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 24, (2) a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 25, and (3) a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 26; d) a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 31, a heavy chain variable domain (VH) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 31, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 33; and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 34, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 36; e) (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 41,(2) a heavy chain variable domain (VH) sequence comprising: (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 42; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 43; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 44; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 45; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 46; f) a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 51; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 53; g) a light chain variable domain (VL) sequence comprising (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 61, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 62, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 63, and a light chain variable domain (VL) sequence comprising (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 65, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 66.

[0096] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain are the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain, respectively, contained in a reference heavy chain variable region. and CDR-H3 domains, wherein the reference heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, and 67, and the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain comprise the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain, respectively, contained in a reference light chain variable region, wherein the reference light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, 58, and 68.

[0097] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain comprise, respectively, the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain contained in a reference heavy chain variable region, wherein the reference heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7; and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain comprise, respectively, the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain comprise, respectively, the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain contained in a reference light chain variable region, wherein the reference light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0098] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain comprise, respectively, the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain contained in a reference heavy chain variable region, wherein the reference heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17; and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain comprise, respectively, the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain comprise, respectively, the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain contained in a reference light chain variable region, wherein the reference light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0099] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain comprise the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain, respectively, contained in a reference heavy chain variable region, wherein the reference heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 27, and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain comprise the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain, respectively, contained in a reference light chain variable region, wherein the reference light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0100] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain comprise, respectively, the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain contained in a reference heavy chain variable region, wherein the reference heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 37, and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain comprise, respectively, the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain comprise, respectively, the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain contained in a reference light chain variable region, wherein the reference light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0101] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain comprise the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain, respectively, contained in a reference heavy chain variable region, wherein the reference heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 47, and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain comprise the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain, respectively, contained in a reference light chain variable region, wherein the reference light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 48.

[0102] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain comprise the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain, respectively, contained in a reference heavy chain variable region, wherein the reference heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 57, and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain comprise the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain, respectively, contained in a reference light chain variable region, wherein the reference light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 58.

[0103] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain comprise, respectively, the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain contained in a reference heavy chain variable region, wherein the reference heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 67, and the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain comprise, respectively, the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain contained in a reference light chain variable region, wherein the reference light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 68.

[0104] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 1; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 4; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 11; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 12; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 13; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 14; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 15; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:21; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:22; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:23; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:24; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:25; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 31; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 32; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 33; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 34; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 35; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 36.In some embodiments, the anti-CD47 antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 41; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 42; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 43; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 44; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 45; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 51; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 53; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 54; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 55; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 61; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 62; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 63; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 65; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 66.

[0105] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, and 67, and the light chain variable region comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, 58, and 68. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, and 67, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, 58, and 68.

[0106] In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:18. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:27 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:37 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:38. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:47 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:48. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:57 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:58. In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:67 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:68.

[0107] In some embodiments, any one of the amino acid sequences contained in the heavy chain variable region may contain at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In some embodiments, the amino acid substitutions are conservative substitutions.

[0108] In some embodiments, the antibody comprises a human framework. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is isolated from a human phage display library.

[0109] In some embodiments, the anti-CD47 antibody does not comprise an Fc region. In some embodiments, the anti-CD47 antibody further comprises an Fc region. In some embodiments, the Fc region comprises a human Fc region. In some embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In some embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In some embodiments, the Fc region comprises an IgG1 Fc region. In some embodiments, the IgG1 Fc region comprises one or more mutations that modulate antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the IgG1 Fc region comprises one or more mutations that reduce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the IgG1 Fc region comprises one or more mutations that enhance antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the Fc region comprises an IgG4 Fc region. In some embodiments, the IgG4 Fc region comprises an S228P mutation. In some embodiments, the Fc region comprises a C-terminal lysine. In some embodiments, the Fc region comprises a deletion of a C-terminal lysine.

[0110] In some embodiments, the anti-CD47 antibody comprises a heavy chain and a light chain, wherein the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 69 and 70, respectively (M1#21). In some embodiments, the anti-CD47 antibody comprises a heavy chain and a light chain, wherein the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 69 and 72, respectively (M1#21P). In some embodiments, the anti-CD47 antibody comprises a heavy chain and a light chain, wherein the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 71 and 70, respectively (M1#21K). In some embodiments, the anti-CD47 antibody comprises a heavy chain and a light chain, wherein the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 71 and 72, respectively (M1#21KP).

[0111] In some embodiments, the anti-CD47 antibody comprises a heavy chain and a light chain, wherein the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 59 and 60, respectively (M1#55). In some embodiments, the anti-CD47 antibody comprises a heavy chain and a light chain, wherein the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 59 and 74, respectively (M1#55P). In some embodiments, the anti-CD47 antibody comprises a heavy chain and a light chain, wherein the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 73 and 60, respectively (M1#55K). In some embodiments, the anti-CD47 antibody comprises a heavy chain and a light chain, wherein the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 73 and 74, respectively (M1#55KP).

[0112] In some embodiments, the anti-CD47 antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, an Fv-Fc fusion, an scFv-Fc fusion, a VHH-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

[0113] In some embodiments, the antibody is comprised in a larger molecule that is an antibody derivative. In some embodiments, the antibody derivative is a multispecific antibody (e.g., a bispecific antibody), wherein the multispecific antibody comprises a second antibody portion that specifically binds to a second antigen. In some embodiments, the second antigen is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is Her-2, B7H3, EGFR, PD-L1, MSLN, c-Met, B-cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial glycoprotein (EGFR), or IL-1. Protein (EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine kinase erb-B2, 3, 4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis A (CA 1.9.9), Lewis Y (LeY), phosphatidylinositol proteoglycan-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), inclusion protein 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), nephroblastoma protein (WT-1), tyrosine kinase transmembrane receptor type 1 (ROR1), PVR, PVRL2, and any combination thereof. In some embodiments, the second antigen is an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is selected from the group consisting of TIGIT, PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof.In some embodiments, binding of the antibody derivative or multispecific antibody to the second antigen inhibits immune checkpoint modulators. In some embodiments, the second antigen is an immune costimulatory molecule or a subunit of the T cell receptor / CD3 complex. In some embodiments, the immune costimulatory molecule is selected from the group consisting of CD28, ICOS, CD27, 4-1BB, OX40, CD40, and any combination thereof. In some embodiments, binding of the antibody derivative or multispecific antibody to the second antigen activates the immune costimulatory molecule. In some embodiments, the subunit of the T cell receptor / CD3 complex is selected from the group consisting of CD3γ, CD3δ, CD3ε, and any combination thereof. In some embodiments, binding of the antibody derivative or multispecific antibody to the second antigen activates the T cell receptor / CD3 complex.

[0114] In some embodiments, the anti-CD47 antibody is linked to the second antigen-binding moiety via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises about 4 to about 30 amino acids. In some embodiments, the peptide linker comprises about 4 to about 15 amino acids. In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-102.

[0115] In some embodiments, the anti-CD47 antibody is conjugated to a therapeutic agent or label. In some embodiments, the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme. In some embodiments, the therapeutic agent is a cytotoxin or a radioisotope.

[0116] 2.2 Antibody affinity In some embodiments, the antibodies or antibody derivatives disclosed herein have high binding affinity for their target antigens. In some embodiments, the antibodies or antibody derivatives have a binding affinity of about 1×10 -7 In some embodiments, the antibody or antibody derivative binds to the target with a KD of about 1 x 10 -8In some embodiments, the antibody or antibody derivative binds to the target with a KD of about 5×10 -9 In some embodiments, the antibody or antibody derivative binds to the target with a KD of about 1 x 10 -9 In some embodiments, the antibody or antibody derivative binds to the target with a KD of about 1 x 10 -10 Binds to target with KD smaller than M.

[0117] In some embodiments, the antibody or antibody derivative is about 1×10 -11 M to approximately 1 x 10 -7 In some embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -10 M to approximately 1 x 10 -7 In some embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -10 M to approximately 1 x 10 -8 In some embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -11 M to approximately 1 x 10 -9 In some embodiments, the antibody or antibody derivative binds to the target with a KD of about 2×10 -10 M to about 5 x 10 -9 In some embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -9 M to about 5 x 10 -8 In some embodiments, the antibody binds to the target with a KD of about 1 x 10 -10 M to approximately 1 x 10 -9 It binds to the target at the KD of M.

[0118] The KD of an antibody or antibody derivative may be determined by methods known in the art, such as Western blot, ELISA test, RIA test, ECL test, IRMA test, EIA test, Octet test, BIACORE test, etc. (登録商標) These include, but are not limited to, peptide scanning and peptide sequencing.

[0119] In some embodiments, the BIACORE (登録商標) Surface plasmon resonance assays may be used to measure KD, e.g., at 25°C on an immobilized antigen CMS chip at approximately 10 response units (RU) using a BIACORE (登録商標) -2000 or BIACORE (登録商標) Measurements are performed using, but not limited to, a Biacore 3000 (Biacore, Piscataway, NJ). In some embodiments, a carboxymethylated dextran synthesizer sensor chip (CMS, Biacore) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / mL (approximately 0.2 μM) using 10 mM sodium acetate, pH 4.8, and injected at a flow rate of 5 μL / min to obtain approximately 10 response units (RU) of conjugated protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μL / min in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. The association rate (k on ) and dissociation rate (k off ) is based on a simple one-to-one Langmuir binding model (BIACORE (登録商標) The equilibrium dissociation constant (KD) is calculated by simultaneously fitting the association and dissociation sensorgrams using the evaluation software (version 3.2). The equilibrium dissociation constant (KD) is calculated as the ratio k off / k on For example, see Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate measured by the surface plasmon resonance assay is 10 6 M -l s -1Above this, the association rate may be determined by a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nM, emission = 340 nM, 16 nM bandpass) of 20 nM anti-antigen antibody (Fab format) in PBS (pH 7.2) solution at 25°C in the presence of increasing antigen concentrations, using, for example, a spectrometer, e.g., a spectrophotometer in pass-cut configuration (Aviv Instruments) or an 8000 series SLM-AMINCO with a stirred absorber pool. (商標) It is measured using a spectrophotometer (ThermoSpectronic).

[0120] 2.3 Antibody fragments In some embodiments, antibodies of the present disclosure include antigen-binding fragments or antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab'), VHH, Fv, and scFv fragments, as well as other fragments described herein. For a review of several antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthin, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab)2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0121] In some embodiments, the antibodies of the present disclosure may be diabodies. Diabodies are antibody fragments with two antigen-binding sites and may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are further described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0122] In some embodiments, antibodies of the present disclosure may comprise single domain antibodies. Single domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In some embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 B1). In some embodiments, a single domain antibody is a camelid single domain antibody. In some embodiments, a single domain antibody is a VHH. In some embodiments, a single domain antibody is a chimeric antibody. In some embodiments, a single domain antibody is a humanized antibody.

[0123] Antibody fragments may be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and recombinant host cell (e.g., E. coli or phage) production, as described herein.

[0124] 2.4 Chimeric and humanized antibodies In some embodiments, the antibody of the present disclosure is a chimeric antibody. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region. In some embodiments, a chimeric antibody is a "class-switched" antibody, in which the class or subclass is altered compared to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0125] In some embodiments, the antibodies of the present disclosure may be humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains, in which the HVRs (e.g., CDRs) (or portions thereof) are derived from a non-human antibody, and one or more framework regions (FRs) (or any portion thereof) are derived from a human antibody sequence. Optionally, the humanized antibody may further comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody derived from the HVR residues), e.g., to restore or improve antibody specificity or affinity.

[0126] Humanized antibodies and methods for their preparation are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and in, for example, Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (SDR (a-CDR) grafting described); Padlan, Mol. Immunol. 28:489-498 (1991) ( These methods are further described in Dall'Acqua et al., Methods 36:43-60 (2005) (where "FR shuffling" is described), Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (where a "guided selection" method for FR shuffling is described).

[0127] Human framework regions that can be used for humanization include framework regions selected by the "best-fit" method (e.g., Sims et al., J. Immunol. 151:2296 (1993)), framework regions derived from the shared sequences of human antibodies of a particular light or heavy chain variable region subclass (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992) and Presta et al., J. Immunol. 151:2623 ... mature (somatic cell proliferation) antibodies, and framework regions derived from human IgG1a and IgG2b. Examples of framework regions include, but are not limited to, naturally mutated framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions obtained by screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0128] 2.5 Human antibodies In some embodiments, an antibody of the present disclosure may be a human antibody (e.g., a human domain antibody or human DAb). Human antibodies may be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. [Current Trends in Pharmacology] 5:368-74 (2001), Lonberg, Curr. Opin. Immunol. [Current Trends in Immunology] 20:450-459 (2008), and Chen, Mol. Immunol. [Molecular Immunology] 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single domain antibodies (or DAbs) are known in the art. See, for example, US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0129] Human antibodies (e.g., human DAbs) can be prepared by administering an immunogen to transgenic animals that have been modified to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, which replace endogenous immunoglobulin loci, or which are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. [Nature-Biotech] 23:1117-1125 (2005). Further, see, e.g., XENOMOUSE. TM US Patent Nos. 6,075,181 and 6,150,584, describing the technology, HuMab (登録商標) U.S. Patent No. 5,770,429, KM MOUSE, describes the technology (登録商標) US Patent No. 7,041,870 describing the technology, and VelociMouse (登録商標)See US Patent No. US2007 / 0061900, which describes the technology. The human variable regions from whole antibodies produced by such animals may be further modified, for example, by attaching them to different human constant regions.

[0130] Human antibodies (e.g., human DAbs) may be prepared using hybridoma-based methods. Human myeloma and murine human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, e.g., Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc. New York, 1987; and Boerner et al., J. Immunol., 147:86 (1991)). Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) further describe human antibodies produced by human B-cell hybridoma technology. Other methods include, for example, those described in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue [Modern Immunology] 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology 27(3):185-91 (2005).

[0131] Human antibodies (e.g., human DAbs) may be generated by isolating Fv clone variable domain sequences selected from a human phage display library. These variable domain sequences may then be combined with desired human constant domains. A description of techniques for selecting human antibodies from antibody libraries follows.

[0132] 2.6 Library-derived antibodies Antibodies of the present disclosure can be isolated by screening combinatorial libraries of antibodies with the desired activity. For example, several methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Humana Press, Totowa, NJ, 2001), McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, eds., Humana Press, Totowa, NJ, 2001). Press, Totowa, NJ (2003), Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004), Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004), and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004). Methods for constructing single domain antibody libraries have been described, see, e.g., U.S. Patent No. 7,371,849.

[0133] In some phage display methods, V H and V LGene libraries can be individually cloned, randomly recombined into phage libraries, and screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. [Annual Comments on Immunology], 12:433-455 (1994). Phages typically display antibody fragments as scFv or Fab fragments. Libraries from immunization can provide high-affinity antibodies to immunogens without constructing hybridomas. Alternatively, natural libraries (e.g., obtained from humans) can be cloned without the need for any immunization, providing a single source of antibodies against a wide range of non-self and self antigens, as described in Griffiths et al., EMBO J. [European Journal of Molecular Biology], 12:725-734 (1993). Finally, natural libraries can be synthesized by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to complete in vitro rearrangement to encode highly variable CDR3 regions, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include U.S. Patent No. 5,750,373 and U.S. Patent Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0134] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

[0135] 2.7 Antibody variants The present disclosure further provides amino acid sequence variants of the disclosed antibodies, which are expected, for example, to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, but are not limited to, deletion and / or insertion and / or substitution of residues within the antibody amino acid sequence. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final (i.e., modified) antibody possesses the desired properties (e.g., antigen binding).

[0136] 2.7.1 Substitution, Insertion, and Deletion Mutants In some embodiments, antibody variants are provided that have one or more amino acid substitutions. Target sites for substitutional mutagenesis include HVRs (or CDRs) and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 2. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 2 and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest and products screened for desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0137] [Table 2]

[0138] Amino acids may be grouped according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) acidic: Asp, Glu, (4) alkaline: His, Lys, Arg, (5) residues that affect chain orientation: Gly, Pro, and (6) aromatic: Trp, Tyr, Phe. In some embodiments, non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

[0139] In some embodiments, one type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). The resulting variants are typically selected for further study and are modified (e.g., improved) with some biological property (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and / or essentially retain some biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be easily generated using affinity maturation techniques (e.g., those described herein), e.g., based on phage display. Briefly, one or more HVR (or CDR) residues are mutated, the variant antibodies are displayed on phage, and variant antibodies with a particular biological activity (e.g., binding affinity) are screened.

[0140] Modifications (e.g., substitutions) can be made in the HVRs (or CDRs) to, for example, improve antibody affinity. Such modifications can be made in HVR (or CDR) "hot spots" (i.e., residues encoded by codons frequently mutated during the somatic maturation process) (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or in the SDRs (a-CDRs), and the resulting variant VH or VL can be tested for binding affinity. Affinity maturation by construction of secondary libraries and reselection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Humana Press, Totowa, NJ, (2001)). In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any one of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves HVR (or CDR)-directed methods, in which a number of HVR (or CDR) residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scanning mutagenesis or modeling can be used to specifically identify HVR (or CDR) residues involved in antigen binding. In particular, CDR-H3 and CDR-L3 are always targeted.

[0141] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs (or CDRs), so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions according to the present specification) may be made in an HVR (or CDR) that do not substantially reduce binding affinity. Such modifications may be located in HVR (or CDR) "hot spots" or outside of a CDR. In some embodiments of the above-described variant VHH sequences, each HVR (or CDR) is unaltered or contains no more than one, two, or three amino acid substitutions.

[0142] As described in Cunningham and Wells (1989) Science 244:1081-1085, a useful method for identifying antibody residues or regions amenable to targeted mutagenesis is called "alanine scanning mutagenesis." In such methods, a target residue or group of target residues (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) is identified, and substitution with neutral or negatively charged amino acids (e.g., alanine or polyalanine) is performed to determine whether the antibody-antigen interaction is affected. Functional sensitivity to the initial substitution can be demonstrated by introducing additional substitutions at the amino acid position. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as substitution candidates. Mutants can be screened to determine whether they contain the desired properties.

[0143] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., used in ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0144] 2.7.2 Glycosylation variants In some embodiments, the antibody portion is modified to increase or decrease the degree of glycosylation of the construct. Addition or deletion of glycosylation sites to an antibody can be readily accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0145] If the antibody comprises an Fc region (e.g., scFv-Fc), the sugars attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched, high-contact angle oligosaccharides, which are typically N-linked to the Fc region C. H The oligosaccharide is linked to Asn297 in the RI-2 domain. See, e.g., Wright et al., TIBTECH [Trends in Biotechnology] 15:26-32 (1997). The oligosaccharide may contain various sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, and fucose linked to GlcNAc in the "stem" of the RI-2 oligosaccharide. In some embodiments, modifications can be made to the oligosaccharide in the antibody to generate antibody variants with improved properties.

[0146] In some embodiments, the antibody has a glycostructure that lacks fucose linked (directly or indirectly) to the Fc region. For example, the fucose content in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 glycochain relative to the sum of all glycostructures (e.g., complex, heterozygous, and high-mannose structures) linked to Asn297 as measured by MALDI-TOF mass spectrometry (e.g., as described in WO 2008 / 077546). Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region; however, due to minor sequence variation in antibodies, Asn297 may be located approximately ±3 amino acid positions upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function (see, e.g., US Patent Nos. US 2003 / 0157108 (Presta, L.) and US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.)). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108; WO 2000 / 61739, WO 2001 / 29246, US 2003 / 0115614, US 2002 / 0164328, US 2004 / 0093621, US 2004 / 0132140, US 2004 / 0110704, US 2004 / 0110282, US 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO2005 / 053742, WO 2002 / 031140, Okazaki et al., J. Mol. Biol. [Journal of Molecular Biology] 336:1239-1249 (2004), Yamane-Ohnuki et al., Biotech. Bioeng. [Biotechnology and Bioengineering] 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. [Biochemistry and Biophysics Publication] 249:533-545 (1986); U.S. Patent No. US 2003 / 0157108 A1, Presta, L, and WO 2004 / 056312 A1, Adams et al.) and knockout cell lines, such as α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (e.g., Yamane-Ohnuki et al., Biotech. Bioeng. [Biotechnology and Bioengineering] 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. [Biotechnology and Bioengineering] 94(4):680-688 (2006) and WO 2003 / 085107).

[0147] In some embodiments, the antibody has a bisected oligosaccharide, for example, where the bicontact angle oligosaccharide linked to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Further provided are antibody variants having at least one half-lactose residue in the oligosaccharide linked to the Fc region. These antibody variants may have improved ADCC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.), and WO 1999 / 22764 (Raju, S.).

[0148] 2.7.3 Fc Region Variants In some embodiments, the Fc region of the presently disclosed antibodies or antibody derivatives may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions. In some embodiments, Fc region variants can be generated by introducing one or more amino acid modifications into the Fc region of an antibody portion (e.g., scFv-Fc or VHH-Fc).

[0149] In some embodiments, the Fc region possesses some, but not all, effector functions, making it a desirable candidate for applications in which in vivo antibody half-life is important but some effector functions (e.g., complement and ADCC) are unnecessary or deleterious. Reduced / depleted CDC and / or ADCC activity can be determined by performing in vitro and / or in vivo cytotoxicity assays. For example, Fc receptor (FcR) binding measurements can be performed to ensure that the antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. [Annual Comments in Immunology] 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA [National Academy of Sciences], 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA [National Academy of Sciences], 82:1499-1502 (1985), and U.S. Patent No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. [Journal of Experimental Medicine], 166:1351-1361 (1987)). Alternatively, non-radioactive assays (e.g., ACTI for flow cytometry) can be used. (商標) Non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA), and CytoTox 96 (登録商標)Non-radioactive cytotoxicity assays (see Promega, Madison, WI) may also be used. Useful effector cells for use in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the target molecule may be assessed in vivo, e.g., in an animal model, as described, e.g., in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie et al., Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays may be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0150] Antibodies with reduced effector function include antibodies (U.S. Pat. No. 6,737,056) in which one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 have been substituted. Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted by alanine (U.S. Pat. No. 7,332,581).

[0151] Several antibody variants with enhanced or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0152] In some embodiments, the Fc region comprises one or more mutations according to the EU numbering of residues. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the IgG1 Fc region comprises an L234A mutation and / or an L235A mutation. In some embodiments, the Fc region is an IgG2 Fc region or an IgG4 Fc region. In some embodiments, the Fc region is an IgG4 Fc region comprising an F234A and / or an L235A mutation.

[0153] In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the IgG1 Fc region comprises one or more mutations that modulate antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the IgG1 Fc region comprises one or more mutations that reduce antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the IgG1 Fc region comprises one or more mutations that enhance antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, Y300L, and P396L. In some embodiments, the IgG1 Fc region comprises the following mutations: S239D, A330L, and I332E. In some embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, and Y300L. In some embodiments, the IgG1 Fc region comprises substitutions at positions 298, 333 and / or 334 of the Fc region.

[0154] In some embodiments, the Fc region comprises an IgG4 Fc region. In some embodiments, the IgG4 Fc region comprises an S228P mutation.

[0155] In some embodiments, the Fc region comprises a C-terminal lysine. In some embodiments, the Fc region comprises a deletion of a C-terminal lysine.

[0156] In some embodiments, modifications are made to the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., 164:4178-4184 (2000).

[0157] In some embodiments, the antibody (e.g., scFv-Fc or VHH-Fc) variants comprise a variant Fc region that contains one or more amino acid substitutions that alter half-life and / or binding to the neonatal Fc receptor (FcRn). Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more amino acid substitutions that alter binding of the Fc region to FcRn. Such Fc variants include Fc variants (US Pat. No. 7,371,826) with substitutions at one or more Fc region residues (eg, substitution at Fc region residue 434).

[0158] See also Duncan and Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0159] 2.7.4 Cysteine ​​Engineered Antibody Variants In some embodiments, it is desirable to generate cysteine ​​engineered antibody moieties, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In some embodiments, the substituted residues are located at accessible sites of the antibody. By replacing these residues with cysteine ​​residues, reactive thiol groups are positioned at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (e.g., drug moieties or linker-drug moieties) to generate immunoconjugates, as further described herein. In some embodiments, any one or more residues of A118 (EU numbering) of the heavy chain and S400 (EU numbering) of the heavy chain Fc region may be substituted with cysteine ​​residues. Cysteine ​​engineered antibody moieties may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0160] 2.8 Antibody derivatives In some embodiments, the antibodies described herein may be further modified into antibody derivatives containing other protein or non-protein moieties known in the art and readily available. Non-protein moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(N-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in formulation due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on the following considerations, including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used for diagnostic purposes under defined conditions, etc.

[0161] In some embodiments, antibodies may be further modified into antibody derivatives that contain one or more biologically active proteins, polypeptides, or fragments thereof. As used interchangeably herein, "biologically active" or "having biological activity" refers to a biological activity exhibited in vivo to perform a specific function. For example, it may mean binding to a specific biological molecule (e.g., protein, DNA, etc.) and promoting or inhibiting the activity of the biological molecule. In some embodiments, biologically active proteins or fragments thereof include proteins or polypeptides administered to a patient as active drug substances for the prevention or treatment of a disease or condition, and proteins and polypeptides for diagnostic purposes (e.g., enzymes used in diagnostic tests or in vitro assays), and proteins and polypeptides administered to a patient to prevent a disease (e.g., vaccines).

[0162] 2.9 Antibody Production Methods Any available or known technique in the art can be used to produce the antibodies and antibody derivatives disclosed herein. For example, but not limited to, recombinant methods and compositions can be used to produce antibodies and antibody derivatives, such as those described in U.S. Patent No. 4,816,567. Detailed processes for producing antibodies and antibody derivatives are described in detail in the Examples below.

[0163] The presently disclosed subject matter further provides isolated nucleic acids encoding the antibodies or antibody derivatives disclosed herein. For example, the isolated nucleic acids can encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody, e.g., the light and / or heavy chains of the antibody.

[0164] In some embodiments, the nucleic acid may be present in one or more vectors (e.g., expression vectors). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid," which can have another DNA segment ligated into its circular double-stranded DNA loop. Another type of vector is a viral vector, in which another DNA segment may be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome. Additionally, some vectors (expression vectors) are capable of directing the expression of genes to which they are operably linked. Generally, expression vectors used in recombinant DNA techniques are always in the form of plasmids (vectors). However, the disclosed subject matter is intended to include other forms of expression vectors that serve equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0165] Different portions of the antibodies or antibody derivatives disclosed herein can be constructed in a single polycistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements for generating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRESs, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Foot and Mouth Disease Virus IRES, Picornanilus IRES, Poliovirus IRES, and Encephalomyocarditis Virus IRES), and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). Retroviral vectors in combination with appropriate packaging lines are also suitable, in which the capsid protein is capable of infecting human cells. Various cell lines that produce amphipathic viruses are known, including, but not limited to, PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437), PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902), and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphipathic particles are also suitable, such as pseudotyped particles having a VSVG, RD114, or GALV envelope, and any other non-amphipathic particles known in the art.

[0166] In some embodiments, a nucleic acid encoding an antibody or antibody derivative of the present disclosure and / or one or more vectors containing the nucleic acid can be introduced into a host cell. In some embodiments, the nucleic acid can be introduced into a cell by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral or phage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. In some embodiments, the host cell can include a host cell transformed with, for example, a vector containing a nucleic acid encoding a single domain antibody and / or an amino acid sequence comprising the VH of the single domain antibody. In some embodiments, the host cell can include a host cell transformed with, for example, (1) a vector containing a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector containing a nucleic acid encoding the amino acid sequence of the VL of the antibody and a second vector containing a nucleic acid encoding the amino acid sequence of the VH of the antibody. In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (eg, YO, NSO, Sp20 cell).

[0167] In some embodiments, methods for preparing an antibody or antibody derivative disclosed in the present disclosure may comprise culturing a host cell into which nucleic acid encoding the antibody or antibody derivative has been introduced under conditions suitable for expression of the antibody or antibody derivative, and optionally recovering the antibody or antibody derivative from the host cell and / or host cell culture medium. In some embodiments, the antibody or antibody derivative is recovered from the host cell by chromatographic techniques.

[0168] To recombinantly produce an antibody or antibody derivative of the present disclosure, for example, nucleic acids encoding the above-described antibody or antibody derivative can be isolated, inserted into one or more vectors, and further cloned and / or expressed in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional processes (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody or antibody derivative). Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies or antibody derivatives can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See further the expression of antibody fragments in E. coli as described in Charlton, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Humana Press, Totowa, NJ), 2003, pp. 245-254.) After expression, the antibody or antibody derivative can be isolated in a soluble fraction from the bacterial cell paste and further purified.

[0169] In addition to prokaryotes, eukaryotic microbes (e.g., filamentous fungi or yeast) are also suitable cloning or expression hosts for antibody-encoding vectors, including "humanized" glycosylation pathways to produce fungal and yeast strains of antibodies or antibody derivatives with partial or fully human glycosylation patterns. See Gemgross, Nat. Biotech. [Nature-Biotech] 22:1409-1414 (2004) and Li et al., Nat. Biotech. [Nature-Biotech] 24:210-215 (2006). Suitable host cells for expressing glycosylated antibodies can be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrates include plant and insect cells. Many baculovirus strains have been identified and can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. In some embodiments, plant cell cultures may be used as host cells. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES FOR PRODUCING ANTIBODIES IN TRANSGENIC PLANTS). (商標) See, for example, the technique described herein.

[0170] In some embodiments, vertebrate cells may be used as host cells. For example, but not limited to, mammalian cell lines adapted for suspension growth may be useful. Non-limiting examples of useful mammalian host cell lines include monkey kidney cell line CV1 transformed with SY40 (COS-7), human embryonic kidney cell lines (293 or 293 cells, e.g., those described in Graham et al., J Gen Viral. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, e.g., those described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African midge kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep 02), mouse mammary tumor (MMT 060562), TRI cells, e.g., those described in Mather et al., Annals

[0004] Examples of useful mammalian host cell lines include those described in NYAcad. Sci. [Annual Issue of the New York Academy of Sciences] 383:44-68 (1982), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFK CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA [Annual Issue of the New York Academy of Sciences] 77:42 I6 (1980)) and myeloma cell lines, such as YO, NSO, and Sp2 / 0. For a review of several mammalian host cell lines suitable for the production of antibodies or antibody derivatives, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0171] In some embodiments, techniques for preparing bispecific and / or multispecific antibodies include, but are not limited to, recombinantly discovering two immunoglobulin heavy chain / light chain pairs with the same specificity, where one or both of the heavy or light chains are fused to an antigen-binding moiety (e.g., a VHH or scFv) with a different specificity, and recombinantly co-expressing the two immunoglobulin heavy chain / light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983); PCT Patent Application No. WO 93 / 08829; and Traunecker et al., EMBO J 10:3655 (1991)), and "punch and die" engineering (see, e.g., U.S. Pat. No. 5,731,168). Bispecific antibodies can be prepared by engineering electrostatic deflection effects to generate antibody Fc-heterodimeric molecules (WO 2009 / 089004A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)), using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 1999, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, Immunol. 148(5):1547-1553 (1992)), preparing bispecific antibody fragments using "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol. 152:5368 (1994)), and preparing trispecific antibodies as described, for example, in Tutt et al., J. Immunol. 147:60 (1991).

[0172] Bispecific and multispecific molecules of the present disclosure may be prepared by chemical techniques (see, e.g., Kranz (1981) Proc. Natl. Acad. Sci. USA [Publication of the National Academy of Sciences of the United States of America] 78:5807), "polyoma" techniques (e.g., U.S. Pat. No. 4,474,893), or recombinant DNA techniques. Bispecific and multispecific molecules of the presently disclosed subject matter may also be prepared by conjugating partial binding specificities (e.g., a first epitope and a second epitope binding specificity) using methods known in the art and described herein. For example, but not limited to, each binding specificity of the bispecific and multispecific molecules may be prepared together using recombinant fusion protein technology, or each may be prepared separately and then conjugated to each other. When the binding specificities are proteins or peptides, covalent conjugation can be achieved using a variety of coupling or cross-linking agents. Non-limiting examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky (1984) J. Exp. Med. 160:1686; Liu (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins. Mitt. (1985) 78th issue, pp. 118-132), Brennan ((1985) Science 229:81-83), and Glennie (1987) J. Immunol. 139:2367-2375). When the binding specificities are antibodies (e.g., two humanized antibodies), they may be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In some embodiments, prior to conjugation, the hinge region may be modified to contain an odd number of sulfhydryl residues (e.g., one).

[0173] In some embodiments, the two binding specificities of a bispecific antibody can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific and multispecific molecule is a MAb x MAb, MAb x Fab, Fab x F(ab')2, or ligand x Fab fusion protein. In some embodiments, the bispecific antibody of the present disclosure can be a single-chain molecule, e.g., a single-chain bispecific antibody, a single-chain bispecific molecule comprising one single-chain antibody and a binding-determining cluster, or a single-chain bispecific molecule comprising two binding-determining clusters. Bispecific and multispecific molecules can be single-chain molecules or can comprise at least two single-chain molecules. Methods for preparing bispecific and multispecific molecules are described, for example, in U.S. Patent No. 5,260,203, U.S. Patent No. 5,455,030, U.S. Patent No. 4,881,175, U.S. Patent No. 5,132,405, U.S. Patent No. 5,091,513, U.S. Patent No. 5,476,786, U.S. Patent No. 5,013,653, U.S. Patent No. 5,258,498, and U.S. Patent No. 5,482,858. This specification includes "octopus antibodies," which further include engineered antibodies with three or more functional antigen binding sites (e.g., epitope binding sites) (see, e.g., US 2006 / 0025576A1).

[0174] In some embodiments, an animal system can be used to produce the antibodies or antibody derivatives of the present disclosure. The animal system for preparing hybridomas is the murine system.

[0175] Hybridoma production in mice is a highly efficient procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known (see, e.g., Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

[0176] 2.10 Assay The antibodies and antibody derivatives of the present disclosure can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a number of assays known in the art and as described herein.

[0177] In some embodiments, the antigen-binding activity of an antibody or antibody derivative of the present disclosure can be tested by known methods (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay). Each of these assays typically detects the presence of a particular target protein-antibody complex by a labeled reagent (e.g., an antibody) that has specificity for the target complex. For example, an antibody or antibody derivative can be detected by, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody or antibody derivative. Alternatively, the antibody or antibody derivative can be detected by any one of several other immunoassays. For example, the antibody or antibody derivative can be radiolabeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated herein by reference). The radioactive isotope can be detected by such means as the use of, for example, a Geiger counter or a scintillation counter or by autoradiography.

[0178] In some embodiments, competition assays may be used to identify antibodies or antibody derivatives that compete with the antibodies of the present disclosure for binding to CD47. In some embodiments, such competing antibodies bind to a similar epitope (e.g., a linear or conformational epitope) as that bound by the antibodies disclosed herein. Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ) provides detailed exemplary methods for mapping epitopes bound by antibodies.

[0179] In a non-limiting example of a competitive assay, immobilized CD47 can be incubated in a solution containing a first labeled antibody or antibody derivative that binds to CD47 and a second unlabeled antibody whose ability to compete with the first antibody for binding to CD47 is tested. The second antibody may be present in hybridoma supernatant. As a control, immobilized CD47 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to CD47, excess unbound antibody is removed and the amount of label associated with the immobilized CD47 is measured. A significant decrease in the amount of label associated with the immobilized CD47 in the test sample compared to the control sample indicates that the second antibody competes with the first antibody for binding to CD47. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0180] The present disclosure provides assays for identifying anti-CD47 antibodies or antibody derivatives thereof that have biological activity. Biological activity may include, for example, activation of immune cells or immune activation reporter genes, such as NFAT reporter genes or NF-κB reporter genes. Antibodies that have such biological activity in vivo and / or in vivo are further provided.

[0181] 2.11 Immunoconjugates The presently disclosed subject matter further provides immunoconjugates comprising an antibody or antibody derivative disclosed herein conjugated to one or more detection probes and / or a cytotoxic agent (e.g., a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof)), or a radioisotope. For example, an antibody or antigen-binding portion of the disclosed subject matter can be operably linked (e.g., by chemical coupling, genetic fusion, noncovalent bonding, or other means) to one or more other binding molecules (e.g., another antibody, an antibody fragment, a peptide, or a binding mimetic).

[0182] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC), in which the antibody is conjugated to one or more drugs, and the drugs are maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 106). 235), auristatins such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298), dolastatins, and carithromycin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. Res. [Cancer Research] 58:2925-2928 (1998)) and anthracyclines such as iodomycin or doxorubicin (Kratz et al., Current Med Chem. [Modern Medicinal Chemistry] 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters [Bioorganic Chemistry and Medicinal Chemistry Communications] 16:358-362 (2006), Torgov et al., Bioconj. Chem. [Bioconjugate Chemistry] 16:717-721 (2005), Nagy et al., Proc. Natl. Acad. Sci. USA [National Academy of Sciences of the United States of America] 97:829-834 (2000), Dubowchik et al., Bioorg. & Med. Chem. Letters [Bioorganic Chemistry and Medicinal Chemistry Communications] 12:1529-1532 (2002), King et al., J Med. Chem. 45:4336-4343 (2002), and U.S. Pat. No. 6,630,579), methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel, trichothecenes, and CC1065.

[0183] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, such as diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-swerin, abrin (Aleurites fordii), dianthin, Phytolaca americana (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, jatrocin, crotin, sapaonaria officinalis inhibitor ... officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecene.

[0184] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. Multiple radioisotopes may be used in the production of radioconjugates. Non-limiting examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When a radioconjugate is used for detection, it may include radioactive atoms used in scintillation studies, such as tc-99m or 1123, or spin labels used in nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-11, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0185] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents (e.g., N-succinimidyl-3-(2-pyridinedimercapto)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminosulfan (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), disazo compounds (e.g., bis(p-azidobenzoyl)hexanediamine), double nitrogen derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., tolylene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene)). For example, ricin immunotoxins may be prepared as described in Vitetta et al., Science, 238:1098 (1987). Carbon-4 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine-pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker may also be a "cleavable linker" that facilitates release of the cytotoxic drug in cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (see Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020).

[0186] Immunoconjugates or ADCs herein expressly cover, but are not limited to, such conjugates prepared using crosslinkers, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA), which are commercially available.

[0187] 2.12 Antigen Recognition Receptor The presently disclosed subject matter further provides an antigen-recognizing receptor comprising an antibody or antibody fragment disclosed herein. An antigen-recognizing receptor is a receptor that can activate, stimulate, or inhibit an immune response cell (e.g., a T cell) in response to binding to an antigen. Non-limiting examples of antigen-recognizing receptors include naturally occurring and recombinant T cell receptors ("TCRs"), chimeric costimulatory receptors (CCRs), chimeric antigen receptors ("CARs"), or inhibitory CARs (iCARs). Methods for designing and using antigen-recognizing receptors are known in the art and are described, for example, in International Publications WO 2018 / 027155, WO 2019 / 099483, WO 2019 / 157454, WO 2019 / 133969, WO 2019 / 099993, WO 2015 / 142314, WO 2018 / 027197, and WO 2014055668.

[0188] In some embodiments, the presently disclosed subject matter provides chimeric antigen receptors (CARs) comprising the antibodies or antibody fragments disclosed herein. CARs are engineered receptors that can graft or confer target specificity onto immune effector cells. In some embodiments, CARs can be used to graft the specificity of a monoclonal antibody onto T cells, with the transfer of its coding sequence facilitated by a vector. In some embodiments, CARs are "first-generation" CARs, typically consisting of an extracellular antigen-binding domain (e.g., scFv or VHH), a transmembrane domain, and a cytoplasmic / intracellular signaling domain, where the extracellular antigen-binding domain (e.g., scFv or VHH) is fused to the transmembrane domain, and the transmembrane domain is fused to the cytoplasmic / intracellular signaling domain. "First-generation" CARs provide de novo antigen recognition and can trigger activation of immune response cells (e.g., CD4+ and CD8+ T cells) via their CD3z chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. In some embodiments, the CAR is a "second generation" CAR that further comprises an intracellular signaling domain from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, CD27, CD40 / My88, and NKGD2) in the cytoplasmic tail of the CAR to provide an additional signal to the immune response cell, thereby "second generation" CARs include CARs that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3z). In some embodiments, the CAR is a "third generation" CAR that comprises multiple costimulatory domains (e.g., CD28 and 4-1BB) and activation (CD3z). In some embodiments, the CAR is a second generation CAR. In some embodiments, the CAR comprises an extracellular antigen-binding domain that binds to an antigen, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the CAR further comprises a hinge / spacer between the extracellular antigen-binding domain and the transmembrane domain. In some embodiments, the extracellular antigen-binding domain comprises an antibody or antibody fragment disclosed herein.In some embodiments, the antibody or antibody fragment comprises a VHH or scFv.

[0189] In some embodiments, the presently disclosed subject matter provides a recombinant TCR comprising an antibody or antibody fragment disclosed herein. A native TCR is a protein complex comprising a disulfide-bonded heterodimeric protein, which consists of two variable chains expressed as part of a complex comprising a CD3 chain molecule. A native TCR is present on the surface of T cells and is responsible for recognizing antigens, which are peptides that bind to major histocompatibility complex (MHC) molecules. In some embodiments, a native TCR comprises an α chain and a β chain (encoded by the TRA and TRB genes, respectively). In some embodiments, a TCR comprises a γ chain and a δ chain (encoded by the TRG and TRD genes, respectively). Each of the α, β, γ, and δ chains comprises two extracellular domains: a variable (V) region and a constant (C) region. The constant region is adjacent to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. Each variable region has three complementarity-determining regions (CDRs). In some embodiments, a TCR comprises a receptor complex having CD3δ, CD3γ, CD3ε, and CD3ζ. When the TCR complex binds to its antigen and MHC (peptide / MHC), a T cell expressing the TCR complex is activated.

[0190] In some embodiments, the recombinant TCR is a non-naturally occurring TCR. In some embodiments, the recombinant TCR comprises a recombinant alpha chain and / or a recombinant b chain, wherein part or all of the variable regions of the recombinant alpha chain and / or the recombinant b chain are replaced by an antibody or antibody fragment disclosed herein. In some embodiments, the antibody or antibody fragment comprises a VHH, VH, VL, or scFv. In some embodiments, the antibody or antibody fragment comprises a VHH. In some embodiments, the recombinant TCR binds to a target antigen in an MHC / HLA-independent manner. In some non-limiting embodiments, antigen binding can activate immune response cells comprising the recombinant TCR.

[0191] The presently disclosed subject matter provides (a) an immune response cell comprising an antigen-recognition receptor (e.g., a CAR or TCR) disclosed herein. In some embodiments, the antigen-recognition receptor can activate the immune response cell. The immune response cell of the presently disclosed subject matter can be a lymphoid cell. The lymphoid system, including B cells, T cells, and natural killer (NK) cells, produces antibodies, regulates the cellular immune system, detects foreign substances in the blood, detects foreign cells in the host, and the like. Non-limiting examples of immune response cells of the lymphoid system include T cells, natural killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., pluripotent stem cells from which lymphoid cells can be differentiated). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. T cells of the presently disclosed subject matter can be any type of T cell, including, but not limited to, accessory T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem cell-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells), regulatory T cells (also called suppressor T cells), natural killer T cells, mucosal-associated constant T cells, and gd T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that induce the death of infected somatic or tumor cells. A patient's own T cells can be genetically engineered to target specific antigens by introducing an antigen-recognition receptor (e.g., a CAR or TCR). In some embodiments, the immune response cells are T cells. The T cells can be CD4+ T cells or CD8+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. Natural killer (NK) cells may be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells can exert their cytotoxic effects on target cells without the need for prior activation.The type of human lymphocytes of the presently disclosed subject matter can be peripheral donor lymphocytes, e.g., Sadelain, M. et al., 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs); Morgan, RA et al., 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express full-length tumor antigen-recognizing T cell receptor complexes containing a and b heterodimers); Panelli, MC et al., 2000 J Immunol 164:495-504; Panelli, MC et al., 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies); and Dupont, J. et al., 2005 Cancer Res 65:5417-5427; Papanicolaou, GA et al., 2003 Blood 102:2498-2505 (disclosed the use of artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells to selectively expand antigen-specific peripheral blood leukocytes ex vivo). In some embodiments, immune response cells (e.g., T cells) may be autologous, non-autologous (e.g., allogeneic), or derived from in vitro engineered progenitor or stem cells.

[0192] 3.How to use The presently disclosed subject matter further provides methods of using the disclosed antibodies and antibody derivatives. In some embodiments, the methods relate to therapeutic uses of the presently disclosed antibodies or antibody derivatives. In some embodiments, the methods relate to diagnostic uses of the presently disclosed antibodies or antibody derivatives.

[0193] 3.1 Treatment method The present disclosure provides methods and uses of the antibodies or antibody derivatives disclosed herein for treating diseases and conditions or enhancing immune responses. In some embodiments, the antibodies or antibody derivatives and / or pharmaceutical compositions comprising same can be administered to a subject (e.g., a mammal, e.g., a human) to treat diseases and conditions or enhance immune responses. In some embodiments, these diseases and conditions are related to immune checkpoint inhibition and / or aberrant CD47 activity. In some embodiments, diseases and conditions treatable by the antibodies or antibody derivatives disclosed herein include, but are not limited to, neoplasia (e.g., cancer).

[0194] In some embodiments, the disclosure provides an antibody or antibody derivative (or fragment thereof) described herein for use in the preparation of a medicament. In some embodiments, the disclosure provides an antibody or antibody derivative (or fragment thereof) described herein for use in the preparation of a medicament for the treatment of cancer. In some embodiments, the disclosure provides an antibody or antibody derivative (or fragment thereof) described herein for use in the treatment of cancer in a subject. In some embodiments, the disclosure provides a pharmaceutical composition comprising an antibody or antibody derivative (or fragment thereof) described herein for use in the treatment of cancer in a subject. In some embodiments, the cancer may be a blood cancer (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, gastric tumor, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various cancers (including prostate cancer and small cell lung cancer). Suitable cancers further include any known cancer in the field of oncology, including astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primary neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchioloalveolar carcinoma, epithelial adenocarcinoma and their liver metastases, lymphangiosarcoma, lymphangioendothelial sarcoma, hepatocellular carcinoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, hidradenoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, pulmonary carcinoma, pulmonary sarcoma ... These include, but are not limited to, bronchial carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, testicular tumor, medulloblastoma, medullopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, breast tumors (e.g., ductal adenocarcinoma and lobular adenocarcinoma), cervical squamous cell carcinoma and adenocarcinoma, uterine epithelial carcinoma and ovarian epithelial carcinoma, prostate cancer, transitional squamous cell carcinoma of the bladder, B and T lymphoma (nodular and dispersed), plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma.

[0195] In some embodiments, the cancer may be melanoma, NSCLC, head and neck cancer, urothelial cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), gastric cancer, cholangiocarcinoma, classical Hodgkin's lymphoma (cHL), non-Hodgkin's lymphoma primary mediastinal B-cell lymphoma (NHL PMBCL), mesothelioma, ovarian cancer, lung cancer (e.g., small cell lung cancer), esophageal cancer, nasopharyngeal carcinoma (NPC), biliary tract cancer, colorectal cancer, cervical cancer, or thyroid cancer.

[0196] In some embodiments, the subject to be treated is a mammal (e.g., a human, a non-human primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is suspected of having cancer, is at risk of having cancer, or has been diagnosed with cancer or any other disease with aberrant CD47 expression or activity.

[0197] Numerous diagnostic methods for cancer or any other disease exhibiting abnormal CD47 activity and the clinical description of these diseases are known in the art. Such methods include, but are not limited to, immunohistochemistry, PCR, and fluorescence in situ hybridization (FISH). Additional details regarding diagnostic methods for abnormal CD47 activity or expression are described, for example, in Gupta et al. (2009) Mod Pathol. [Modern Pathology] 22(1):128-133; Lopez-Rios et al. (2013) J Clin Pathol. [Journal of Clinical Pathology] 66(5):381-385; Ellison et al. (2013) J Clin Pathol. [Journal of Clinical Pathology] 66(2):79-89; and Guha et al. (2013) PLoS ONE [Public Science Library Integrated] 8(6):e67782.

[0198] It may be administered by any suitable route, including, for example, intravenously, intramuscularly, or subcutaneously. In some embodiments, an antibody or antibody derivative (or fragment thereof) and / or composition provided herein is administered in combination with a second, third, or fourth agent (including, for example, an anti-tumor agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent) to treat a disease or condition associated with aberrant CD47 activity. Such agents include, for example, docetaxel, gefitinib, FOLFIRI (irinotecan, 5-fluorouracil, and folinic acid), irinotecan, cisplatin, carboplatin, paclitaxel, bevacizumab (an anti-VEGF antibody), FOLFOX-4, infused fluorouracil, folinic acid and oxaliplatin, alfaltinib, gemcitabine, capecitabine, pemetrexed, tecartinib, everolimus, CpG-ODN, rapamycin, lenalidomide, belofinil, endostatin, lapatinib, PX-866, Imprime PGG, and irinotinib. In some embodiments, the antibody or antibody derivative (or fragment thereof) is conjugated to another agent.

[0199] In some embodiments, an antibody or antibody derivative (or fragment thereof) and / or composition provided herein is administered in combination with one or more other therapies (e.g., radiation therapy, surgery, chemotherapy, and / or targeted therapy). In some embodiments, an antibody, antibody derivative (or fragment thereof) and / or composition provided herein is administered in combination with radiation therapy. In some embodiments, an antibody, antibody derivative (or fragment thereof) and / or composition provided herein is used in combination with radiation therapy to treat a neoplasm or cancer as disclosed herein.

[0200] Depending on the indication to be treated and administration-related factors well known to those skilled in the art, the antibodies or antibody derivatives herein are administered in a dosage effective to treat the indication while minimizing toxicity and side effects. For cancer treatment, a typical dosage may be, for example, in the range of 0.001 μg to 1000 μg, although dosages lower or higher than these exemplary ranges are within the scope of the present invention. A daily dosage may be from about 0.1 μg / kg to about 100 mg / kg of total body weight, from about 0.1 μg / kg to about 100 μg / kg of total body weight, or from about 1 μg / kg to about 100 μg / kg of total body weight. As described above, therapeutic or prophylactic efficacy can be monitored by periodic evaluation of the treated patient. For repeated administration over several days or longer, treatments are repeated depending on symptoms until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the present invention. The desired dosage may be delivered by a single bolus administration of the composition, multiple bolus administrations of the composition, or continuous infusion of the composition.

[0201] Pharmaceutical compositions containing the antibodies or antibody derivatives disclosed herein may be administered once, twice, three times, or four times daily. The compositions may also be administered less frequently than daily, for example, six times weekly, five times weekly, four times weekly, three times weekly, twice weekly, once weekly, once every two weeks, once every three weeks, once monthly, once every two months, once every three months, or once every six months. The compositions may also be administered in sustained-release formulations, for example, in implants that gradually release the composition for use over a period of time and allow for less frequent administration of the composition, for example, once monthly, once every two to six months, once yearly, or even once daily. Sustained-release devices (e.g., pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) may be administered by injection or surgical implantation at various locations.

[0202] Cancer treatment may be evaluated by, for example, but not limited to, tumor regression, tumor weight or size reduction, time to progression, survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression and / or activity. Methods for determining efficacy of treatment can be used, including, for example, measuring response by radiological imaging.

[0203] In some embodiments, the therapeutic effect is measured as percentage tumor growth inhibition (%TGI) and is calculated using the equation 100-(T / C x 100), where T is the mean relative tumor volume of treated tumors and C is the mean relative tumor volume of untreated tumors. In some embodiments, the %TGI may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, or greater than 95%.

[0204] 3.2 Diagnostic and imaging methods Labeled antibodies or antibody derivatives are used diagnostically to detect, diagnose, or monitor diseases and / or conditions associated with CD47 expression, aberrant expression, and / or activity. For example, antibodies and antibody derivatives provided herein may be used in in situ, in vivo, ex vivo, and in vitro diagnostic or imaging assays. Methods for detecting CD47 polypeptide expression include (a) measuring polypeptide expression in cells (e.g., tissues) or body fluids of an individual using one or more antibodies or antibody derivatives, and (b) comparing the gene expression level with a standard gene expression level, wherein an increase or decrease in the measured gene expression level relative to the standard expression level indicates aberrant expression.

[0205] Another embodiment herein includes methods for diagnosing a disease or condition associated with CD47 expression or aberrant expression in an animal (e.g., a mammal, e.g., a human). These methods include detecting CD47 molecules in the mammal. In some embodiments, diagnosis includes (a) administering to the mammal an effective amount of a labeled antibody or antibody derivative; (b) waiting a period of time after administration to allow preferential concentration of the labeled antibody or antibody derivative at sites in the subject expressing CD47 molecules (and removing unbound labeled molecules to background levels); (c) determining the background level; and (d) detecting the labeled molecule in the subject, with detected labeled molecules higher than the background level indicating that the subject has a particular disease or condition associated with CD47 expression or aberrant expression. The background level may be determined in different ways, and these methods include comparing the amount of detected labeled molecule to a standard value previously determined for a particular system.

[0206] The antibodies and antibody derivatives herein may be used to measure protein levels in biological samples using classical immunohistology methods well known to those skilled in the art (see, e.g., Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods available for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase and iodine ( 131 I, 125 I, 123 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115m In, 113m In,112 In, 111 In), and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F). 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 These include radioisotopes such as Ru, luminol, and fluorescent labels such as fluorescein, rhodamine, and biotin.

[0207] Techniques known in the art can be applied to labeling antibodies (or fragments thereof) according to the present disclosure, including, but not limited to, the use of bifunctional conjugates (see, e.g., U.S. Patent Nos. 5,756,065, 5,714,631, 5,696,239, 5,652,361, 5,505,931, 5,489,425, 5,435,990, 5,428,139, 5,342,604, 5,274,119, 4,994,560, and 5,808,003).

[0208] Alternatively, or additionally, levels of nucleic acid or mRNA encoding a CD47 polypeptide in cells may be measured using, for example, fluorescence in situ hybridization (FISH; see WO 98 / 45479, published October 1998), DNA blotting, RNA blotting, or polymerase chain reaction (PCR) techniques (e.g., real-time quantitative PCR (RT-PCR)) using nucleic acid-based probes corresponding to the nucleic acid encoding CD47 or its complementary sequence. For example, antibody-based assays can be used to study CD47 overexpression by measuring shed antigen in biological fluids (e.g., serum). (See, further, e.g., U.S. Pat. No. 4,933,294, published June 12, 1990; WO 91 / 05264, published April 18, 1991; U.S. Pat. No. 5,401,638, published March 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). In addition to the above assays, various in vivo and ex vivo assays are available to those skilled in the art. For example, cells within a mammal can be exposed to an antibody, optionally labeled with a detectable label (e.g., a radioisotope), and binding of the antibody to the cells assessed, for example, by radioactive external scanning or by analysis of a sample (e.g., a biopsy or other biological sample) taken from the mammal previously exposed to the antibody.

[0209] 4. Drug Formulations The presently disclosed subject matter further provides a pharmaceutical formulation comprising an antibody or antibody derivative disclosed herein and a pharmaceutically acceptable carrier agent. In some embodiments, a pharmaceutical composition may comprise a combination of multiple (e.g., two or more) antibodies and / or antibody derivatives of the presently disclosed subject matter.

[0210] In some embodiments, the disclosed drug formulations may be prepared by combining an antibody or antibody derivative having a desired purity with one or more pharmaceutically acceptable carrier agents (see Remington's Pharmaceutical Sciences, 16th ed., edited by Osol, A. (1980)), and may be in the form of a lyophilized formulation or an aqueous solution. For example, lyophilized antibody formulations are described in, but not limited to, U.S. Pat. No. 6,267,958. In some embodiments, aqueous antibody formulations may include those described in U.S. Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations containing a histidine-acetate buffer. In some embodiments, the antibody or antibody derivative may have a purity of greater than about 80%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8%, or greater than about 99.9%.

[0211] Pharmaceutically acceptable carrier materials are typically nontoxic to recipients at the dosages and concentrations employed, and may include buffers (e.g., phosphate, citrate, and other organic acids), antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl parabens (e.g., methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and meta-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, Examples of suitable carriers include, but are not limited to, proteins (e.g., serum albumin, gelatin, or immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone), amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine), monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrins, chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose, or sorbitol), counterions (e.g., sodium) forming salts, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants (e.g., polyethylene glycol (PEG)). Exemplary pharmaceutically acceptable carrier agents herein include mesenchymal drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX). (登録商標) , Baxter International Inc. Some exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Nos. 2005 / 0260186 and 2006 / 0104968. In some embodiments, a sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases).

[0212] The carrier agent may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (e.g., anti-CD47 antibody) may be coated in a material to protect the compound from the effects of acids and other natural conditions that may inactivate the compound.

[0213] The pharmaceutical compositions of the present disclosure may be administered in combination therapy, i.e., co-administered with other drugs. In some embodiments, the pharmaceutical compositions disclosed herein may further comprise more than one active ingredient essential for the indication being treated, e.g., active ingredients whose complementary activities do not adversely affect each other. In some embodiments, the pharmaceutical formulation may also comprise a second active ingredient for treating the same disease being treated by the first therapeutic agent. Such active ingredients are present in a suitable combination in amounts effective for the desired purpose. For example, the formulations of the present disclosure may further comprise more than one active ingredient essential for the particular indication being treated, preferably with active ingredients whose complementary activities do not adversely affect each other. For example, it may be desirable to further provide a second therapeutic agent for the same disease. Such active ingredients are present in a suitable combination in amounts effective for the desired purpose.

[0214] The compositions of the present disclosure may be administered in a variety of ways known in the art. The route and / or mode of administration will vary depending on the desired results. The active compounds may be prepared using carriers that protect the compound from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may also be used. Many methods for preparing such formulations are described, for example, in "Sustained and Controlled Release Drug Delivery Systems," edited by JR Robinson, Marcel Dekker, Inc., New York, 1978. In some embodiments, the pharmaceutical compositions are produced in accordance with U.S. Food and Drug Administration Good Manufacturing Practice (GMP).

[0215] Sustained-release formulations containing the disclosed antibodies or antibody derivatives may also be prepared. Suitable examples of sustained-release formulations include semipermeable matrices containing the antibody or antibody derivative in solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films, or microcapsules. In some embodiments, the active ingredient can be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or crude emulsions, prepared, for example, by coacervation techniques or interfacial polymerization. Such techniques are disclosed in "Remington's Pharmaceutical Sciences," 16th ed., edited by Osol, A. (1980).

[0216] To administer an antibody or antibody derivative of the present disclosure by some routes of administration, it may be necessary to coat the compound with, or co-administer, a material that prevents compound inactivation. For example, the compound may be administered to a subject in a suitable carrier agent (e.g., liposomes) or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

[0217] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The substances used for such media and agents for pharmaceutical activity are well known in the art.

[0218] Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present disclosure is contemplated. Supplementary active compounds may also be doped into the compositions.

[0219] Therapeutic compositions must usually be sterile, essentially isotonic, and stable under the conditions of preparation and storage. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable to high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, a coating (e.g., lecithin) can be used to maintain the required particle size in the case of dispersions, and surfactants can be used to maintain proper fluidity. In many cases, it is preferable to include an isotonic agent, for example, a sugar, a polyol (e.g., mannitol, sorbitol), or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including an absorption-delaying agent (e.g., monostearate and gelatin) in the composition.

[0220] Sterile injectable solutions may be prepared by doping the required amount of one or more antibodies or antibody derivatives disclosed herein with a suitable solvent and one or more of the ingredients listed above, as required, followed by sterilization microfiltration (e.g., filtration through a sterile filtration membrane). Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed 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 (lyophilization), which yield a powder of the active ingredient and any other desired ingredients from a previously sterile-filtered solution thereof.

[0221] Therapeutic compositions can also be administered using medical devices known in the art. For example, the therapeutic compositions of the present disclosure may be administered with a needleless hypodermic injection device, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of implants and modules that can be used in the present disclosure include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate, U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin, U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at precise infusion rates, U.S. Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for continuous drug delivery, U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments, and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many such implants, delivery systems, and modules are known.

[0222] For therapeutic compositions, the formulations of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. These formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of antibody or antibody derivative that can be combined with carrier materials to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of antibody or antibody derivative that can be combined with carrier materials to produce a single dosage form will typically be that amount of the composition that produces a therapeutic effect. Typically, this amount will be, in percentage, from about 0.01% to about 99% active ingredient, from about 0.1% to about 70% active ingredient, or from about 1% to about 30% active ingredient.

[0223] Dosage forms for topical or transdermal administration of the compositions of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0224] The phrases "parenteral administration" and "administration by parenteral route" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0225] These compositions may further contain auxiliary agents, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by the above-mentioned sterilization procedures and by the addition of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to add isotonic agents, such as sugars, sodium chloride, and the like, to the compositions. Furthermore, prolonged absorption of the injectable drug form can be achieved by adding agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0226] In some embodiments, when the antibodies or antibody derivatives of the present disclosure are administered to humans and animals as drugs, they may be administered alone or in combination with a pharmaceutically acceptable carrier in the form of a pharmaceutical composition, which contains, for example, from about 0.01% to about 99.5% (or from about 0.1% to 90%) of the antibody or antibody derivative.

[0227] 5.Products The presently disclosed subject matter further provides articles of manufacture (eg, kits) containing materials for use in the treatment, prevention, and / or diagnosis of the above-described conditions.

[0228] In some embodiments, the article of manufacture / kit includes a container and a label or packaging insert on or associated with the container. Non-limiting examples of suitable containers include bottles, vials, syringes, intravenous solution bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for treating, preventing, and / or diagnosing a medical condition (by itself or in combination with another composition) and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle).

[0229] In some embodiments, at least one active agent in the composition is an antibody or antibody derivative of the present disclosure. The label or package insert can indicate that the composition is used for treating a selected condition.

[0230] In some embodiments, the article of manufacture / kit may include (a) a first container containing a composition comprising an antibody or antibody derivative of the present disclosure, and (b) a second container containing a composition comprising another cytotoxic or therapeutic agent. In some embodiments, the article of manufacture / kit may further include a packaging insert indicating that the composition can be used to treat a particular medical condition.

[0231] Alternatively, or additionally, the article / kit may further include another container (e.g., a second or third container) containing a pharmaceutically acceptable buffer, such as, but not limited to, bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and glucose solution. The article / kit may also include other materials necessary from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0232] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

[0233] The following examples are merely illustrative of the presently disclosed subject matter and should not be construed as limiting in any way. [Example]

[0234] Example 1: Identification of anti-CD47 antibodies with reduced off-target binding Anti-CD47 clones were isolated from an in-house synthesized naive human Fab phage library and screened for the CD47 N-terminal ECD by enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS). A naive human Fab phage library was generated using PBMC samples isolated from eight healthy donors. The VL and VH nucleotide sequences of the resulting clones were then fused to the constant region of human IgG4 using standard assembly PCR techniques, and the resulting clones were used to produce full-length antibodies. Two leader clones, M1 and M2, were identified from the naive human Fab phage library.

[0235] The total cell binding ability of anti-CD47 antibodies was then tested by incubating CD47-expressing cells (Raji cells, B-cell lymphoma) with serially diluted anti-CD47 monoclonal antibodies in FACS buffer (1x PBS containing 2% FBS) for 30 minutes at 4°C. Cells were washed with FACS buffer, and binding was redetected with goat anti-human IgG (H+L) FITC antibody or goat anti-mouse IgG (H+L) FITC antibody for 30 minutes at 4°C. Flow cytometry analysis was performed using a Cytomics FC 500 (Beckman Coulter Inc.). An anti-CD47 reference antibody (an in-house synthesized magrolimab analogue based on the sequence information disclosed in Liu et al. (2015), PLOS ONE [Public Science Library and Integration] 10(9):e0137345) served as a positive control. IgG isotype control (anti-PD1 antibody) and IgG isotype control (anti-PD1 antibody) served as negative controls. As shown in Figure 1, the binding of antibody clones M1 and M2 to CD47-expressing Raji cells was detected by flow cytometry.

[0236] Following standard procedures, antibody clone M1 was subjected to in vitro phage display-based affinity maturation to enhance its affinity for the CD47 antigen. Briefly, one or more CDR residues were mutated, the mutant antibodies were displayed on phage, and mutant antibodies with better binding ability to the CD47 protein were screened by enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS).

[0237] Using the method described above, we tested the whole-cell binding ability of M1 variants to Jurkat cells. An anti-CD47 reference antibody (a magrolimab analog) served as a positive control. Bevacizumab (an anti-VEGF-A antibody) served as a negative control. As shown in Figure 2A, clone M1 and its variants (M1#11, M1#21, M1#25, M1#33, M1#46, and M1#55) were able to bind to Jurkat cells, a human leukemia cell line that naturally expresses CD47.

[0238] CD47 is expressed by many types of tumor tissues to evade the immune system, but it is also expressed on red blood cells (RBCs). We screened for anti-CD47 antibodies with reduced binding to normal tissues (e.g., RBCs). Using the method described above, we tested the whole-cell binding ability of M1 variants to RBCs. An anti-CD47 reference antibody (magrolimab analog) served as a positive control. Bevacizumab (anti-VEGF-A antibody) served as a negative control. As shown in Figure 2B, at pharmaceutically acceptable concentrations, clone M1 and its variants (M1#11, M1#21, M1#25, M1#33, M1#46, and M1#55) all showed reduced RBC binding compared to the magrolimab analog. These results suggest that clone M1 and its variants can target CD47-expressing tumor tissues and have reduced off-target binding to RBCs compared to the magrolimab analog.

[0239] We also tested M1 variants with modifications in the constant region. For example, M1#21K contains an additional C-terminal lysine compared to M1#21, and M1#21KP contains one amino acid substitution in the light chain constant region of M1#21K. Using the methods described above, we tested the whole-cell binding ability of these constant region variants to Jurkat cells and RBCs. An anti-CD47 reference antibody (magrolimab analog) served as a positive control. Trastuzumab (anti-HER2 antibody) served as a negative control. As shown in Figure 3A, the constant region variants (M1#21K and M1#21KP) can bind to Jurkat cells (a human leukemia cell line that naturally expresses CD47) in the same manner as M1#21. As shown in Figure 3B, similar to M1#21, both constant region variants (M1#21K and M1#21KP) showed reduced binding to RBCs compared to the magrolimab analog. These results suggest that clone M1 and its variants can target CD47-expressing tumor tissue and have reduced off-target binding to RBCs compared to the magrolimab analog. These results also suggest that modifications in the constant region do not alter the ability of the M1 variants to target CD47-expressing tumor tissue or their reduced off-target binding to RBCs compared to the magrolimab analog.

[0240] Example 2: Inhibition of human SIRPα binding to CD47 by anti-CD47 antibodies To test the effect of anti-CD47 antibodies on inhibiting CD47-mediated signaling pathways, we tested the ability of the antibodies to block CD47 binding to SIRPα (the CD47 ligand). CD47-expressing Jurkat cells (1E5 cells / well) were washed and resuspended in FACS buffer (1x PBS containing 2% FBS). Different concentrations of test antibodies were mixed with a predetermined concentration of biotin-human SIRPα-Fc and incubated with the cells for 30 minutes at 4°C. Unbound antibody and biotin-human SIRPα-Fc were washed away, and the cells were stained with streptavidin-PE for 30 minutes at 4°C. Flow cytometry analysis was performed using a Cytomics FC 500 (Beckman Coulter Inc.). An anti-CD47 reference antibody (magrolimab analog) and an IgG isotype control (anti-PD1 antibody) served as positive and negative controls, respectively.

[0241] As shown in Figure 4, clone M1 and its variants exhibited similar abilities to block CD47 binding to SIRPα compared to the magrolimab analogs, suggesting that clone M1 and its variants function normally as antagonistic antibodies against CD47.

[0242] Example 3: Reduced off-target effects of anti-CD47 antibodies on erythrocytes CD47 is expressed by many types of tumor tissues to evade the immune system, but it is also expressed on red blood cells (RBCs). To assess the off-target effects of anti-CD47 antibodies on RBCs, we detected their induction of blood coagulation responses. Human RBCs were washed twice with 0.9% NaCl buffer, diluted to 10% in 0.9% NaCl buffer, and incubated with serially diluted antibodies in a round-bottom 96-well plate at 37°C for 2 hours or overnight. Aggregated RBCs uniformly covered the bottom of the well, whereas non-aggregated cells formed a red dot at the bottom of the well. An anti-CD47 reference antibody (magrolimab analog) served as a positive control. An isotype control (anti-PD1 IgG4) served as a negative control.

[0243] As shown in Figure 5, the anti-CD47 reference antibody (magrolimab analog) induced RBC aggregation in a dose-dependent manner, whereas clone M1 and its variants, similar to the IgG isotype control (anti-PD1 antibody), did not induce any RBC aggregation.

[0244] Previous studies have also suggested that anti-CD47 antibodies can exert their antitumor effects by inducing macrophage-mediated phagocytosis of tumor cells. To assess the off-target effects of anti-CD47 antibodies on RBCs, we detected macrophage-mediated phagocytosis of RBCs and tumor cells. Raw264.7 cells (immortalized macrophages) and macrophages isolated from human peripheral blood mononuclear cells (PBMCs) were seeded into 48-well plates, allowed to adhere, and cultured overnight. The human leukemia cell line Jurkat was used as a target cell due to its high expression of CD47.

[0245] Jurkat cells or RBCs were labeled with CFSE for 10 minutes at 37°C, washed with complete RPMI-1640 medium, and then incubated with serially diluted clone M1 and its variants, an anti-CD47 reference antibody (magrolimab analog), or an isotype control antibody (anti-PD1 antibody, negative control) for 30 minutes at 37°C. The antibody-target cell mixture was then incubated with Raw264.7 cells or PBMC-derived macrophages at a 1:1 ratio (for Jurkat cells) or a 10:1 ratio (for RBCs) for 2 hours. Unengulfed target cells were then removed. Remaining phagocytic cells were scraped and stained with PE-Cyanine 7-conjugated F4 / 80 or CD14 antibodies (eBioscience) and analyzed by flow cytometry. Phagocytic activity was calculated as the percentage of F4 / 80+CFSE+ cells among total F4 / 80+ cells or the percentage of CD14+CFSE+ cells among total CD14+ cells.

[0246] Magrolimab analogs, clone M1, and its variants all induced phagocytosis of Jurkat cells via Raw264.7 cells (Figure 6A) and PBMC-derived macrophages (Figure 6C). However, in contrast to magrolimab analogs, clone M1 and its variants did not induce phagocytosis of RBCs via Raw264.7 cells (Figure 6B) or PBMC-derived macrophages (Figure 6D). These results suggest that, compared with magrolimab analogs, clone M1 and its variants exhibited reduced off-target effects on normal tissues but retained their antitumor effect of inducing macrophage-mediated phagocytosis of tumor cells.

[0247] Example 4: Antitumor effects of anti-CD47 antibodies in a WiDr (human colon cancer) xenograft tumor mouse model All in vivo studies were performed in NOD / SCID (non-obese diabetic / severe combined immunodeficiency) mice because SIRPα expressed in these mice strongly binds to human CD47, simulating the binding of human SIRPα to human CD47. In vivo studies were performed in accordance with established guidelines.

[0248] The in vivo antitumor effect of anti-CD47 antibodies was evaluated in a WiDr (human colon cancer) xenograft tumor mouse model using NOD / SCID mice. 6 WiDr human colon cancer cells were implanted subcutaneously. Seven days after tumor inoculation, mice were treated intraperitoneally with a solvent control (placebo), a CD47 reference antibody (magrolimab analog), and the M1 variant M1#21 at doses of 3 mg / kg and 10 mg / kg twice weekly for three weeks. Tumor formation was monitored twice weekly. Tumor volume was calculated as TV (tumor volume) = (length × width). 2 ) / 2.

[0249] The tumor growth curves are shown in Figure 7. The data show that both the magrolimab analog and M1#21 exhibited significant tumor growth inhibition against NCI-H82 cancer cells at two different dose regimens, and M1#21 exhibited better tumor growth inhibition than the magrolimab analog at each of the two dose regimens.

[0250] Example 5: Antitumor effects of anti-CD47 antibodies in NCI-H82 (human small cell lung cancer) xenograft tumor mouse model In addition, the in vivo antitumor effect of anti-CD47 antibodies was evaluated in a mouse model of NCI-H82 (human small cell lung cancer) xenograft tumors using NOD / SCID mice. 6 Mice were subcutaneously implanted with human CD47-expressing small cell lung cancer NCI-H82. On days 7, 0, 3, 7, and 14 after tumor inoculation, mice were treated intraperitoneally with a solvent control (placebo), a CD47 reference antibody (magrolimab analog), clone M1, and its variants (M1#21 and M1#55). Tumors were observed and measured twice weekly. Tumor volume was calculated as TV (tumor volume) = (length × width). 2 ) / 2.

[0251] The tumor growth curves are shown in Figure 8. The data show that the magrolimab analogs, clone M1 and its variants M1#21 and M1#55, both exhibited significant tumor growth inhibition against NCI-H82 cancer cells, and M1#21 and M1#55 both exhibited enhanced tumor growth inhibition compared to the magrolimab analogs.

[0252] In addition to the various embodiments described and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, specific features presented herein may be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of features disclosed herein. The foregoing descriptions of specific embodiments of the disclosed subject matter are provided for purposes of illustration and description. The foregoing descriptions are not intended to be exhaustive or to limit the disclosed subject matter to those examples disclosed.

[0253] It will be apparent to those skilled in the art that various modifications and variations can be made in the structure and methodology of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Thus, it is intended that the disclosed subject matter cover modifications and variations that come within the scope of the appended claims and their equivalents.

[0254] Various publications, patents and patent applications are cited herein, the contents of which are incorporated by reference in their entireties. The present disclosure may provide the following aspects. [Section 1] An antibody that binds to CD47, the antibody comprising: a) a heavy chain variable region; and b) a light chain variable region; The heavy chain variable region comprises: (1) a heavy chain variable region CDR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 1, 11, 21, 31, 41, 51, and 61, or a variant thereof comprising up to about three amino acid substitutions; (2) a heavy chain variable region CDR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 2, 12, 22, 32, 42, 52, and 62, or a variant thereof comprising up to about three amino acid substitutions; (3) a heavy chain variable region CDR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 3, 13, 23, 33, 43, 53, and 63, or a variant thereof comprising up to about three amino acid substitutions; The light chain variable region comprises: (1) a light chain variable region CDR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 14, 24, 34, 44, 54, and 64, or a variant thereof comprising up to about three amino acid substitutions; (2) a light chain variable region CDR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 15, 25, 35, 45, 55, and 65, or a variant thereof comprising up to about three amino acid substitutions; (3) An antibody comprising a light chain variable region CDR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 6, 16, 26, 36, 46, 56 and 66, or a variant thereof comprising up to about three amino acid substitutions. [Section 2] The antibody was 1×10 -8 The antibody of item 1, which binds to CD47 with a KD of M or smaller. [Section 3] The antibody was 5×10 -9 The antibody of item 1 or 2, which binds to CD47 with a KD of M or smaller. [Section 4] The antibody is about 1×10 -11 M to approximately 1 x 10 -8 The antibody according to any one of Items 1 to 3, which binds to CD47 with a KD of M. [Section 5] The antibody is about 1×10 -10 M to approximately 1 x 10 -8 Item 5. The antibody according to any one of Items 1 to 4, which binds to CD47 with a KD of M. [Section 6] The antibody cross-competes with a reference anti-CD47 antibody, the reference anti-CD47 antibody being: a) a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 1; (2) a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 2; and (3) a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 4; (2) a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 5; and (3) a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 6; b) a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 11; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 12; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 13; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 14; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 15; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 16; c) a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 21; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 22; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 23; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 24; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 25; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 26; d) a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 31; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 32; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 33; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 35; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 36; e) a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 41; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 42; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 43; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 44; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 45; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 46; f) a heavy chain variable domain (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 51; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 53; and a light chain variable domain (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 54; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 55; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 56; or g) The antibody according to any one of items 1 to 5, comprising a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 61; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 62; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 63; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 64; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 65; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 66. [Section 7] the antibody comprises: a) a heavy chain variable region; and b) a light chain variable region; the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain respectively comprise the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain contained in a reference heavy chain variable region, and the reference heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, and 67; The antibody of any one of Aspects 1 to 6, wherein the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain comprise the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain, respectively, contained in a reference light chain variable region, and the reference light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, 58, and 68. [Section 8] The antibody according to any one of Items 1 to 7, comprising a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 1; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 2; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 4; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 5; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 6. [Section 9] The antibody according to any one of Items 1 to 7, comprising a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 11; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 12; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 13; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 14; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 15; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 16. [Section 10] The antibody according to any one of Items 1 to 7, comprising a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 21; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 22; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 23; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 24; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 25; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 26. [Section 11] The antibody according to any one of Items 1 to 7, comprising a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 31; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 32; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 33; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 35; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 36. [Section 12] The antibody according to any one of Items 1 to 7, comprising a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 41; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 42; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 43; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 44; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 45; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 46. [Section 13] The antibody according to any one of Items 1 to 7, comprising a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 51; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 52; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 53; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 54; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 55; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 56. [Section 14] The antibody according to any one of Items 1 to 7, comprising a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 61; (2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 62; and (3) CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 63; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 64; (2) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 65; and (3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 66. [Section 15] Item 15. The antibody according to any one of Items 1 to 14, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8. [Section 16] Item 15. The antibody according to any one of Items 1 to 14, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18. [Section 17] Item 15. The antibody according to any one of Items 1 to 14, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28. [Section 18] Item 15. The antibody according to any one of Items 1 to 14, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 38. [Section 19] Item 15. The antibody according to any one of Items 1 to 14, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 47 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 48. [Section 20] Item 15. The antibody according to any one of Items 1 to 14, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 58. [Section 21] Item 15. The antibody according to any one of Items 1 to 14, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 68. [Section 22] Item 22. The antibody according to any one of Items 1 to 21, wherein the antibody comprises a human framework. [Section 23] Item 23. The antibody according to any one of Items 1 to 22, wherein the antibody is a human antibody. [Section 24] The antibodies may be full-length immunoglobulins, single-chain Fv (scFv) fragments, Fab fragments, Fab' fragments, F(ab') 2 , Fv fragment, disulfide bond stabilized Fv fragment (dsFv), (dsFv) 2 24. The antibody according to any one of items 1 to 23, comprising an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof. [Section 25] Item 25. The antibody according to any one of Items 1 to 24, wherein the antibody comprises an Fc region. [Section 26] Item 26. The antibody according to any one of Items 1 to 25, wherein the Fc region comprises a human Fc region. [Section 27] 27. The antibody according to any one of Items 1 to 26, wherein the Fc region comprises an Fc region selected from the group consisting of Fc regions of IgG, IgA, IgD, IgE, and IgM. [Section 28] 28. The antibody according to any one of Items 1 to 27, wherein the Fc region comprises an Fc region selected from the group consisting of Fc regions of IgG1, IgG2, IgG3, and IgG4. [Section 29] Item 29. The antibody according to any one of Items 1 to 28, wherein the Fc region comprises an IgG1 Fc region. [Section 30] Item 29. The antibody according to any one of Items 1 to 28, wherein the Fc region comprises an IgG4 Fc region. [Section 31] The antibody of clause 30, wherein the IgG4 Fc region comprises an S228P mutation. [Section 32] Item 32. The antibody according to any one of Items 1 to 31, wherein the Fc region comprises a C-terminal lysine. [Section 33] Item 32. The antibody according to any one of Items 1 to 31, wherein the Fc region comprises a deletion of a C-terminal lysine. [Section 34] Item 34. The antibody according to any one of Items 1 to 33, wherein the antibody is comprised in a multispecific antibody, for example, a bispecific antibody, wherein the multispecific antibody comprises a second antibody portion that specifically binds to a second antigen. [Section 35] Item 35. The antibody of Item 34, wherein the second antigen is a tumor-associated antigen. [Section 36] The tumor-associated antigens include Her-2, EGFR, PDL1, MSLN, c-Met, B-cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial glycoprotein (EGF), and EGFR. 2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine kinase erb-B2, 3, 4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis 36. The antibody of item 35, selected from the group consisting of phosphoinositide A (CA 1.9.9), Lewis Y (LeY), phosphatidylinositol proteoglycan-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), inclusion protein 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), nephroblastoma protein (WT-1), tyrosine kinase transmembrane receptor 1 (ROR1), PVR, PVRL2, and any combination thereof. [Section 37] 37. The antibody of claim 36, wherein the second antigen is an immune checkpoint modulator. [Section 38] 38. The antibody of claim 37, wherein the immune checkpoint modulator is selected from the group consisting of TIGIT, PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof. [Section 39] 37. The antibody of claim 36, wherein the second antigen is an immune co-stimulatory molecule or a subunit of the T cell receptor / CD3 complex. [Section 40] 40. The antibody of claim 39, wherein the immune co-stimulatory molecule is selected from the group consisting of CD28, ICOS, CD27, 4-1BB, OX40, and CD40, and any combination thereof. [Section 41] 40. The antibody of claim 39, wherein the subunit of the T cell receptor / CD3 complex is selected from the group consisting of CD3γ, CD3δ, CD3ε, and any combination thereof. [Section 42] 42. An immunoconjugate comprising the antibody of any one of items 1 to 41 linked to a therapeutic agent or a label. [Section 43] 43. The immunoconjugate of paragraph 42, wherein the therapeutic agent is a cytotoxin or a radioisotope. [Section 44] 43. The immunoconjugate of claim 42, wherein the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme. [Section 45] A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising the antibody according to any one of Items 1 to 41. [Section 46] The CAR of clause 45, wherein the antibody is an scFv. [Section 47] An immune response cell comprising the CAR of paragraph 45 or 46. [Section 48] 48. The immune response cell of claim 47, wherein the immune response cell is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, and myeloid cells. [Section 49] The immune response cell of paragraph 48, wherein the immune response cell is a T cell. [Section 50] A pharmaceutical composition comprising: a) the antibody according to any one of Items 1 to 41, the immunoconjugate according to any one of Items 42 to 44, or the immune response cell according to any one of Items 47 to 49; and b) a pharmaceutically acceptable carrier agent. [Section 51] A nucleic acid encoding the antibody according to any one of Items 1 to 41. [Section 52] A vector comprising the nucleic acid according to Item 51. [Section 53] A host cell comprising the nucleic acid of Paragraph 51 or the vector of Paragraph 52. [Section 54] A method for preparing the antibody of any one of Aspects 1 to 41, comprising expressing the antibody in a host cell of Aspect 53 and isolating the antibody from the host cell. [Section 55] A method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of the antibody of any one of Items 1 to 41, the immunoconjugate of any one of Items 42 to 44, or the pharmaceutical composition of Item 50. [Section 56] 56. The method of paragraph 55, wherein said method reduces the number of tumor cells. [Section 57] 57. The method of paragraph 55 or 56, wherein the method reduces tumor size. [Section 58] Item 58. The method according to any one of Items 55 to 57, wherein the method eradicates a tumor in the subject. [Section 59] Item 59. The method according to any one of Items 55 to 58, wherein the tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof. [Section 60] A method for treating and / or preventing a neoplasm, comprising administering to a subject an effective amount of the antibody of any one of Items 1 to 41, the immunoconjugate of any one of Items 42 to 44, or the pharmaceutical composition of Item 50. [Section 61] A method for extending the survival time of a subject having a neoplasm, the method comprising administering to the subject an effective amount of the antibody of any one of Items 1 to 41, the immunoconjugate of any one of Items 42 to 44, or the pharmaceutical composition of Item 50. [Section 62] Item 62. The method of item 60 or 61, wherein the neoplasm is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof. [Section 63] Item 42. The antibody according to any one of Items 1 to 41, which is used as a drug. [Section 64] Item 42. The antibody according to any one of Items 1 to 41, which is used in the treatment of cancer. [Section 65] Item 51. The pharmaceutical composition according to Item 50, which is used as a drug. [Section 66] Item 51. The pharmaceutical composition according to Item 50, which is used in the treatment of cancer. [Section 67] Item 67. The antibody of Item 64 or the pharmaceutical composition of Item 66, wherein the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof. [Section 68] A kit comprising the antibody according to any one of Items 1 to 41, the immunoconjugate according to any one of Items 42 to 44, the pharmaceutical composition according to Item 50, the nucleic acid according to Item 51, the vector according to Item 52, or the immune response cell according to Items 47 to 49. [Section 69] Item 69. The kit of Item 68, further comprising a manual for treating and / or preventing a neoplasm.

Claims

1. An antibody that binds to CD47, a) a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 51; (2) CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52; and (3) CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 53; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 54; (2) CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 55; and (3) CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 56; or b) comprising a heavy chain variable domain (VH) sequence comprising: (1) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 61; (2) CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 62; and (3) CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 63; and a light chain variable domain (VL) sequence comprising: (1) CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64; (2) CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 65; and (3) CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 66; antibody.

2. The antibody a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 57, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 58; or b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 68; The antibody described in claim 1.

3. The antibody may be a full-length immunoglobulin, a single chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab') fragment, or a Fab' fragment. ２ , Fv fragment, disulfide bond stabilized Fv fragment (dsFv), (dsFv) ２ 3. The antibody of claim 1 or 2, comprising an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

4. The antibody of claim 1 or 2, wherein the antibody comprises an Fc region.

5. The antibody of claim 4, wherein the Fc region comprises an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions.

6. The antibody of claim 4 , wherein the Fc region comprises an IgG1 Fc region.

7. The antibody of claim 4 , wherein the Fc region comprises an IgG4 Fc region.

8. The antibody of claim 7, wherein the IgG4 Fc region comprises the S228P mutation.

9. The antibody of claim 4, wherein the Fc region comprises a C-terminal lysine.

10. The antibody of claim 4, wherein the Fc region comprises a deletion of a C-terminal lysine.

11. An immunoconjugate comprising the antibody of any one of claims 1 to 10 linked to a therapeutic agent or a label.

12. The immunoconjugate of claim 11 , wherein the therapeutic agent is a cytotoxin or a radioisotope.

13. The immunoconjugate of claim 11 , wherein the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

14. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising the antibody of any one of claims 1 to 10.

15. The CAR of claim 14, wherein the antibody is an scFv.

16. An immune response cell comprising the CAR of claim 14 or 15.

17. The immune response cell of claim 16, wherein the immune response cell is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, and myeloid cells.

18. The immune response cell of claim 17 , wherein the immune response cell is a T cell.

19. 19. A pharmaceutical composition comprising: a) an antibody according to any one of claims 1 to 10, an immunoconjugate according to any one of claims 11 to 13, or an immune response cell according to any one of claims 16 to 18; and b) a pharmaceutically acceptable carrier agent.

20. A nucleic acid encoding the antibody of any one of claims 1 to 10.

21. A vector comprising the nucleic acid of claim 20.

22. 22. A host cell comprising the nucleic acid of claim 20 or the vector of claim 21.

23. 23. A method for preparing an antibody according to any one of claims 1 to 10, comprising expressing said antibody in a host cell according to claim 22 and isolating said antibody from said host cell.

24. 20. The pharmaceutical composition of claim 19 for use as a medicine.

25. 20. The pharmaceutical composition of claim 19, for use in the treatment of cancer.

26. 26. The pharmaceutical composition of claim 25, wherein the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof.

27. 20. A kit comprising an antibody according to any one of claims 1 to 10, an immunoconjugate according to any one of claims 11 to 13, a pharmaceutical composition according to claim 19, a nucleic acid according to claim 20, a vector according to claim 21, or an immune response cell according to any one of claims 16 to 18.

28. 28. The kit of claim 27, further comprising instructions for the treatment and / or prevention of neoplasia.

Images