Bispecific antibodies for the treatment of CD47-related diseases

Abstract

This article discloses bispecific antibodies for the treatment of CD47-related diseases, specifically bispecific antibodies comprising a first targeting portion specifically binding to CD47 and a second targeting portion specifically binding to EpCAM or EGFR, as well as pharmaceutical compositions and methods comprising said bispecific antibodies.

Description

[0001] This application is a divisional application of Chinese patent application No. 202180045565.X, entitled "Bispecific Antibody for Treating CD47-Related Diseases" (the corresponding PCT application was filed on April 23, 2021, with application number PCT / CN2021 / 089397). 1. Cross-referencing This application claims the benefit of International Patent Application No. PCT / CN2020 / 086815, filed on April 24, 2020, which is incorporated herein by reference in its entirety for all purposes.

[0002] 1.1 Sequence List This application contains a sequence list electronically submitted in ASCII format and incorporated herein by reference in its entirety. The ASCII copy created on April 21, 2021, is named 55429-708_602_SL.txt and has a size of 157,978 bytes. 2. Summary of the Invention This disclosure provides bispecific proteins (e.g., bispecific antibodies) having a CD47-binding domain and an EpCAM-binding domain. In some embodiments, the various domains of the bispecific protein have human-derived sequences and may therefore induce a weaker immune response than similar domains having non-human sequences. These bispecific proteins can bind to CD47 on the cell surface and can block CD47-SIRPα interactions, allowing phagocytosis by CD47+ cells. In some embodiments, the CD47-binding domain preferentially binds to CD47 on malignant cells rather than CD47 on RBCs or platelets.

[0003] EpCAM is a tumor-associated glycoprotein that is highly expressed in some cancer cells, but at low or zero levels in some normal cells. On some polarized epithelial cells, EpCAM is expressed on apical surfaces inaccessible to circulating therapeutic proteins. Its expression pattern in cancer is altered to strong, uniform membrane overexpression.

[0004] In some embodiments, the bispecific CD47×EpCAM antibody blocks the "don't-eat-me" signaling pathway, thereby increasing phagocytosis by CD47+ / EpCAM+ cells. In some embodiments, the bispecific CD47×EpCAM antibody includes an IgG1 Fc domain that mediates antibody-dependent cytotoxicity (ADCC) of CD47+ / EpCAM+ cells.

[0005] In some embodiments, compositions comprising bispecific antibodies are described herein, wherein the bispecific antibody includes a CD47-binding domain and an EpCAM-binding domain. In some embodiments, the bispecific antibody further includes an Fc domain. In some embodiments, the bispecific antibody has a higher affinity for CD47 expressed on the surface of tumor cells than for CD47 expressed on the surface of erythrocytes or platelets. In some embodiments, the tumor cells express EpCAM. In some embodiments, the concentration of the bispecific antibody required for half-maximal binding to human erythrocytes is greater than 500 nM. In some embodiments, the bispecific antibody binds to human CD47 with a KD of less than 100 nM. In some embodiments, the bispecific antibody binds to human CD47 with a KD of 1 nM to 100 nM, 1 nM to 50 nM, or 5 nM to 50 nM. In some embodiments, the bispecific antibody binds to EpCAM with a KD of less than 500 nM. In some embodiments, the bispecific antibody binds to EpCAM with a KD of less than 25 nM. In some embodiments, the bispecific antibody binds to EpCAM with a KD of 0.2 nM to 500 nM, 1 nM to 300 nM, 5 nM to 200 nM, or 10 nM to 150 nM. In some embodiments, the KD is determined by surface plasmon resonance. In some embodiments, the CD47 binding domain is a human or engineered human CD47 binding domain. In some embodiments, the CD47 binding domain includes a heavy chain variable domain and a light chain variable domain. In some embodiments, the CD47 binding domain includes scFv. In some embodiments, the EpCAM binding domain includes a heavy chain variable domain and a light chain variable domain. In some embodiments, the EpCAM binding domain includes scFv. In some embodiments, the Fc domain is a human Fc domain. In some embodiments, the isotype of the human Fc domain is IgG1 or IgG4. In some embodiments, the Fc domain is a heterodimeric Fc domain, wherein the heterodimeric Fc region includes a knock chain and a hole chain, forming a knock-in-hole (KiH) structure. In some embodiments, the knock chain includes the mutant T366W, and the hole chain includes the mutants T366S, L368A, and Y407V, wherein the amino acid positions are numbered according to the EU index of Kabat et al. In some embodiments, the bispecific antibody has an asymmetric triple-chain knock-in-hole structure. In some embodiments, the CD47 binding domain is scFv. In some embodiments, the EpCAM binding domain is scFv. In some embodiments, the CD47 binding domain includes three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3;And three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein LC-CDR1 comprises SEQ ID NO: 15, LC-CDR2 comprises SEQ ID NO: 16, LC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 17, LC-CDR1 comprises SEQ ID NO: 18, LC-CDR2 comprises SEQ ID NO: 19, and LC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 20. In some embodiments, HC-CDR3 comprises amino acid substitutions at one or more of K94, E95, G96, S97, F98, G99, V100b, D101, and P102; and LC-CDR3 comprises amino acid substitutions at one or more of Y89, S90, T91, D92, I93, S94, G95, N95a, H95b, W96, and V97. In some embodiments, the heavy chain variable domain of the CD47 binding domain includes a sequence having at least 90% identity with SEQ ID NO: 5, SEQ ID NO: 10 or SEQ ID NO: 13, and the light chain of the CD47 binding domain includes a sequence having at least 90% identity with SEQ ID NO: 8, SEQ ID NO: 11 or SEQ ID NO: 14. In some embodiments, the EpCAM binding domain includes three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein the CDRs are defined by an ordered set of sequences listed as HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3; and wherein the CDRs are selected from sequences having at least 90% identity with the following sequences: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83; SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86; SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 83. ID NO: 53, SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89;SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92; SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 95; SEQ ID NO: 60. SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98; SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101; SEQ ID NO: 66, SEQ ID NO: 67. SEQ ID NO: 68, SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104; SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107; SEQ ID NO: 72, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110; SEQ ID NO: 75. SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113; and SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116. In some embodiments, the EpCAM binding domain includes: a sequence that is at least 90% identical to SEQ ID NO: 21 and a sequence that is at least 90% identical to SEQ ID NO: 33; a sequence that is at least 90% identical to SEQ ID NO: 22 and a sequence that is at least 90% identical to SEQ ID NO: 34;Or a sequence at least 90% identical to SEQ ID NO: 24 and at least 90% identical to SEQ ID NO: 36. In some embodiments, a bispecific antibody of less than 1 nM or less than 0.1 nM increases the percentage of A431 cells phagocytosed by macrophages by at least 4-fold compared to a nonspecific IgG1 antibody control. In some embodiments, the concentration of the antibody required to mediate antibody-dependent phagocytosis of EpCAM-positive, CD47-positive tumor cells by macrophages is between 0.01 nM and -3 nM. In some embodiments, the EpCAM-positive, CD47-positive tumor cells are OVISE cells or A431 cells. In some embodiments, the EpCAM-positive, CD47-positive tumor cells are selected from pancreatic ductal adenocarcinoma cells, ovarian clear cell adenocarcinoma cells, colon adenocarcinoma cells, lung adenocarcinoma cells, ovarian adenocarcinoma cells, and vulvar squamous cell carcinoma cells. In some embodiments, a 100 nM bispecific antibody inhibits the binding of SIRPα to CD47 on the cell surface by at least 30%. In some embodiments, the cells are CD47+EpCAM+ tumor cells. In some embodiments, the cells express at least as much EpCAM protein on their surface as CFPAC-1 cells or OVISE cells. In some embodiments, the cells express at least 100,000, at least 300,000, at least 600,000, or at least 1,500,000 EpCAM proteins on their surface. In some embodiments, a 400 nM bispecific antibody does not induce hemolysis of erythrocytes in a hemagglutination assay.

[0006] Some embodiments provide a complex comprising the bispecific antibody according to any one of the above embodiments and CD47+EpCAM+ target cells. In some embodiments, the target cells express at least as much EpCAM protein on their surface as HCC-44 cells. In some embodiments, the target cells express at least 100,000, at least 300,000, at least 600,000, or at least 1,500,000 EpCAM proteins on their surface. In some embodiments, the CD47+EpCAM+ target cells are cancer cells. In some embodiments, the cancer cells are pancreatic ductal adenocarcinoma cells, ovarian clear cell adenocarcinoma cells, colon adenocarcinoma cells, lung adenocarcinoma cells, ovarian adenocarcinoma cells, or vulvar squamous cell carcinoma cells.

[0007] One embodiment provides a method for inducing phagocytosis in CD47+EpCAM+ target cells, the method comprising administering the bispecific antibody according to any of the above embodiments. In some embodiments, the bispecific antibody is administered at concentrations of less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.1 nM, or less than 0.05 nM.

[0008] Some embodiments provide a method for killing CD47+EpCAM+ target cells, the method comprising administering the bispecific antibody according to any of the above embodiments, wherein the bispecific antibody inhibits the binding of SIRPα to CD47 on the surface of CD47+EpCAM+ target cells.

[0009] Some embodiments provide a method for killing CD47+EpCAM+ target cells, the method comprising administering a bispecific antibody according to any one of the above embodiments, wherein the bispecific antibody induces antibody-dependent cytotoxicity in killing CD47+EpCAM+ target cells. In some embodiments, the antibody is administered at concentrations of 0.01 to 1 nM, 0.01 to 0.5 nM, 0.01 to 0.25 nM, 0.01 to 0.1 nM, 0.01 to 0.05 nM, or less than 0.01 nM. In some embodiments, the CD47+EpCAM+ target cells are selected from A431 cells, HCC-44 cells, SKOV-3 cells, OVISE cells, or CFPAC-1 cells. In some embodiments, the CD47+EpCAM+ target cells are selected from pancreatic ductal adenocarcinoma cells, ovarian clear cell adenocarcinoma cells, colon adenocarcinoma cells, lung adenocarcinoma cells, ovarian adenocarcinoma cells, or vulvar squamous cell carcinoma cells.

[0010] Some embodiments provide a medicine comprising the bispecific antibody according to any one of the above embodiments. Another embodiment provides a method of treating an individual with cancer, the method comprising administering the pharmaceutical composition. In some embodiments, the cancer is pancreatic ductal adenocarcinoma, ovarian clear cell adenocarcinoma, colonic adenocarcinoma, lung adenocarcinoma, ovarian adenocarcinoma, or vulvar squamous cell carcinoma. 3. Description of the attached drawings Figure 1 A schematic diagram of the structure of the triple-chain EpCAM×CD47 bispecific antibody.

[0011] Figure 2 Antibody-dependent phagocytosis (ADCP) of CFPAC-1 pancreatic adenocarcinoma cells as determined by flow cytometry.

[0012] Figure 3 αCD47 antibody VIR47 heavy chain ( Figure 3 A) and light chains ( Figure 3 B) Sequence alignment of alanine scanning mutants. Figure 3 A discloses SEQ ID NO: 5 and 141-151 in order of appearance. Figure 3 B discloses SEQ ID NO: 152-163 in appearance order.

[0013] Figures 4A-4B Bispecific antibody Bi-1, identified by ELISA, interacts with human CD47 (…). Figure 4A ) and human EpCAM ( Figure 4B The combination of ).

[0014] Figures 5A-5B Inhibit SIRPα and OVISE cells with bispecific or bivalent anti-CD47 antibodies. Figure 5A ) or Raji cells ( Figure 5B The combination of ).

[0015] Figure 6 Antibody-dependent cytophagy (ADCP) of A431 cells treated with specified bispecific, bivalent, and / or monovalent antibodies.

[0016] Figure 7 Hemagglutination of human erythrocytes was performed using a specified bispecific divalent antibody.

[0017] Figures 8A-8B Bispecific bivalent antibodies and red blood cells ( Figure 8A ) and platelets ( Figure 8B The combination of ).

[0018] Figures 9A-9B A schematic diagram of the structure of the triple-chain mortar anti-EGFRxCD47 bispecific antibody.

[0019] Figures 10A-10B . Through ELISA huEGFR ( Figure 10A ) and human CD47 ( Figure 10B The binding of anti-CD47 / EGFR bispecific antibody to the target.

[0020] Figure 11 Compared with various controls, the binding of SIRPα to A431 cells was inhibited by the anti-CD47 / EGFR bispecific antibody.

[0021] Figure 12 Compared with various controls, antibody-dependent cytophagy (ADCP) of A431 cells was enhanced by anti-CD47 / EGFR bispecific antibody.

[0022] Figures 13A-13BCompared with various controls, the anti-CD47 / EGFR bispecific antibody showed better results with human RBC cells ( Figure 13A ) and human platelet cells ( Figure 13B The combination of ).

[0023] Figure 14 Compared with various controls, the in vivo antitumor activity of the anti-CD47 / EGFR bispecific antibody in the subcutaneous gastric cancer model SNU-5 was demonstrated. 4. Detailed Implementation Differentiation cluster 47 (CD47), also known as integrin-associated protein (IAP), is a membrane glycoprotein of approximately 50 kDa from the immunoglobulin superfamily that is overexpressed in a variety of hematologic malignancies and solid tumors. High CD47 expression is often associated with more aggressive disease and poorer clinical outcomes.

[0024] CD47 on the surface of CD47+ cells interacts with signal regulatory protein α (SIRPα), which is expressed on cells of the innate and adaptive immune system, such as macrophages and dendritic cells. This interaction sends a "don't eat me" signal that inhibits phagocytosis, allowing CD47+ cells to evade immune surveillance.

[0025] These data suggest that CD47 can serve as an immune checkpoint and that blocking the CD47-SIRPα interaction by shutting down the "don't eat me" signal may have therapeutic value. Therefore, blocking CD47 has become a promising therapeutic strategy, with numerous studies demonstrating that disrupting the CD47-SIRPα signaling pathway promotes antitumor activity against human cancers both in vitro and in vivo.

[0026] Several anti-CD47 monoclonal antibodies (mAbs) have been shown to increase the phagocytic activity of acute myeloid leukemia cells, non-Hodgkin's lymphoma cells, breast cancer cells, and ovarian cancer cells. In clinical studies, CD47 mAbs have enhanced the antitumor activity of other therapeutic antibodies. At least six anti-CD47 mAbs and three SIRPα fusion proteins are in active Phase I or II clinical trials for the treatment of human hematologic malignancies and solid tumors.

[0027] The efficacy of anti-CD47 mAbs is limited by their interaction with red blood cells (RBCs) that also express CD47. RBCs act as receivers to sequester anti-CD47 antibodies, preventing them from binding to malignant CD47-expressing (CD47+) cells. Furthermore, the binding of anti-CD47 mAbs to RBCs leads to hemagglutination and lysis of the RBCs, resulting in anemia. Therefore, there is a need for improved approaches to treat CD47+ cell-mediated malignancies with reduced extratumor effects.

[0028] Epithelial cell adhesion molecule (EpCAM), also known as CD236, is a type I transmembrane glycoprotein of approximately 35 kDa that acts as an isotropic Ca2+-independent intercellular adhesion molecule. EpCAM is also involved in cell signaling, proliferation, differentiation, and the formation and maintenance of organ morphology. Overexpression of EpCAM has been found in many metastatic epithelial cancers such as breast cancer, prostate cancer, ovarian cancer, lung cancer, colon cancer, kidney cancer, and gastric cancer, highlighting its potential as an ideal target for immunotherapy (Spizzo et al., Breast Cancer Res Treat, 86:207-213).

[0029] Epidermal growth factor receptor (EGFR), also known as ErbB-1 or HER1, is a transmembrane protein that acts as a receptor for members of the epidermal growth factor family of extracellular protein ligands. EGFR is a member of the ErbB receptor family, a subfamily of four closely related receptor tyrosine kinases: EGFR, HER2 / neu, Her3, and Her4. Overexpression of EGFR is associated with many cancers, including lung adenocarcinoma, anal cancer, glioblastoma, and head and neck epithelial tumors.

[0030] In some embodiments, this document discloses anti-CD47 antibodies, anti-EpCAM antibodies, and multispecific antibodies that include a targeting portion for CD47 and a targeting portion for EpCAM, or a combination thereof. In some embodiments, this document also describes bispecific antibodies that include a first targeting portion for CD47 and a second targeting portion for EpCAM. Further embodiments described herein include methods for treating cancer with anti-CD47 antibodies, anti-EpCAM antibodies, and multispecific antibodies (e.g., bispecific CD47 / EpCAM antibodies).

[0031] In some embodiments, this document discloses anti-CD47 antibodies, anti-EGFR antibodies, and multispecific antibodies comprising a targeting portion for CD47 and a targeting portion for EGFR, or a combination thereof. In some embodiments, this document also describes bispecific antibodies comprising a first targeting portion for CD47 and a second targeting portion for EGFR. Further embodiments described herein include methods for treating cancer with anti-CD47 antibodies, anti-EGFR antibodies, and multispecific antibodies (e.g., bispecific CD47 / EGFR antibodies).

[0032] 4.1 Anti-CD47 antibody In some embodiments, an anti-CD47 antibody is disclosed herein. In some embodiments, the anti-CD47 antibody described herein is a full-length antibody comprising a heavy chain (HC) and a light chain (LC). In some cases, the HC comprises sequences selected from Table 5. In some cases, the light chain comprises sequences selected from Table 5. In some cases, the anti-CD47 antibody comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises a CDR1 sequence selected from SEQ ID NO: 15; a CDR2 sequence selected from SEQ ID NO: 16; and a CDR3 sequence selected from SEQ ID NO: 17; and wherein the VL region comprises CDR1, CDR2, and CDR3 sequences SEQ ID NO: 18, 19, and 20, respectively.

[0033] In some embodiments, VH-CDR3 and / or VL-CDR3 comprises one or more amino acid substitutions that have binding affinity for human CD47. In some cases, the amino acid substitution is alanine (A). In some cases, VH-CDR3 includes K 94 E 95 G 96 S 97 F 98 G 99 E 100 G 100a V 100b D 101 and P 102 Amino acid substitutions at the position. In one case, VH-CDR3 includes at least one alanine substitution selected from the following amino acid positions: K 94 E 95 G 96 S 97 F 98 G 99 E 100 G 100a V 100b D 101 and P 102 In other cases, VL-CDR3 includes position Y. 89 S 90 T 91 D 92 I 93 S 94 G 95 N 95a H 95b W 96 and V 97 Amino acid substitutions at the position. In one case, VL-CDR3 includes at least one alanine substitution selected from the following amino acid positions: Y 89 S90 T 91 D 92 I 93 S 94 G 95 N 95a H 95b W 96 and V 97 .

[0034] In some embodiments, the anti-CD47 antibody includes a VH region, wherein the sequence of the VH region comprises approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5, 10, and 13.

[0035] In some embodiments, the anti-CD47 antibody is modified at one or more amino acid positions to modify its binding, immunogenicity, or other properties. In some cases, the mutation is in the heavy chain. In others, the mutation is in the light chain. In some cases, the mutation is in both the heavy and light chains. In some embodiments, the modified amino acid residues of the heavy chain are selected from I... 67 F 79 and G 82b In some embodiments, the amino acid residues modifying the light chain are selected from E. 103 And M4. In some cases, the heavy chain includes at least one selected from I. 67 F, F 79 Y, G 82b Mutations in S. In some cases, the light chain includes at least one component selected from E. 103 Mutations in K and M4L. In some implementations, the heavy chain includes mutation I. 67 F, F 79 Y, G 82b S. In some implementations, the light chain includes the mutant E. 103 K and M4L.

[0036] In some embodiments, the anti-CD47 antibody is a full-length antibody. In other embodiments, the anti-CD47 antibody is a binding fragment. In some cases, the anti-CD47 antibody includes an antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a humanized antibody or a binding fragment thereof, or an engineered antibody or a binding fragment thereof. In some cases, the anti-CD47 antibody includes monovalent Fab, bivalent Fab'2, single-chain variable fragment (scFv), or a binding fragment thereof.

[0037] In some embodiments, the anti-CD47 antibody described herein has an EC50 of about 0.02 nM to about 2.27 nM. In some embodiments, the anti-CD47 antibody described herein has an EC50 of at least about 0.02 nM. In some embodiments, the anti-CD47 antibody described herein has an EC50 of at most about 2.27 nM. In some embodiments, the anti-CD47 antibody described herein has a concentration of about 0.02 nM to about 0.16 nM, about 0.02 nM to about 0.21 nM, about 0.02 nM to about 1.03 nM, about 0.02 nM to about 1.2 nM, about 0.02 nM to about 2.27 nM, about 0.16 nM to about 0.21 nM, about 0.16 nM to about 1.03 nM, about 0.16 nM to about 1.2 nM, about 0.16 nM to about 2.27 nM, about 0.21 nM to about 1.03 nM, about 0.21 nM to about 1.2 nM, about 0.21 nM to about 2.27 nM, about 1.03 nM to about 1.2 nM, about 1.03 nM to about 2.27 nM, or about 1.2 nM to about 2.27 nM in an in vitro phagocytosis assay. The EC50 of nM is used to determine ADCP activity, for example, by using macrophages and targeting cancer cells, such as metastatic cancer cells from pancreatic cancer (e.g., CFPAC-1 cells) or metastatic cancer cells from ovarian cancer (e.g., OVISE cells).

[0038] In some embodiments, the anti-CD47 antibody described herein has an EC50 of about 0.002 nM to about 0.138 nM. In some embodiments, the anti-CD47 antibody described herein has an EC50 of at least about 0.002 nM. In some embodiments, the anti-CD47 antibody described herein has an EC50 of at most about 0.138 nM. In some embodiments, the anti-CD47 antibody described herein exhibits the following concentrations in in vitro cytotoxicity assays: about 0.002 nM to about 0.012 nM, about 0.002 nM to about 0.053 nM, about 0.002 nM to about 0.108 nM, about 0.002 nM to about 0.138 nM, about 0.012 nM to about 0.053 nM, about 0.012 nM to about 0.108 nM, about 0.012 nM to about 0.138 nM, about 0.053 nM to about 0.108 nM, about 0.053 nM to about 0.138 nM, or about 0.108 nM to about 0.138 nM. The EC50 of nM is used to determine ADCP activity, for example, by using NK92 / CD16a176V effector cells and targeting cancer cells, such as metastatic cancer cells from pancreatic cancer (e.g., CFPAC-1 cells) or metastatic cancer cells from ovarian cancer (e.g., OVISE cells).

[0039] In some implementations, the anti-CD47 antibody described herein has reduced erythrocyte (RBC) binding compared to CD47 BMK-1. In some cases, the reduced RBC binding compared to the reference antibody CD47 BMK-1 is a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.

[0040] In some embodiments, the anti-CD47 antibody described herein has an improved serum half-life compared to the reference antibody CD47 BMK-2, CD47 BMK-1, or CD47 BMK-4. In some embodiments, the improved serum half-life is at least 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or longer than that of the reference antibody CD47 BMK-2, CD47 BMK-1, or CD47 BMK-4. In some cases, the improved serum half-life is at least 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days or longer than that of the reference antibody CD47 BMK-2 or CD47 BMK-1.

[0041] In some cases, the serum half-life of the anti-CD47 antibody described herein is at least 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or longer. In other cases, the serum half-life of the anti-CD47 antibody described herein is approximately 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or longer.

[0042] 4.2 Anti-EPCAM antibody In some embodiments, an anti-EpCAM antibody is disclosed herein. In some embodiments, the anti-EpCAM antibody described herein is a full-length antibody comprising a heavy chain (HC) and a light chain (LC). In some cases, the HC comprises sequences selected from Table 8. In some cases, the light chain comprises sequences selected from Table 9. In some cases, anti-EpCAM antibodies include a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region includes a CDR1 sequence selected from SEQ ID NO: 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78; a CDR2 sequence selected from SEQ ID NO: 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79; and a CDR3 sequence selected from SEQ ID NO: 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, and wherein the VL region includes a CDR1 sequence selected from SEQ ID NO: 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, and 114; and a CDR3 sequence selected from SEQ ID NO: CDR2 sequences 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112 and 115; and CDR3 sequences selected from SEQ ID NO: 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113 and 116.

[0043] In some embodiments, VH-CDR3 and / or VL-CDR3 include one or more amino acid substitutions that modify binding to human EpCAM, immunogenicity, or some other characteristic. In some cases, the amino acid substitution is alanine (A). In some embodiments, VH-CDR3 is SEQ ID NO: 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80. In some embodiments, VH-CDR3 includes at least one alanine substitution. In some embodiments, the alanine substitution ablates binding to human EpCAM.

[0044] In some embodiments, the anti-EpCAM antibody includes a VH region and a VL region, wherein the sequence of the VH region comprises approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 21-32, and the sequence of the VL region comprises approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 33-44. In some embodiments, the anti-EpCAM antibody is a full-length antibody. In other embodiments, the anti-EpCAM antibody is a binding fragment. In some cases, the anti-EpCAM antibody includes an antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, or a humanized antibody or a binding fragment thereof. In some cases, the anti-EpCAM antibody includes monovalent Fab, bivalent Fab'2, single-chain variable fragment (scFv), or a binding fragment thereof.

[0045] In some embodiments, the anti-EpCAM antibody described herein has enhanced binding to the human EpCAM protein compared to the reference antibody EpCAM BMK-6. In some cases, the enhanced binding compared to EpCAM BMK-6 is a reduction in KD of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0046] In some embodiments, the anti-EpCAM antibody described herein has an EC50 of about 0.023 nM to about 3.07 nM. In some embodiments, the anti-EpCAM antibody described herein has an EC50 of at least about 0.023 nM. In some embodiments, the anti-EpCAM antibody described herein has an EC50 of at most about 3.07 nM. In some embodiments, the anti-EpCAM antibody described herein exhibits the following values ​​in in vitro cytotoxicity assays: about 0.023 nM to about 0.024 nM, about 0.023 nM to about 0.036 nM, about 0.023 nM to about 0.038 nM, about 0.023 nM to about 0.04 nM, about 0.023 nM to about 0.059 nM, about 0.023 nM to about 0.069 nM, about 0.023 nM to about 0.086 nM, about 0.023 nM to about 3.07 nM, about 0.024 nM to about 0.036 nM, about 0.024 nM to about 0.038 nM, about 0.024 nM to about 0.04 nM, about 0.024 nM to about 0.059 nM, about 0.024 nM to about 0.069 nM, and about 0.024 nM to about 0.024 nM. nM to about 0.086 nM, about 0.024 nM to about 3.07 nM, about 0.036 nM to about 0.038 nM, about 0.036 nM to about 0.04 nM, about 0.036 nM to about 0.059 nM, about 0.036 nM to about 0.069 nM, about 0.036 nM to about 0.086 nM, about 0.036 nM to about 3.07 nM, about 0.038 nM to about 0.04 nM, about 0.038 nM to about 0.059 nM, about 0.038 nM to about 0.069 nM, about 0.038 nM to about 0.086 nM, about 0.038 nM to about 3.07 nM, about 0.04 nM to about 0.059 nM, about 0.04 nM to about 0.069 nM EC50 values ​​of approximately 0.04 nM to approximately 0.086 nM, approximately 0.04 nM to approximately 3.07 nM, approximately 0.059 nM to approximately 0.069 nM, approximately 0.059 nM to approximately 0.086 nM, approximately 0.059 nM to approximately 3.07 nM, approximately 0.069 nM to approximately 0.086 nM, approximately 0.069 nM to approximately 3.07 nM, or approximately 0.086 nM to approximately 3.07 nM are used to determine ADCC activity, for example, by using NK92 / CD16a176V effector cells and targeting cancer cells, such as metastatic cancer cells from vulvar squamous cell carcinoma, such as A431 cells.

[0047] In some embodiments, the first anti-EpCAM antibody described herein inhibits the binding of a second anti-EpCAM antibody or a fragment thereof to the human EpCAM protein. In some embodiments, the first anti-EpCAM antibody will bind with 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 95%, or 100% inhibition.

[0048] 4.3 Anti-EGFR antibodies In some embodiments, an anti-EGFR antibody is disclosed herein. In some embodiments, the anti-EGFR antibody described herein is a full-length antibody comprising a heavy chain (HC) and a light chain (LC). In some cases, the HC comprises sequences selected from Tables 15-17. In some cases, the light chain comprises sequences selected from Tables 15-17. In some cases, the anti-EGFR antibody comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises CDR1 according to SEQ ID NO: 121; CDR2 according to SEQ ID NO: 122; and CDR3 according to SEQ ID NO: 123, and wherein the VL region comprises CDR1, CDR2, and CDR3 sequences SEQ ID NO: 124, 125, and 126, respectively.

[0049] In some embodiments, VH-CDR3 and / or VL-CDR3 include one or more amino acid substitutions that modify binding to human EGFR, immunogenicity, or some other characteristic. In some cases, the amino acid substitution is alanine (A). In some embodiments, VH-CDR3 includes at least one alanine substitution. In some embodiments, the alanine substitution ablates binding to human EGFR.

[0050] In some embodiments, the anti-EGFR antibody includes a VH region and a VL region, wherein the sequence of the VH region comprises approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 127, and the sequence of the VL region comprises approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 128.

[0051] In some embodiments, the anti-EGFR antibody is a full-length antibody. In other embodiments, the anti-EGFR antibody is a binding fragment. In some cases, the anti-EGFR antibody includes an antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, or a humanized antibody or a binding fragment thereof. In some cases, the anti-EGFR antibody includes monovalent Fab, bivalent Fab'2, single-chain variable fragment (scFv), or a binding fragment thereof.

[0052] In some implementations, the anti-EGFR antibody described herein has enhanced binding to human EGFR protein compared to a reference antibody. In some cases, enhanced binding is a reduction in KD of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference antibody.

[0053] In some embodiments, the anti-EGFR antibody described herein has an EC50 of about 0.023 nM to about 3.07 nM. In some embodiments, the anti-EGFR antibody described herein has an EC50 of at least about 0.023 nM. In some embodiments, the anti-EGFR antibody described herein has an EC50 of at most about 3.07 nM. In some embodiments, the anti-EGFR antibody described herein exhibits the following values ​​in in vitro cytotoxicity assays: about 0.023 nM to about 0.024 nM, about 0.023 nM to about 0.036 nM, about 0.023 nM to about 0.038 nM, about 0.023 nM to about 0.04 nM, about 0.023 nM to about 0.059 nM, about 0.023 nM to about 0.069 nM, about 0.023 nM to about 0.086 nM, about 0.023 nM to about 3.07 nM, about 0.024 nM to about 0.036 nM, about 0.024 nM to about 0.038 nM, about 0.024 nM to about 0.04 nM, about 0.024 nM to about 0.059 nM, about 0.024 nM to about 0.069 nM, and about 0.024 nM to about 0.024 nM. nM to about 0.086 nM, about 0.024 nM to about 3.07 nM, about 0.036 nM to about 0.038 nM, about 0.036 nM to about 0.04 nM, about 0.036 nM to about 0.059 nM, about 0.036 nM to about 0.069 nM, about 0.036 nM to about 0.086 nM, about 0.036 nM to about 3.07 nM, about 0.038 nM to about 0.04 nM, about 0.038 nM to about 0.059 nM, about 0.038 nM to about 0.069 nM, about 0.038 nM to about 0.086 nM, about 0.038 nM to about 3.07 nM, about 0.04 nM to about 0.059 nM, about 0.04 nM to about 0.069 nM EC50 values ​​of approximately 0.04 nM to approximately 0.086 nM, approximately 0.04 nM to approximately 3.07 nM, approximately 0.059 nM to approximately 0.069 nM, approximately 0.059 nM to approximately 0.086 nM, approximately 0.059 nM to approximately 3.07 nM, approximately 0.069 nM to approximately 0.086 nM, approximately 0.069 nM to approximately 3.07 nM, or approximately 0.086 nM to approximately 3.07 nM are used to determine ADCC activity, for example, by using NK92 / CD16a176V effector cells and targeting cancer cells, such as metastatic cancer cells from vulvar squamous cell carcinoma, such as A431 cells.

[0054] In some embodiments, the first anti-EGFR antibody described herein inhibits the binding of a second anti-EGFR antibody or a fragment thereof to the human EGFR protein. In some embodiments, the first anti-EGFR antibody will bind with 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 95%, or 100% inhibition.

[0055] 4.4 Bispecific antibodies In some embodiments, the bispecific antibody includes a first targeting portion specific to CD47 and a second targeting portion specific to tumor cells. In some embodiments, the second targeting portion is specific to an antigen expressed by tumor cells. In some cases, the antigen expressed by tumor cells is a tumor-associated antigen (TAA). In some embodiments, tumor-associated antigens include, but are not limited to: ACVR2, HER2 / neu, CD20, EGFR, CD3, CD22, CD80, CD23, EpCAM, CD2, CD3, CD19, mesothelin, Mum-1, β -Catalytic protein, CDK4, p53, Ras, CDC27 α - Actin-4, TRP1 / gp75, Wilm, EphA3, prostatic acid phosphatase (PAP), alpha-fetoprotein (AFP), 9D7, cyclin-B1, carcinoembryonic antigen (CEA), gp100 / pmel17, BRCA1 / 2, VEGFR, TGF-β RII, MUC-1, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), carbonic anhydrase IX, cytotoxic T-lymphocyte antigen 4, folate-binding protein A-33, prostate-specific antigen (PSA), survivin, EGFRvIII, melanocyte-derived peptides, multiple melanoma-associated peptides, cervical cancer antigen HPV-16-E7, PRAME, SSX-2, CA125, MART, CS-1, BING-4, fibronectin, CML66, MC1R, calcium-activated chloride channel 2, immature laminin receptor, and hTERT. In some embodiments, TAAs are expressed on the cell surface.

[0056] In some embodiments, this document describes a bispecific anti-CD47 and anti-EpCAM antibody or a binding fragment thereof. In some cases, the bispecific anti-CD47 and anti-EpCAM antibody includes at least one targeting portion specifically binding to CD47 and at least one targeting portion specifically binding to EpCAM. In some cases, the bispecific antibody is bivalent, trivalent, tetravalent, or more than tetravalent. In some cases, the bispecific antibody has more than one binding site for binding to CD47. In some cases, the bispecific antibody has more than one binding site for binding to EpCAM. In some cases, the bispecific antibody or a binding fragment thereof is a bispecific antibody conjugate, hybrid bispecific IgG, variable domain-only bispecific antibody, CH1 / CL fusion protein, Fab fusion protein, non-immunoglobulin fusion protein, Fc-modified IgG, additional and Fc-modified IgG, modified Fc and CH3 fusion protein, additional IgG-HC fusion protein, Fc fusion protein, CH3 fusion protein, IgE / IgM CH2 fusion protein, or F(ab')2 fusion protein.

[0057] In some embodiments, this document describes bispecific anti-CD47 and anti-EGFR antibodies or binding fragments thereof. In some cases, the bispecific anti-CD47 and anti-EGFR antibodies include at least one targeting portion specifically binding to CD47 and at least one targeting portion specifically binding to EGFR. In some cases, the bispecific antibody is bivalent, trivalent, tetravalent, or more than tetravalent. In some cases, the bispecific antibody has more than one binding site for binding to CD47. In some cases, the bispecific antibody has more than one binding site for binding to EGFR. In some cases, the bispecific antibody or its binding fragment is a bispecific antibody conjugate, hybrid bispecific IgG, variable domain-only bispecific antibody, CH1 / CL fusion protein, Fab fusion protein, non-immunoglobulin fusion protein, Fc-modified IgG, additional and Fc-modified IgG, modified Fc and CH3 fusion protein, additional IgG-HC fusion protein, Fc fusion protein, CH3 fusion protein, IgE / IgM CH2 fusion protein, or F(ab')2 fusion protein.

[0058] In some cases, bispecific antibodies also include one or more mutations in the framework region (e.g., in the CH1 domain, CH2 domain, CH3 domain, hinge region, or combinations thereof). In some cases, one or more mutations are intended to stabilize the antibody and / or increase its half-life. In some cases, one or more mutations are intended to modulate Fc receptor interactions, increase ADCC, or antibody-dependent phagocytosis (ADCP). In other cases, one or more mutations are intended to reduce or eliminate Fc effector functions, such as Fc... γ R-binding, ADCC, or ADCP. In other cases, one or more mutations are designed to regulate glycosylation. In some cases, one or more mutations enhance stability, increase half-life, reduce glycosylation, and / or regulate Fc receptor interactions, such as increasing or decreasing ADCC and / or ADCP.

[0059] In some cases, bispecific antibodies comprise the IgG1 framework. In some embodiments, the constant region of anti-CD47 or anti-EpCAM or EGFR antibodies is modified at one or more amino acid positions to alter Fc receptor interactions. Exemplary residues that modulate or alter Fc receptor interactions include, but are not limited to, G236, S239, T250, M252, S254, T256, K326, A330, I332, E333A, M428, H433, or N434 (Kabat number; EU index of Sequences of Proteins of Immunological Interest, Kabat et al., 1991). In some cases, mutations include G236A, S239D, T250Q, M252Y, S254T, T256E, K326W, A330L, I332E, E333A, E333S, M428L, H433K, or N434F.

[0060] In some embodiments, modifications at one or more amino acid sites in the constant region of IgG1 that alter Fc receptor interaction result in an increased half-life. In some cases, modifications at one or more amino acid sites include T250, M252, S254, T256, M428, H433, N434, or combinations thereof; for example, including T250Q / M428L or M252Y / S254T / T256E and H433K / N434F.

[0061] In some embodiments, the bispecific antibody described above comprises a mortar and pestle (KIH) form. In some cases, the KIH is located in the Fc region, wherein the residues within the CH3 domain are optionally modified based on the disclosures of WO96 / 027011; Ridgway et al., Protein Eng. 9 (1996) 617-621; or Merchant et al., Nat. Biotechnol. 16 (1998) 677-681. In some cases, one of the CH3 domain pairs is a "mortar" chain and the other is a "pestle" chain, and additional disulfide bonds are optionally introduced to further stabilize the antibody and / or increase yield.

[0062] In some cases, the bispecific antibody is IgG1, and the CH3 domain of the "pestle" chain includes the T366W mutation, while the CH3 domain of the "mortar" chain includes the mutations T366S, L368A, and Y407V. In some cases, the CH3 domain of the "pestle" chain also includes the Y349C mutation, which forms an interchain disulfide bond with E356C or S354C in the CH3 domain of the "mortar" chain.

[0063] In some cases, the CH3 domain of the "pestle" chain includes the R409D and K370E mutations, and the CH3 domain of the "mortar" chain includes the D399K and E357K mutations. In some cases, the CH3 domain of the "pestle" chain also includes the T366W mutation, and the CH3 domain of the "mortar" chain also includes the mutations T366S, L368A, and Y407V.

[0064] In some embodiments, modifications at one or more amino acid positions in the IgG1 constant region that alter Fc receptor interaction result in increased ADCC and / or ADCP. In some cases, modifications at one or more amino acid positions include S239, K326, A330, I332, E333, or combinations thereof. In some cases, modifications at one or more amino acid positions for increased ADCC and / or ADCP include, for example, E333A, S239D / A330L / I332E, or K326W / E333S. In some cases, modifications at one or more amino acid positions for increased ADCC include S239D / A330L / I332E. In some cases, modifications at one or more amino acid positions for increased ADCP include K326W / E333S.

[0065] In some embodiments, the IgG1 constant region is afucosylated. In other embodiments, IgG1 is expressed in cells that cannot undergo fucosylation. In some embodiments, the cells are mammalian cells, such as the Chinese hamster ovary cell line. In some embodiments, the cells do not express fucosyltransferase 8 (FUT8).

[0066] In some embodiments, the bispecific antibody comprises an IgG2 framework. In some cases, one or more amino acid positions in the IgG2 framework are modified to alter Fc receptor interactions, e.g., to increase ADCC and / or CDC. In some cases, one or more amino acid positions in the IgG2 framework are modified to stabilize the antibody and / or increase its half-life. In some cases, one or more amino acid positions in the IgG2 framework are modified to regulate glycosylation. In some cases, the IgG2 constant region is non-fucosylated. In some embodiments, the IgG2 constant region is expressed in cells that cannot undergo fucosylation. In some embodiments, the cells are mammalian cells, such as the Chinese hamster ovary cell line. In some embodiments, the cells do not express fucosyltransferase 8 (FUT8).

[0067] In some embodiments, the bispecific antibody comprises an IgG3 framework. In some cases, one or more amino acid positions in the IgG3 framework are modified to alter Fc receptor interactions, e.g., to increase ADCC and / or ADCP. In some cases, one or more amino acid positions in the IgG3 framework are modified to stabilize the antibody and / or increase its half-life. In some cases, one or more amino acid positions in the IgG3 framework are modified to regulate glycosylation. In some cases, the constant region of the antibody is modified at amino acid R435 to prolong its half-life, e.g., R435H (Kabat number). In some cases, the constant region is deglycosylated at residue N297. In some cases, the IgG3 constant region is expressed in cells that cannot undergo fucosylation. In some embodiments, the cells are mammalian cells, such as the Chinese hamster ovary cell line. In some embodiments, the cells do not express fucosyltransferase 8 (FUT8).

[0068] In some embodiments, the bispecific antibody comprises an IgG4 framework. In some cases, one or more amino acid positions in the IgG4 framework are modified to alter Fc receptor interactions, for example, to increase ADCC and / or ADCP. For example, in some embodiments, mutations increasing ADCC include S239D, I332E, and A330L (amino acid numbers according to the EU index of Kabat et al.), as described in U.S. Patent No. 8,093,359. In some cases, one or more amino acid positions in the IgG4 framework are modified to stabilize the antibody and / or increase its half-life. In some cases, one or more amino acid positions in the IgG4 framework are modified to regulate glycosylation. In some cases, the constant region is modified at the hinge region to prevent or reduce chain exchange. In some cases, the modified amino acid is S228 (e.g., S228P). In some cases, the IgG4 constant region is expressed in cells that cannot undergo fucosylation. In some embodiments, the cells are mammalian cells, such as the Chinese hamster ovary cell line. In some embodiments, the cells cannot express fucosyltransferase 8 (FUT8).

[0069] In some implementations, the human IgG constant region is modified to alter its ADCC and / or ADCP activities, for example, using Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010; Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul, 48: 152-164; Alegre et al., 1992 J Immunol, 148: Amino acid modifications described in 3461-3468; reviewed in Kaneko and Niwa, Biodrugs, 2011, 25(1): 1-11.

[0070] In some implementations, the constant region of human IgG is modified to induce heterodimerization. For example, an amino acid modification at Thr366 within the CH3 domain (which, when substituted by a larger amino acid such as Trp (T366W)) can preferentially pair with a second CH3 domain modified with a smaller amino acid at Thr366, Leu368, and Tyr407, such as Ser, Ala, and Val (T366S / L368A / Y407V), respectively. In some cases, heterodimerization via CH3 modification is further stabilized by the introduction of disulfide bonds, for example by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on the opposite CH3 domains (reviewed in Carter, 2001 Journal of Immunological Methods, 248: 7-15).

[0071] In some cases, the bispecific antibodies described herein have reduced or absent glycosylation, but are not modified at amino acid Asn297 (Kabat number). In these cases, glycosylation is eliminated, for example, by generating antibodies in host cells lacking post-translational glycosylation capacity, such as bacterial or yeast-derived systems or modified mammalian cell expression systems. In some embodiments, the cells are mammalian cells, such as the Chinese hamster ovary cell line. In some embodiments, the cells do not express fucosyltransferase 8 (FUT8). In some aspects, such systems are cell-free expression systems.

[0072] In some implementations, bispecific proteins comprising a first component that binds CD47 and a second component that binds EpCAM have different affinities (KD) for their respective target antigens, as measured by surface plasmon resonance.

[0073] In some cases, the first component is expressed in the following quantities: about 0.1 nM to about 100 nM, about 0.15 nM to about 95 nM, about 0.2 nM to about 90 nM, about 0.25 nM to about 85 nM, about 0.3 nM to about 80 nM, about 0.35 nM to about 75 nM, about 0.4 nM to about 70 nM, about 0.5 nM to about 70 nM, about 0.6 nM to about 60 nM, about 0.7 nM to about 50 nM, about 0.8 nM to about 40 nM, about 0.9 nM to about 30 nM, about 1 nM to about 20 nM, about 1.5 nM to about 10 nM, about 0.01 nM to about 25 nM, about 0.01 nM to about 20 nM, about 0.01 nM to about 10 nM, about 0.01 nM to about 5 nM, about 0.02 nM to about 20 nM, about 0.04 nM. KD bound to human CD47 at concentrations of nM to about 20 nM, about 0.06 nM to about 20 nM, about 0.08 nM to about 20 nM, or about 0.1 nM to about 20 nM.

[0074] In some cases, the second component is bound to human EpCAM in the form of KD at a concentration of about 0.1 nM to about 500 nM, about 0.2 nM to about 500 nM, about 1 nM to about 300 nM, about 5 nM to about 200 nM, or about 10 nM to about 150 nM.

[0075] 4.5 Humanization In some embodiments, the bispecific antibody and its binding fragment are derived from non-human (e.g., rabbit or mouse) antibodies. In some cases, the humanized form of the non-human antibody contains a minimal non-human sequence that maintains the specificity of the original antigen. In some cases, the humanized antibody is a human immunoglobulin (receptor antibody) in which the CDR of the receptor antibody is replaced by residues of the CDR of a non-human immunoglobulin (donor antibody), such as a rat, rabbit, or mouse donor, which has the desired specificity, affinity, avidity, binding kinetics, and / or ability. In some cases, one or more frame region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues of the donor antibody.

[0076] 4.6 Bispecific antibodies that bind to target cells In some embodiments, the bispecific antibody of this disclosure comprising a first component that binds to CD47 and a second component that binds to EpCAM binds to cells expressing a target antigen of a bispecific protein on their surface with an affinity at least 2-50 times, 10-100 times, 2 times, 5 times, 10 times, 25 times, 50 times, or 100 times higher than that of a bivalent antibody that binds to cells only with one of CD47 or EpCAM (e.g., preferential binding).

[0077] In some embodiments, the bispecific protein provided herein binds to target cells that express EpCAM at higher levels than CD47 on their surface. For example, the ratio of EpCAM to CD47 protein expression on the target cell surface is about 1, 1.5, 2.0, 2.5, 5, 10, 15, 20, 25, 50, 100, or greater than 200, as measured by flow cytometry.

[0078] In some embodiments, the bispecific protein provided herein binds to target cells that express CD47 at higher levels than EpCAM on their surface. For example, the ratio of CD47 to EpCAM protein expression on the target cell surface is about 1, 1.5, 2.0, 2.5, 5, 10, 15, 20, 50, 100, or greater than 200, as measured by flow cytometry.

[0079] In some embodiments, the bispecific proteins provided herein bind to target cells that express equal levels of CD47 and EpCAM on their surface. For example, the ratio of CD47 to EpCAM protein expression on the target cell surface is approximately 1, as measured by flow cytometry.

[0080] In some cases, cells express approximately 35,000 to approximately 250,000 CD47 proteins. In some cases, cells express at least approximately 35,000 CD47 proteins. In some cases, cells express at most approximately 250,000 CD47 proteins. In some cases, cells express approximately 35,000 to approximately 40,000 CD47 proteins, approximately 35,000 to approximately 45,000 CD47 proteins, approximately 35,000 to approximately 60,000 CD47 proteins, approximately 35,000 to approximately 100,000 CD47 proteins, approximately 35,000 to approximately 150,000 CD47 proteins, and approximately... 35,000 CD47 proteins to approximately 250,000 CD47 proteins, approximately 40,000 CD47 proteins to approximately 45,000 CD47 proteins, approximately 40,000 CD47 proteins to approximately 60,000 CD47 proteins, approximately 40,000 CD47 proteins to approximately 120,000 CD47 proteins, approximately 40,000 CD47 proteins to approximately 150,000 CD47 proteins, approximately 40,000 CD47 proteins Approximately 250,000 CD47 proteins, approximately 45,000 CD47 proteins to approximately 60,000 CD47 proteins, approximately 45,000 CD47 proteins to approximately 120,000 CD47 proteins, approximately 45,000 CD47 proteins to approximately 150,000 CD47 proteins, approximately 45,000 CD47 proteins to approximately 250,000 CD47 proteins, approximately 60,000 CD47 proteins to approximately 120,000 CD47 protein, approximately 60,000 to approximately 150,000 CD47 proteins, approximately 60,000 to approximately 250,000 CD47 proteins, approximately 120,000 to approximately 150,000 CD47 proteins, approximately 120,000 to approximately 250,000 CD47 proteins, or approximately 150,000 to approximately 250,000 CD47 proteins. In some cases, CD47 protein is expressed on the cell surface. In some cases, the cells are tumor cells. In some cases, the cells originate from pancreatic ductal adenocarcinoma, ovarian clear cell adenocarcinoma, colonic adenocarcinoma, lung adenocarcinoma, ovarian adenocarcinoma, or vulvar squamous cell carcinoma. In some cases, the amount of CD47 protein expressed on the cells is measured by flow cytometry. In some cases, the amount of CD47 protein expressed on the cells is measured by quantitative flow cytometry.

[0081] In some cases, cells express approximately 35,000 EpCAM proteins to approximately 2 × 10⁻⁶. 7There are approximately 35,000 EpCAM proteins. In some cases, cells express at least approximately 35,000 EpCAM proteins. In other cases, cells express up to approximately 2 × 10⁶ EpCAM proteins. 7 EpCAM protein. In some cases, cells express approximately 35,000 to approximately 85,000 EpCAM proteins, approximately 35,000 to approximately 170,000 EpCAM proteins, approximately 35,000 to approximately 300,000 EpCAM proteins, approximately 35,000 to approximately 400,000 EpCAM proteins, approximately 35,000 to approximately 650,000 EpCAM proteins, approximately 35,000 to approximately 750,000 EpCAM proteins, approximately 35,000 to approximately 1,500,000 EpCAM proteins, approximately 35,000 to approximately 1,900,000 EpCAM proteins, and approximately 35,000 to approximately 4 × 10⁻⁶ EpCAM proteins. 6 One EpCAM protein, approximately 35,000 EpCAM proteins to approximately 6 × 10 6 One EpCAM protein, approximately 35,000 EpCAM proteins to approximately 7 × 10 6 One EpCAM protein, approximately 35,000 EpCAM proteins to approximately 15 × 10 6 One EpCAM protein, approximately 35,000 EpCAM proteins to approximately 2 × 10 7 1 EpCAM protein, approximately 85,000 EpCAM proteins to approximately 170,000 EpCAM proteins, approximately 85,000 EpCAM proteins to approximately 300,000 EpCAM proteins, approximately 85,000 EpCAM proteins to approximately 400,000 EpCAM proteins, approximately 85,000 EpCAM proteins to approximately 650,000 EpCAM proteins, approximately 85,000 EpCAM proteins to approximately 750,000 EpCAM proteins, approximately 85,000 EpCAM proteins to approximately 1,500,000 EpCAM proteins, approximately 85,000 EpCAM proteins to approximately 1,900,000 EpCAM proteins, approximately 85,000 EpCAM proteins to approximately 4 × 10 6 One EpCAM protein, approximately 85,000 EpCAM proteins, approximately 6 × 10 6 One EpCAM protein, approximately 85,000 EpCAM proteins to approximately 7 × 10 6 One EpCAM protein, approximately 85,000 EpCAM proteins, approximately 15 × 10⁻⁶ 6 One EpCAM protein, approximately 85,000 EpCAM proteins to approximately 2 × 107 170,000 EpCAM proteins to approximately 300,000 EpCAM proteins, approximately 170,000 EpCAM proteins to approximately 400,000 EpCAM proteins, approximately 170,000 EpCAM proteins to approximately 650,000 EpCAM proteins, approximately 170,000 EpCAM proteins to approximately 750,000 EpCAM proteins, approximately 170,000 EpCAM proteins to approximately 1,500,000 EpCAM proteins, approximately 170,000 EpCAM proteins to approximately 1,900,000 EpCAM proteins, approximately 170,000 EpCAM proteins to approximately 4 × 10 6 One EpCAM protein, approximately 170,000 EpCAM proteins to approximately 6 × 10 6 One EpCAM protein, approximately 170,000 EpCAM proteins to approximately 7 × 10 6 One EpCAM protein, approximately 170,000 EpCAM proteins to approximately 15 × 10⁻⁶ 6 One EpCAM protein, approximately 170,000 EpCAM proteins to approximately 2 × 10⁻⁶ 7 Approximately 300,000 EpCAM proteins to approximately 400,000 EpCAM proteins, approximately 300,000 EpCAM proteins to approximately 650,000 EpCAM proteins, approximately 300,000 EpCAM proteins to approximately 750,000 EpCAM proteins, approximately 300,000 EpCAM proteins to approximately 1,500,000 EpCAM proteins, approximately 300,000 EpCAM proteins to approximately 1,900,000 EpCAM proteins, approximately 300,000 EpCAM proteins to approximately 4 × 10⁻⁶ EpCAM proteins. 6 One EpCAM protein, approximately 300,000 EpCAM proteins to approximately 6 × 10 6 One EpCAM protein, approximately 300,000 EpCAM proteins to approximately 7 × 10 6 One EpCAM protein, approximately 300,000 EpCAM proteins to approximately 15 × 10⁻⁶ 6 One EpCAM protein, approximately 300,000 EpCAM proteins to approximately 2 × 10⁻⁶ 7Approximately 400,000 EpCAM proteins to approximately 650,000 EpCAM proteins, approximately 400,000 EpCAM proteins to approximately 750,000 EpCAM proteins, approximately 400,000 EpCAM proteins to approximately 1,500,000 EpCAM proteins, approximately 400,000 EpCAM proteins to approximately 1,900,000 EpCAM proteins, approximately 400,000 EpCAM proteins to approximately 4 × 10⁻⁶ EpCAM proteins. 6 One EpCAM protein, approximately 400,000 EpCAM proteins to approximately 6 × 10 6 One EpCAM protein, approximately 400,000 EpCAM proteins to approximately 7 × 10 6 One EpCAM protein, approximately 400,000 EpCAM proteins to approximately 15 × 10⁻⁶ 6 One EpCAM protein, approximately 400,000 EpCAM proteins to approximately 2 × 10⁻⁶ 7 1 EpCAM protein, approximately 650,000 EpCAM proteins to approximately 750,000 EpCAM proteins, approximately 650,000 EpCAM proteins to approximately 1,500,000 EpCAM proteins, approximately 650,000 EpCAM proteins to approximately 1,900,000 EpCAM proteins, approximately 650,000 EpCAM proteins to approximately 4 × 10 6 One EpCAM protein, approximately 650,000 EpCAM proteins to approximately 6 × 10 6 One EpCAM protein, approximately 650,000 EpCAM proteins to approximately 7 × 10 6 One EpCAM protein, approximately 650,000 EpCAM proteins, approximately 15 × 10⁻⁶ 6 One EpCAM protein, approximately 650,000 EpCAM proteins, approximately 2 × 10⁻⁶ 7 1 EpCAM protein, approximately 750,000 EpCAM proteins to approximately 1,500,000 EpCAM proteins, approximately 750,000 EpCAM proteins to approximately 1,900,000 EpCAM proteins, approximately 750,000 EpCAM proteins to approximately 4 × 10 6 One EpCAM protein, approximately 750,000 EpCAM proteins to approximately 6 × 10 6 One EpCAM protein, approximately 750,000 EpCAM proteins to approximately 7 × 10 6 One EpCAM protein, approximately 750,000 EpCAM proteins to approximately 15 × 10⁻⁶ 6 One EpCAM protein, approximately 750,000 EpCAM proteins, approximately 2 × 10⁻⁶ 7One EpCAM protein, approximately 1,500,000 EpCAM proteins to approximately 1,900,000 EpCAM proteins, approximately 1,500,000 EpCAM proteins to approximately 4 × 10 6 One EpCAM protein, approximately 1,500,000 EpCAM proteins to approximately 6 × 10⁻⁶ 6 One EpCAM protein, approximately 1,500,000 EpCAM proteins to approximately 7 × 10⁻⁶ 6 One EpCAM protein, approximately 1,500,000 EpCAM proteins to approximately 15 × 10⁻⁶ 6 One EpCAM protein, approximately 1,500,000 EpCAM proteins to approximately 2 × 10⁻⁶ 7 One EpCAM protein, approximately 1,900,000 EpCAM proteins to approximately 4 × 10⁻⁶ 6 One EpCAM protein, approximately 1,900,000 EpCAM proteins to approximately 6 × 10⁻⁶ 6 One EpCAM protein, approximately 1,900,000 EpCAM proteins to approximately 7 × 10⁻⁶ 6 One EpCAM protein, approximately 1,900,000 EpCAM proteins to approximately 15 × 10⁻⁶ 6 One EpCAM protein, approximately 1,900,000 EpCAM proteins to approximately 2 × 10⁻⁶ 7 One EpCAM protein, approximately 4 × 10 6 EpCAM protein approximately 6 × 10 6 One EpCAM protein, approximately 4 × 10 6 EpCAM protein approximately 7 × 10 6 One EpCAM protein, approximately 4 × 10 6 One EpCAM protein is approximately 15 × 10⁻⁶. 6 One EpCAM protein, approximately 4 × 10 6 One EpCAM protein to approximately 2 × 10 7 One EpCAM protein, approximately 6 × 10 6 EpCAM protein approximately 7 × 10 6 One EpCAM protein, approximately 6 × 10 6 One EpCAM protein is approximately 15 × 10⁻⁶. 6 One EpCAM protein, approximately 6 × 10 6 One EpCAM protein to approximately 2 × 10 7 One EpCAM protein or approximately 15 × 10 6 One EpCAM protein to approximately 2 × 10 7The EpCAM protein is expressed on the surface of cells in several ways. In some cases, the cells are tumor cells. In others, the cells originate from pancreatic ductal adenocarcinoma, ovarian clear cell adenocarcinoma, colonic adenocarcinoma, lung adenocarcinoma, ovarian adenocarcinoma, or vulvar squamous cell carcinoma. In some cases, the amount of EpCAM protein expressed on the cells is measured by flow cytometry. In still others, the amount of EpCAM protein expressed on the cells is measured by quantitative flow cytometry.

[0082] In some embodiments, bispecific proteins with both CD47-binding and EpCAM-binding domains exhibit enhanced affinity for cells expressing both CD47 and EpCAM compared to bivalent proteins with one or more CD47-binding domains and / or bivalent proteins with one or more EpCAM-binding domains. In some cases, the bispecific protein exhibits 1.5, 2, 3, 4, 5, or 10-fold higher affinity for cells expressing CD47 than a bivalent protein binding to CD47 or EpCAM. In some embodiments, bispecific proteins with both CD47-binding and EpCAM-binding domains exhibit enhanced affinity for cells expressing higher levels of CD47 than EpCAM compared to bivalent proteins with a CD47-binding domain. In some cases, the bispecific protein exhibits 1.5, 2, 3, 4, 5, or 10-fold higher affinity for cells expressing EpCAM than CD47 compared to a bivalent protein binding to CD47.

[0083] In some embodiments, bispecific antibodies having both a CD47 binding domain and an EpCAM binding domain exhibit reduced erythrocyte (RBC) binding compared to CD47 BMK-1, CD47 BMK-2, or CD47 BMK-4. In some cases, the reduced RBC binding compared to CD47 BMK-1 is a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In some cases, the reduced RBC binding compared to CD47 BMK-2 is a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In some cases, the reduced RBC binding compared to CD47 BMK-4 is a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.

[0084] In some embodiments, the bispecific antibody of this disclosure comprising a first component that binds to CD47 and a second component that binds to EGFR binds to cells expressing a target antigen of a bispecific protein on their surface with an affinity at least 2-50 times, 10-100 times, 2 times, 5 times, 10 times, 25 times, 50 times, or 100 times higher than that of a bivalent antibody that binds to cells only with one of CD47 or EGFR (e.g., preferential binding).

[0085] In some embodiments, the bispecific protein provided herein binds to target cells that express EGFR at higher levels than CD47 on their surface. For example, the ratio of EGFR to CD47 protein expression on the target cell surface is about 1, 1.5, 2.0, 2.5, 5, 10, 15, 20, 25, 50, 100, or greater than 200, as measured by flow cytometry.

[0086] In some embodiments, the bispecific protein provided herein binds to target cells on which CD47 is expressed at higher levels than EGFR. For example, the ratio of CD47 to EGFR protein expression on the target cell surface is about 1, 1.5, 2.0, 2.5, 5, 10, 15, 20, 50, 100, or greater than 200, as measured by flow cytometry.

[0087] In some embodiments, the bispecific protein provided herein binds to target cells that express equal levels of CD47 and EGFR on their surface. For example, the ratio of CD47 to EGFR protein expression on the target cell surface is approximately 1, as measured by flow cytometry.

[0088] In some cases, cells express approximately 35,000 to approximately 250,000 CD47 proteins. In some cases, cells express at least approximately 35,000 CD47 proteins. In some cases, cells express at most approximately 250,000 CD47 proteins. In some cases, cells express approximately 35,000 to approximately 40,000 CD47 proteins, approximately 35,000 to approximately 45,000 CD47 proteins, approximately 35,000 to approximately 60,000 CD47 proteins, approximately 35,000 to approximately 100,000 CD47 proteins, approximately 35,000 to approximately 150,000 CD47 proteins, and approximately... 35,000 CD47 proteins to approximately 250,000 CD47 proteins, approximately 40,000 CD47 proteins to approximately 45,000 CD47 proteins, approximately 40,000 CD47 proteins to approximately 60,000 CD47 proteins, approximately 40,000 CD47 proteins to approximately 120,000 CD47 proteins, approximately 40,000 CD47 proteins to approximately 150,000 CD47 proteins, approximately 40,000 CD47 proteins Approximately 250,000 CD47 proteins, approximately 45,000 CD47 proteins to approximately 60,000 CD47 proteins, approximately 45,000 CD47 proteins to approximately 120,000 CD47 proteins, approximately 45,000 CD47 proteins to approximately 150,000 CD47 proteins, approximately 45,000 CD47 proteins to approximately 250,000 CD47 proteins, approximately 60,000 CD47 proteins to approximately 120,000 CD47 protein, approximately 60,000 to approximately 150,000 CD47 proteins, approximately 60,000 to approximately 250,000 CD47 proteins, approximately 120,000 to approximately 150,000 CD47 proteins, approximately 120,000 to approximately 250,000 CD47 proteins, or approximately 150,000 to approximately 250,000 CD47 proteins. In some cases, CD47 protein is expressed on the cell surface. In some cases, the cells are tumor cells. In some cases, the cells originate from pancreatic ductal adenocarcinoma, ovarian clear cell adenocarcinoma, colonic adenocarcinoma, lung adenocarcinoma, ovarian adenocarcinoma, or vulvar squamous cell carcinoma. In some cases, the amount of CD47 protein expressed on the cells is measured by flow cytometry. In some cases, the amount of CD47 protein expressed on the cells is measured by quantitative flow cytometry.

[0089] In some cases, cells express approximately 35,000 EGFR proteins to approximately 2 × 10⁻⁶. 7There are approximately 35,000 EGFR proteins. In some cases, cells express at least approximately 35,000 EGFR proteins. In other cases, cells express up to approximately 2 × 10⁶ EGFR proteins. 7 EGFR proteins. In some cases, cells express approximately 35,000 to approximately 85,000 EGFR proteins, approximately 35,000 to approximately 170,000 EGFR proteins, approximately 35,000 to approximately 300,000 EGFR proteins, approximately 35,000 to approximately 400,000 EGFR proteins, approximately 35,000 to approximately 650,000 EGFR proteins, approximately 35,000 to approximately 750,000 EGFR proteins, approximately 35,000 to approximately 1,500,000 EGFR proteins, approximately 35,000 to approximately 1,900,000 EGFR proteins, and approximately 35,000 to approximately 4 × 10⁻⁶ EGFR proteins. 6 One EGFR protein, approximately 35,000 EGFR proteins to approximately 6 × 10 6 One EGFR protein, approximately 35,000 EGFR proteins to approximately 7 × 10 6 One EGFR protein, approximately 35,000 EGFR proteins, approximately 15 × 10⁻⁶ 6 One EGFR protein, approximately 35,000 EGFR proteins to approximately 2 × 10 7 Approximately 85,000 EGFR proteins to approximately 170,000 EGFR proteins, approximately 85,000 EGFR proteins to approximately 300,000 EGFR proteins, approximately 85,000 EGFR proteins to approximately 400,000 EGFR proteins, approximately 85,000 EGFR proteins to approximately 650,000 EGFR proteins, approximately 85,000 EGFR proteins to approximately 750,000 EGFR proteins, approximately 85,000 EGFR proteins to approximately 1,500,000 EGFR proteins, approximately 85,000 EGFR proteins to approximately 1,900,000 EGFR proteins, approximately 85,000 EGFR proteins to approximately 4 × 10⁻⁶ EGFR proteins. 6 One EGFR protein, approximately 85,000 EGFR proteins, approximately 6 × 10⁻⁶ 6 One EGFR protein, approximately 85,000 EGFR proteins to approximately 7 × 10 6 One EGFR protein, approximately 85,000 EGFR proteins to approximately 15 × 10⁻⁶ 6 One EGFR protein, approximately 85,000 EGFR proteins to approximately 2 × 10 7Approximately 170,000 EGFR proteins to approximately 300,000 EGFR proteins, approximately 170,000 EGFR proteins to approximately 400,000 EGFR proteins, approximately 170,000 EGFR proteins to approximately 650,000 EGFR proteins, approximately 170,000 EGFR proteins to approximately 750,000 EGFR proteins, approximately 170,000 EGFR proteins to approximately 1,500,000 EGFR proteins, approximately 170,000 EGFR proteins to approximately 1,900,000 EGFR proteins, approximately 170,000 EGFR proteins to approximately 4 × 10⁻⁶ EGFR proteins. 6 One EGFR protein, approximately 170,000 EGFR proteins to approximately 6 × 10⁻⁶ 6 One EGFR protein, approximately 170,000 EGFR proteins to approximately 7 × 10⁻⁶ 6 One EGFR protein, approximately 170,000 EGFR proteins to approximately 15 × 10⁻⁶ 6 One EGFR protein, approximately 170,000 EGFR proteins to approximately 2 × 10⁻⁶ 7 Approximately 300,000 EGFR proteins to approximately 400,000 EGFR proteins, approximately 300,000 EGFR proteins to approximately 650,000 EGFR proteins, approximately 300,000 EGFR proteins to approximately 750,000 EGFR proteins, approximately 300,000 EGFR proteins to approximately 1,500,000 EGFR proteins, approximately 300,000 EGFR proteins to approximately 1,900,000 EGFR proteins, approximately 300,000 EGFR proteins to approximately 4 × 10⁻⁶ EGFR proteins. 6 One EGFR protein, approximately 300,000 EGFR proteins to approximately 6 × 10⁻⁶ 6 One EGFR protein, approximately 300,000 EGFR proteins to approximately 7 × 10⁻⁶ 6 One EGFR protein, approximately 300,000 EGFR proteins to approximately 15 × 10⁻⁶ 6 One EGFR protein, approximately 300,000 EGFR proteins to approximately 2 × 10⁻⁶ 7 Approximately 400,000 EGFR proteins to approximately 650,000 EGFR proteins, approximately 400,000 EGFR proteins to approximately 750,000 EGFR proteins, approximately 400,000 EGFR proteins to approximately 1,500,000 EGFR proteins, approximately 400,000 EGFR proteins to approximately 1,900,000 EGFR proteins, approximately 400,000 EGFR proteins to approximately 4 × 10⁻⁶ EGFR proteins. 6 One EGFR protein, approximately 400,000 EGFR proteins to approximately 6 × 10⁻⁶ 6One EGFR protein, approximately 400,000 EGFR proteins to approximately 7 × 10⁻⁶ 6 One EGFR protein, approximately 400,000 EGFR proteins to approximately 15 × 10⁻⁶ 6 One EGFR protein, approximately 400,000 EGFR proteins to approximately 2 × 10⁻⁶ 7 Approximately 650,000 EGFR proteins to approximately 750,000 EGFR proteins, approximately 650,000 EGFR proteins to approximately 1,500,000 EGFR proteins, approximately 650,000 EGFR proteins to approximately 1,900,000 EGFR proteins, approximately 650,000 EGFR proteins to approximately 4 × 10⁻⁶ EGFR proteins. 6 One EGFR protein, approximately 650,000 EGFR proteins to approximately 6 × 10⁻⁶ 6 One EGFR protein, approximately 650,000 EGFR proteins to approximately 7 × 10⁻⁶ 6 One EGFR protein, approximately 650,000 EGFR proteins, approximately 15 × 10⁻⁶ 6 One EGFR protein, approximately 650,000 EGFR proteins, approximately 2 × 10⁻⁶ 7 1 EGFR protein, approximately 750,000 EGFR proteins to approximately 1,500,000 EGFR proteins, approximately 750,000 EGFR proteins to approximately 1,900,000 EGFR proteins, approximately 750,000 EGFR proteins to approximately 4 × 10 6 One EGFR protein, approximately 750,000 EGFR proteins to approximately 6 × 10⁻⁶ 6 One EGFR protein, approximately 750,000 EGFR proteins to approximately 7 × 10 6 One EGFR protein, approximately 750,000 EGFR proteins, approximately 15 × 10⁻⁶ 6 One EGFR protein, approximately 750,000 EGFR proteins to approximately 2 × 10⁻⁶ 7 One EGFR protein, approximately 1,500,000 EGFR proteins to approximately 1,900,000 EGFR proteins, approximately 1,500,000 EGFR proteins to approximately 4 × 10 6 One EGFR protein, approximately 1,500,000 EGFR proteins to approximately 6 × 10⁻⁶ 6 One EGFR protein, approximately 1,500,000 EGFR proteins to approximately 7 × 10⁻⁶ 6 One EGFR protein, approximately 1,500,000 EGFR proteins to approximately 15 × 10⁻⁶ 6 One EGFR protein, approximately 1,500,000 EGFR proteins to approximately 2 × 10⁻⁶ 7One EGFR protein, approximately 1,900,000 EGFR proteins to approximately 4 × 10⁻⁶ 6 One EGFR protein, approximately 1,900,000 EGFR proteins to approximately 6 × 10⁻⁶ 6 One EGFR protein, approximately 1,900,000 EGFR proteins to approximately 7 × 10⁻⁶ 6 One EGFR protein, approximately 1,900,000 EGFR proteins to approximately 15 × 10⁻⁶ 6 One EGFR protein, approximately 1,900,000 EGFR proteins to approximately 2 × 10⁻⁶ 7 One EGFR protein, approximately 4 × 10 6 One EGFR protein to approximately 6 × 10 6 One EGFR protein, approximately 4 × 10 6 EGFR protein approximately 7 × 10 6 One EGFR protein, approximately 4 × 10 6 One EGFR protein to approximately 15 × 10 6 One EGFR protein, approximately 4 × 10 6 One EGFR protein to approximately 2 × 10 7 One EGFR protein, approximately 6 × 10 6 EGFR protein approximately 7 × 10 6 One EGFR protein, approximately 6 × 10 6 One EGFR protein to approximately 15 × 10 6 One EGFR protein, approximately 6 × 10 6 One EGFR protein to approximately 2 × 10 7 One EGFR protein or approximately 15 × 10 6 One EGFR protein to approximately 2 × 10 7 There are several types of EGFR protein. In some cases, EGFR protein is expressed on the cell surface. In some cases, the cells are tumor cells. In some cases, the cells originate from pancreatic ductal adenocarcinoma, ovarian clear cell adenocarcinoma, colon adenocarcinoma, lung adenocarcinoma, ovarian adenocarcinoma, or vulvar squamous cell carcinoma. In some cases, the amount of EGFR protein expressed on the cells is measured by flow cytometry. In some cases, the amount of EGFR protein expressed on the cells is measured by quantitative flow cytometry.

[0090] In some embodiments, bispecific proteins with both CD47-binding and EGFR-binding domains exhibit enhanced affinity for cells expressing both CD47 and EGFR compared to bivalent proteins with one or more CD47-binding domains and / or bivalent proteins with one or more EGFR-binding domains. In some cases, the bispecific protein shows 1.5, 2, 3, 4, 5, or 10-fold higher affinity for cells expressing CD47 than a bivalent protein binding to CD47 or to EGFR. In some embodiments, bispecific proteins with both CD47-binding and EGFR-binding domains exhibit enhanced affinity for cells expressing CD47 at higher levels than EGFR compared to bivalent proteins with a CD47-binding domain. In some cases, the bispecific protein shows 1.5, 2, 3, 4, 5, or 10-fold higher affinity for cells expressing EGFR than CD47 compared to a bivalent protein binding to CD47.

[0091] In some embodiments, bispecific antibodies having both a CD47-binding domain and an EGFR-binding domain exhibit reduced erythrocyte (RBC) binding compared to CD47 BMK-1 or CD47 BMK-4. In some cases, the reduced RBC binding compared to CD47 BMK-1 is a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In some cases, the reduced RBC binding compared to CD47 BMK-4 is a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.

[0092] 4.7 Immunological activity of bispecific antibodies against target cells In some embodiments, bispecific antibodies with both CD47-binding and EpCAM-binding domains exhibit higher immunomodulatory activity against CD47-expressing cells compared to bivalent antibodies with CD47-binding and / or bivalent antibodies with EpCAM-binding domains. In some cases, the bispecific antibody exhibits 1.5, 2, 3, 4, 5, or 10-fold higher immunomodulatory activity than bivalent antibodies with CD47-binding and / or bivalent antibodies with EpCAM-binding domains. The various immunomodulatory activities of the bispecific antibody can be measured in vitro in assays such as ADCC and ADCP assays. In some cases, the bispecific antibody exhibits 1.5, 2, 3, 4, 5, or 10-fold higher ADCC activity than bivalent antibodies with CD47-binding and / or bivalent antibodies with EpCAM-binding domains. In some cases, the bispecific antibody exhibits 1.5, 2, 3, 4, 5, or 10-fold higher ADCP activity than bivalent antibodies with CD47-binding and / or bivalent antibodies with EpCAM-binding domains.

[0093] In some embodiments, bispecific antibodies with both CD47-binding and EpCAM-binding domains exhibit higher immunomodulatory activity against CD47-expressing cells compared to bivalent antibodies with CD47-binding and / or bivalent antibodies with EpCAM-binding domains. In some cases, the bispecific antibody exhibits 10-20%, 21-30%, 31-40%, 41-50%, or at least 51% higher immunomodulatory activity than bivalent antibodies with CD47-binding and / or bivalent antibodies with EpCAM-binding domains. The various immunomodulatory activities of the bispecific antibody can be measured in vitro in assays such as ADCC and ADCP assays. In some cases, the bispecific antibody exhibits 10-20%, 21-30%, 31-40%, 41-50%, or at least 51% higher ADCC activity than bivalent antibodies with CD47-binding and / or bivalent antibodies with EpCAM-binding domains. In some cases, bispecific antibodies have 10-20%, 21-30%, 31-40%, 41-50%, or at least 51% higher ADCP activity than bivalent antibodies with a CD47 binding domain and / or bivalent antibodies with an EpCAM binding domain.

[0094] In some embodiments, this document describes a bispecific antibody comprising a first targeting moiety specifically binding to CD47 and a second targeting moiety specifically binding to EpCAM. In some cases, the bispecific antibody further includes an enhanced ADCP effect compared to the ADCP effect of the reference antibody CD47 BMK-1. In some cases, the bispecific antibody further includes an enhanced ADCC effect compared to the ADCC effect of the reference antibody CD47 BMK-1. In some cases, the enhanced ADCP is at least 2, 3, 4, or more times higher than the ADCP effect of the reference antibody CD47 BMK-1. In some cases, the enhanced ADCP is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more times higher than the CD47 BMK-1 effect of the reference antibody CD47 BMK-1. In some cases, the enhanced ADCC is at least 2, 3, 4, 5, or more times higher than the ADCC effect of the reference antibody CD47 BMK-1. In some cases, the enhanced ADCC is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more higher than the ADCC effect of the reference antibody CD47 BMK-1.

[0095] In some embodiments, this document describes a bispecific antibody comprising a first component that specifically binds to CD47 and a second component that specifically binds to EpCAM, wherein the bispecific antibody mediates ADCC more effectively than a bivalent antibody comprising either the first or second component, wherein ADCC activity is determined using an in vitro cytotoxicity assay. In some embodiments, the bispecific antibody induces at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or at least about 100% higher maximum cytotoxicity than a bivalent antibody comprising either the first or second component in an in vitro ADCC assay. In some embodiments, the bispecific antibody induces at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold higher maximum cytotoxicity than a bivalent antibody comprising either the first or second component in an in vitro ADCC assay.

[0096] In some embodiments, this document describes a bispecific antibody comprising a first component that specifically binds to CD47 and a second component that specifically binds to EpCAM, wherein the bispecific antibody mediates antibody-dependent phagocytosis (ADCP) more effectively than a bivalent antibody comprising either the first or second component, wherein ADCP activity is determined using an in vitro FACS-based phagocytosis assay. In some embodiments, the bispecific antibody induces at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or at least about 100% higher maximum phagocytosis than a bivalent antibody comprising either the first or second component in an in vitro ADCP assay.

[0097] In some cases, bispecific antibodies also include reduced cellular killing by immune cells compared to the killing effect of immune cells via the reference antibody CD47 BMK-1. In some cases, cellular killing is increased by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher compared to immune cell viability in the presence of the reference antibody CD47 BMK-1. In some cases, the immune cells are natural killer cells. In some cases, the immune cells are phagocytes. In some cases, the immune cells are macrophages.

[0098] Immunological activity can also be measured in cell line-derived xenograft assays, in which transformed cells are injected into mice and tumors form. In some cases, bispecific proteins with both CD47-binding and EpCAM-binding domains more significantly inhibit tumor growth, including cells expressing CD47, compared to bispecific proteins with the same CD47-binding domain and / or bispecific proteins with the same EpCAM-binding domain. In some cases, bispecific proteins exhibit 1.5, 2, 3, 4, 5, or 10-fold greater inhibition of xenograft tumor growth compared to bispecific proteins with the same CD47-binding and / or bispecific proteins with the same EpCAM-binding domain.

[0099] In some embodiments, bispecific antibodies with both CD47-binding and EGFR-binding domains exhibit higher immunomodulatory activity against CD47-expressing cells compared to bivalent antibodies with CD47-binding and / or bivalent antibodies with EGFR-binding domains. In some cases, the bispecific antibody exhibits 1.5, 2, 3, 4, 5, or 10-fold higher immunomodulatory activity than bivalent antibodies with CD47-binding and / or bivalent antibodies with EGFR-binding domains. The various immunomodulatory activities of the bispecific antibody can be measured in vitro in assays such as ADCC and ADCP assays. In some cases, the bispecific antibody exhibits 1.5, 2, 3, 4, 5, or 10-fold higher ADCC activity than bivalent antibodies with CD47-binding and / or bivalent antibodies with EGFR-binding domains. In some cases, the bispecific antibody exhibits 1.5, 2, 3, 4, 5, or 10-fold higher ADCP activity than bivalent antibodies with CD47-binding and / or bivalent antibodies with EGFR-binding domains.

[0100] In some embodiments, bispecific antibodies with both CD47-binding and EGFR-binding domains exhibit higher immunomodulatory activity against CD47-expressing cells compared to bivalent antibodies with CD47-binding and / or bivalent antibodies with EGFR-binding domains. In some cases, the bispecific antibody exhibits 10-20%, 21-30%, 31-40%, 41-50%, or at least 51% higher immunomodulatory activity than bivalent antibodies with CD47-binding and / or bivalent antibodies with EGFR-binding domains. The various immunomodulatory activities of the bispecific antibody can be measured in vitro in assays such as ADCC and ADCP assays. In some cases, the bispecific antibody exhibits 10-20%, 21-30%, 31-40%, 41-50%, or at least 51% higher ADCC activity than bivalent antibodies with CD47-binding and / or bivalent antibodies with EGFR-binding domains. In some cases, bispecific antibodies have 10-20%, 21-30%, 31-40%, 41-50%, or at least 51% higher ADCP activity than bivalent antibodies with a CD47-binding domain and / or bivalent antibodies with an EGFR-binding domain.

[0101] In some embodiments, this document describes a bispecific antibody comprising a first targeting portion specifically binding to CD47 and a second targeting portion specifically binding to EGFR. In some cases, the bispecific antibody further includes an enhanced ADCP effect compared to the ADCP effect of the reference antibody CD47 BMK-1. In some cases, the bispecific antibody further includes an enhanced ADCC effect compared to the ADCC effect of the reference antibody CD47 BMK-1. In some cases, the enhanced ADCP is at least 2, 3, 4, or more times higher than the ADCP effect of the reference antibody CD47 BMK-1. In some cases, the enhanced ADCP is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more times higher than the CD47 BMK-1 effect of the reference antibody CD47 BMK-1. In some cases, the enhanced ADCC is at least 2, 3, 4, 5, or more times higher than the ADCC effect of the reference antibody CD47 BMK-1. In some cases, the enhanced ADCC is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more higher than the ADCC effect of the reference antibody CD47 BMK-1.

[0102] In some embodiments, this document describes a bispecific antibody comprising a first component that specifically binds to CD47 and a second component that specifically binds to EGFR, wherein the bispecific antibody mediates ADCC more effectively than a bivalent antibody comprising either the first or second component, wherein ADCC activity is determined using an in vitro cytotoxicity assay. In some embodiments, the bispecific antibody induces at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or at least about 100% higher maximum cytotoxicity than a bivalent antibody comprising either the first or second component in an in vitro ADCC assay. In some embodiments, the bispecific antibody induces at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold higher maximum cytotoxicity than a bivalent antibody comprising either the first or second component in an in vitro ADCC assay.

[0103] In some embodiments, this document describes a bispecific antibody comprising a first component that specifically binds to CD47 and a second component that specifically binds to EGFR, wherein the bispecific antibody mediates antibody-dependent phagocytosis (ADCP) more effectively than a bivalent antibody comprising either the first or second component, wherein ADCP activity is determined using an in vitro FACS-based phagocytosis assay. In some embodiments, the bispecific antibody induces at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or at least about 100% higher maximum phagocytosis than a bivalent antibody comprising either the first or second component in an in vitro ADCP assay.

[0104] In some cases, bispecific antibodies also include reduced cellular killing by immune cells compared to the killing effect of immune cells via the reference antibody CD47 BMK-1. In some cases, cellular killing is increased by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher compared to immune cell viability in the presence of the reference antibody CD47 BMK-1. In some cases, the immune cells are natural killer cells. In some cases, the immune cells are phagocytes. In some cases, the immune cells are macrophages.

[0105] Immunological activity can also be measured in cell line-derived xenograft assays, in which transformed cells are injected into mice and tumors form. In some cases, bispecific proteins with both CD47-binding and EGFR-binding domains more significantly inhibit tumor growth, including cells expressing CD47, compared to bispecific proteins with the same CD47-binding domain and / or bispecific proteins with the same EGFR-binding domain. In some cases, bispecific proteins exhibit 1.5, 2, 3, 4, 5, or 10-fold greater inhibition of xenograft tumor growth compared to bispecific proteins with the same CD47-binding domain and / or bispecific proteins with the same EGFR-binding domain.

[0106] As used herein, “antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize binding antibodies on target cells and subsequently cause lysis of the target cells. In one embodiment, the target cells are human cells, such as tumor cells (e.g., myeloma cells). In some embodiments, the tumor cells are derived from adenocarcinoma, lymphoma, or carcinoma. In some cases, the adenocarcinoma is pancreatic ductal adenocarcinoma, ovarian clear cell adenocarcinoma, colonic adenocarcinoma, lung adenocarcinoma, or ovarian adenocarcinoma. In other cases, the carcinoma is vulvar squamous cell carcinoma. While it is not desirable to be bound to any particular mechanism of action, cytotoxic cells mediating ADCC typically express Fc receptors (FcRs). NK cells used to mediate ADCC express FcγRIII, while monocytes express FcγRI, FcγRII, FcγRIII, and / or FcγRIV. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991).

[0107] To assess the ADCC activity of the bispecific proteins described herein, in vitro ADCC assays are performed in some embodiments, such as cytotoxicity assays using cancer cell lines. Useful effector cells for such assays include, but are not limited to, peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, in some embodiments, the ADCC activity of the bispecific protein of interest is assessed in vivo (e.g., in animals).

[0108] "Antibody-dependent phagocytosis" or "ADCP" refers to the ability of phagocytes (such as macrophages) to eliminate antibody-coated target cells. In some implementations, phagocytosis assays are used to measure ADCP effects.

[0109] In some embodiments, the bispecific protein described herein, which binds to CD47 and EpCAM, mediates antibody-dependent phagocytosis in at least 50% of cells in an exponentially growing population of CD47+ / EpCAM+ cancer cells. In some embodiments, the tumor cells are derived from adenocarcinoma, lymphoma, or carcinoma. In some cases, the adenocarcinoma is pancreatic ductal adenocarcinoma, ovarian clear cell adenocarcinoma, colonic adenocarcinoma, lung adenocarcinoma, or ovarian adenocarcinoma.

[0110] In some embodiments, the bispecific protein described herein that binds to CD47 and EpCAM mediates the inhibition of CD47-SIRPα interaction. In some embodiments, the bispecific antibody inhibits the binding of SIRPα to CD47 by at least 50%. In some embodiments, binding inhibition is measured by ELISA. In some embodiments, binding inhibition is measured using CD47+ cells. In some embodiments, CD47+ cells are CD47+EpCAM+ tumor cells.

[0111] In some implementations, the bispecific antibody exhibits reduced binding to CD47+ non-tumor cells such as erythrocytes and platelets. In some cases, the binding to CD47+ non-tumor cells is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference antibody CD47 BMK-1. In some cases, the binding of the bispecific antibody to CD47+ non-tumor cells is measured by erythrocyte (RBC) binding assay or flow cytometry.

[0112] In some implementations, the concentration of the bispecific antibody required to induce RBC lysis is at least 2, 3, 4, or higher than the hemolytic activity of the reference antibody CD47BMK-1.

[0113] 4.8 Production of antibodies or their binding fragments In some embodiments, the polypeptides described herein (e.g., antibodies and their binding fragments) are produced using any method known in the art that can be used to synthesize polypeptides (e.g., antibodies), particularly by chemical synthesis or by recombinant expression, and preferably by recombinant expression technology.

[0114] In some cases, antibodies or their binding fragments are recombinantly expressed, and the nucleic acids encoding the antibodies or their binding fragments are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242). This involves synthesizing overlapping oligonucleotides containing the sequence portion encoding the antibody, annealing and linking these oligonucleotides, and then amplifying the linked oligonucleotides by PCR.

[0115] Alternatively, the nucleic acid molecule encoding the antibody may be generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library produced from any tissue or cell expressing an immunoglobulin) by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequence or by cloning an oligonucleotide probe that is specific to the specific gene sequence.

[0116] In some cases, antibodies or their conjugates are optionally produced by immunizing animals (such as mice) to generate polyclonal antibodies, or more preferably by producing monoclonal antibodies, for example, as described by Kohler and Milstein (1975, Nature 256:495-497), or as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, clones encoding at least the Fab portion of an antibody may be obtained by screening Fab expression libraries for clones of Fab fragments that bind to specific antigens (e.g., as described in Huse et al., 1989, Science 246:1275-1281), or by screening antibody libraries (see, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0117] In some implementations, techniques for producing “chimeric antibodies” are used, developed by splicing together genes from mouse antibody molecules specific to the appropriate antigen with genes from human antibody molecules of appropriate biological activity (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454). Chimeric antibodies are molecules in which different parts are derived from different animal species, such as those with variable regions from mouse monoclonal antibodies and constant regions from human immunoglobulins.

[0118] In some embodiments, techniques described for producing single-chain antibodies (US Patent No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are suitable for producing single-chain antibodies. Single-chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via amino acid bridges, resulting in single-chain polypeptides. Alternatively, techniques for assembling functional Fv fragments in *E. coli* (Skerra et al., 1988, Science 242:1038-1041) may also be used.

[0119] In some embodiments, an expression vector containing the antibody's nucleotide sequence or the antibody's nucleotide sequence is transferred into host cells using conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured using conventional techniques to produce the antibody. In specific embodiments, antibody expression is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0120] In some implementations, a variety of host-expression vector systems are used to express the antibodies or their binding fragments described herein. Such host expression systems represent vectors through which the coding sequence of the antibody is generated and subsequently purified, but also represent cells that express the antibody or its binding fragment in situ when transformed or transfected with a suitable nucleotide coding sequence. These include, but are not limited to, microorganisms such as bacteria (e.g., *Escherichia coli* and *Bacillus subtilis*) transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing sequences encoding antibodies or their binding fragments; yeast (e.g., *Saccharomyces pichia*) transformed with recombinant yeast expression vectors containing sequences encoding antibodies or their binding fragments; insect cell systems infected with recombinant viral expression vectors (e.g., baculoviruses); plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors containing sequences encoding antibodies or their binding fragments (e.g., Ti plasmids); or mammalian cell systems carrying recombinant expression constructs (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters).

[0121] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines stably expressing antibodies are optionally engineered. Host cells are transformed with DNA controlled by suitable expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectivity markers, rather than using expression vectors containing viral origins of replication. After the introduction of exogenous DNA, the engineered cells are allowed to grow in enriched media for 1–2 days, and then transferred to selective media. Selectivity markers in the recombinant plasmid confer resistance to the selected medium and allow cells to stably integrate the plasmid into their chromosomes and grow to form foci, which are then cloned and amplified into the cell line. This method can be advantageously used for the engineering of cell lines expressing antibodies or their binding fragments.

[0122] In some cases, multiple selection systems are used, including but not limited to those for herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202) and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes for tk cells, hgprt cells, or aprt cells, respectively. In addition, antimetabolite resistance has been used as a basis for selecting the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIB TECH 11(5):155-215) and hygro, which conferred resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Well-known methods in the field of recombinant DNA technology that can be used are described in Ausubel et al. (eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York; Kriegler, Gene Transfer and Expression, Laboratory Manual, Stockton Press, New York; and Dracopoli et al. (eds.), Current Protocols in Human Genetics, Chapters 12 and 13, John Wiley & Sons, New York; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0123] In some cases, antibody expression levels are increased by vector amplification (for a review, see Bebbington and Hentschel, The Use of Vectors Based on Gene Amplification for the Expression of Cloned Genes in Mammalian Cells in DNA Cloning, Vol. 3 (Academic Press, New York, 1987)). When the marker in the antibody-expressing vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Because the amplified region is associated with the antibody's nucleotide sequence, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0124] In some cases, any method known in the art for purifying antibodies is used, such as by chromatography (e.g., ion exchange, affinity (particularly by affinity for the specific antigen after protein A) and size column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins.

[0125] 4.9 Expression Vectors In some implementations, the vector includes any suitable vector derived from eukaryotic or prokaryotic sources. In some cases, the vector is obtained from bacteria (e.g., *Escherichia coli*), insects, yeast (e.g., *Pichia pastoris*), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, the pBAD vector series, the pBADM vector series, the pET vector series, the pETM vector series, the pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, the pQE vector series, pRSET A, pRSET B, pRSET C, the pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0126] Exemplary insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[0127] In some cases, yeast vectors include Gateway® pDEST™ 14 vector, Gateway® pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® target vector, pAO815 Pichia pastoris vector, pFLD1 Pichia pastoris vector, pGAPZA Pichia pastoris vector, pGAPZB Pichia pastoris vector, pGAPZC Pichia pastoris vector, pPIC3.5K Pichia pastoris vector, pPIC6A Pichia pastoris vector, pPIC6B Pichia pastoris vector, pPIC6C Pichia pastoris vector, pPIC9K Pichia pastoris vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A yeast vector, pYES2 / NT B yeast vector, and pYES2 / NT C yeast vector or pYES3 / CT yeast vector.

[0128] Exemplary algal vectors include the pChlamy-4 vector or the MCS vector.

[0129] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Transient mammalian expression vectors may include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a, pFLAG-CMV 6b, pFLAG-CMV 6c, pFLAG-CMV 5.1, pFLAG-CMV 5a, pFLAG-CMV 5b, pFLAG-CMV 5c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Stable mammalian expression vectors may include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0130] In some cases, cell-free systems are mixtures of cytoplasmic and / or nuclear components derived from cells and are used for in vitro nucleic acid synthesis. In other cases, cell-free systems utilize prokaryotic or eukaryotic cell components. Sometimes, nucleic acid synthesis is achieved in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, or HeLa cells. Exemplary cell-free systems include, but are not limited to, the E. coli S30 Extract system, the E. coli T7 S30 system, or PURExpress®.

[0131] 4.10 Host Cell In some implementations, the host cell includes any suitable cell, such as naturally derived cells or genetically modified cells. In some cases, the host cell is a production host cell. In some cases, the host cell is a eukaryotic cell. In other cases, the host cell is a prokaryotic cell. In some cases, eukaryotic cells include fungi (e.g., yeast cells), animal cells, or plant cells. In some cases, prokaryotic cells are bacterial cells. Examples of bacterial cells include Gram-positive bacteria or Gram-negative bacteria. Sometimes, Gram-negative bacteria are anaerobic, rod-shaped, or both.

[0132] In some cases, Gram-positive bacteria include Actinobacteria, Firmicutes, or Tenericulates. In other cases, Gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi / Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia / Chlamydiae (PVC group), Proteobacteria, Spirochaetes, or Synergistetes. Other bacteria can be from the phyla Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. Bacterial cells can be *Escherichia coli*, *Clostridium botulinum*, or *Coli bacilli*.

[0133] Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10 / P3, ccdBSurvival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0134] In some cases, animal cells include cells derived from vertebrates or invertebrates. In others, animal cells include cells derived from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In still others, fungal cells include yeast cells, such as brewer's yeast, baker's yeast, or wine yeast.

[0135] Fungi include ascomycetes, such as yeasts, molds, filamentous fungi, basidiomycetes, or zygomycetes. In some cases, yeasts include either the phylum Ascomycota or Basidiomycota. In some cases, the phylum Ascomycota includes the subphylum Saccharomycotina (true yeasts, such as Saccharomyces cerevisiae (baker's yeast)) or the subphylum Taphrinomycotina (e.g., Schizosaccharomycetes). In some cases, the subphylum Basidiomycota includes the subphylum Agaricomycotina (e.g., Tremellomycetes) or the subphylum Pucciniomycotina (e.g., Microbotryomycetes).

[0136] Exemplary yeasts or filamentous fungi include, for example, the genera *Saccharomyces*, *Schizosaccharomyces*, *Candida*, *Pichia*, *Hansenula*, *Kluyveromyces*, *Zygosaccharomyces*, *Yarrowia*, *Trichosporon*, *Rhodosporidi*, *Aspergillus*, *Fusarium*, or *Trichoderma*.Exemplary yeasts or filamentous fungi include, for example, the following species: *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, *Candida utilis*, *Candida booidini*, *Candida albicans*, *Candida tropicalis*, *Candida stellatoidea*, *Candida glabrata*, *Candida krusei*, *Candida parapsilosis*, *Candida guilliermondii*, *Candida viswanathii*, *Candida lusitaniae*, *Rhodotorula mucilaginosa*, *Pichia metanolica*, *Pichia angusta*, *Pichia pastoris*, *Pichia anomala*, *Hansenula polymorpha*, and *Kluyveromyces lactis*. *Zygosaccharomyces rouxii*, *Yarrowia lipolytica*, *Trichosporon pullulans*, *Rhodosporidium toru-Aspergillus niger*, *Aspergillus nidulans*, *Aspergillus awamori*, *Aspergillus oryzae*, *Trichodermareesei*, *Zygosaccharomyces bailii*, *Cryptococcus neoformans*, *Cryptococcus garterii* (gattii) or Saccharomyces boulardii.

[0137] Exemplary yeast host cells include, but are not limited to, Pichia pastoris strains such as GS115, KM71H, SMD1168, SMD1168H and X-33; and Saccharomyces cerevisiae strains such as INVSC1.

[0138] In some cases, the additional animal cells include cells derived from mollusks, arthropods, annelids, or sponges. In others, the additional animal cells are mammalian cells, such as those from primates, apes, horses, cattle, pigs, dogs, cats, or rodents. In still others, rodents include mice, rats, hamsters, gerbils, gray squirrels, fancy rats, or guinea pigs.

[0139] Exemplary mammalian host cells include, but are not limited to, the 293A cell line, 293FT cell line, 293F cell line, 293H cell line, CHO DG44 cell line, CHO-S cell line, CHO-K1 cell line, Expi293F™ cell line, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp-In™-Jurkat cell line, FreeStyle™ 293-F cell line, FreeStyle™ CHO-S cell line, GripTite™ 293 MSR cell line, GS-CHO cell line, HepaRG™ cell line, and T-REx™ cell line. Jurkat cell line, Per.C6 cell line, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line.

[0140] In some cases, mammalian host cells are stable cell lines, or cell lines that have incorporated the genetic material of interest into their own genome and have the ability to express the product of the genetic material after many generations of cell division. In other cases, mammalian host cells are transient cell lines, or cell lines that have not yet incorporated the genetic material of interest into their own genome and do not have the ability to express the product of the genetic material after many generations of cell division.

[0141] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

[0142] In some cases, plant cells include cells derived from algae. Exemplary insect cell lines include, but are not limited to, strains from *Chlamydomonas reinhardtii* 137c or *Synechococcuselongatus* PPC 7942.

[0143] 4.11 Certain Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. It should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and do not limit any of the claimed subject matter. In this application, the singular is used to include the plural unless otherwise specified. It must be noted that, as used in the specification and appended claims, the singular forms “a,” “an,” and “the” include the plural referent unless the context clearly indicates otherwise. In this application, the use of “or” means “and / or” unless otherwise stated. Furthermore, the use of the term “including” and other forms such as “include,” “includes,” and “included” is not restrictive.

[0144] As used herein, ranges and quantities can be expressed as “about” a specific value or range. “About” also includes an exact quantity. Therefore, “about 5 µL” means both “about 5 µL” and “5 µL”. Typically, the term “about” includes quantities that are expected to be within the experimental error range.

[0145] The chapter titles used in this article are for organizational purposes only and are not to be construed as limiting the subject matter.

[0146] "Antibody" and "immunoglobulin" (IgS) are glycoproteins with the same structural features. These terms are used synonymously. In some cases, the antigen specificity of immunoglobulins is known.

[0147] The term “antibody” is used in the broadest sense and covers fully assembled antibodies, antibody fragments that can bind to antigens (e.g., Fab, F(ab')2, Fv, single-chain antibodies, biantibodies, antibody chimeras, hybrid antibodies, bispecific antibodies, etc.), and recombinant peptides including the foregoing.

[0148] As used herein, the terms “monoclonal antibody” and “mAb” refer to antibodies obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in small amounts.

[0149] The term "bispecific antibody" is used to refer to an antibody that specifically binds to at least two different antigens, and includes antibodies that can specifically bind to only two different antigens, and further includes antibodies that can specifically bind to two different antigens, and further includes one or more additional binding domains that specifically bind to a third, fourth or more antigens or conjugate to one or more additional binding domains that specifically bind to a third, fourth or more antigens.

[0150] "Natural antibodies" and "natural immunoglobulins" are typically heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a covalent disulfide bond, the number of which varies between heavy chains of different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domains of the light chain are aligned with the first constant domain of the heavy chain, and the variable domains of the light chain are aligned with the variable domains of the heavy chain. Specific amino acid residues are thought to form the interface between the variable domains of the light and heavy chains.

[0151] The term "variable" refers to the fact that certain portions of the variable domain differ significantly in sequence between antibodies. The variable region imparts antigen-binding specificity. However, the variation is not uniformly distributed throughout the entire variable domain of the antibody. It is concentrated in three segments in both the light and heavy chain variable domains, known as complementarity-determining regions (CDRs) or hypervariable regions. More conserved portions of the variable domain reside in the framework (FR) regions. The variable domains of the native heavy and light chains each comprise four FR regions, predominantly employing a β-sheet structure, linked by three CDRs that form loops connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. The CDRs in each chain remain tightly bound together through the FR regions and, together with CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see, Kabat et al. (1991), NIH PubL., Vol. 91-3242, pp. 647-669). The constant domain does not directly participate in the binding of antibodies to antigens, but it exhibits various effector functions, such as Fc receptor (FcR) binding, antibody participation in antibody-dependent cytotoxicity, initiation of complement-dependent cytotoxicity, and mast cell degranulation.

[0152] The term "hypervariate region" as used in this article refers to the amino acid residues in the antibody responsible for antigen binding. The hypervariable region includes amino acid residues from the “complementarity-determining region” or “CDR” (i.e., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD) and / or residues from the “hypervariable ring” (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues (H1), 53-55 (H2), and 96-101 (13) in the heavy chain variable domain; Clothia and Lesk, (1987) J. Mol. Biol, 196:901-917). “Frame” or “FR” residues are variable domain residues other than hypervariable region residues, as considered in this paper.

[0153] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fab, F(ab')2, and Fv fragments; biantibodies; linear antibodies (Zapata et al., (1995) Protein Eng. 10:1057-1062); single-chain antibody molecules; and bispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, referred to as "Fab" fragments, each with a single antigen-binding site; and a residual "Fc" fragment, the name reflecting its tendency to crystallize. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and remains capable of cross-linking antigens.

[0154] The “Fv” is the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a heavy-chain variable domain and a light-chain variable domain, tightly bound together by nonvalent association. It is in this configuration that the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. In general, the six CDRs confer antigen-binding specificity against the antibody. However, even a single variable domain (or half of an Fv containing only the three CDRs specific to the antigen) has the ability to recognize and bind to the antigen, although with less affinity than the entire binding site.

[0155] The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment in that it has several residues added at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the name for Fab' in this paper, where the cysteine ​​residues of the constant domain have free thiol groups. The Fab' fragment is generated by reducing the heavy chain disulfide bonds of the F(ab')2 fragment. Other chemical conjugations of antibody fragments are also known.

[0156] Based on the amino acid sequence of their constant structural domains, the “light chains” of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two distinct types, known as κ (κ) and λ (λ).

[0157] Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, IgM, and IgY, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known. Different isotypes possess different effector functions. For example, human IgG1 and IgG3 isotypes possess ADCC (antibody-dependent cell-mediated cytotoxicity) activity.

[0158] In some cases, the CDR of an antibody is determined according to (i) the Kabat numbering system (Kabat et al. (197) Ann. NYAcad. Sci. 190:382-391 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication, pp. 91-3242); or (ii) the Chothia numbering scheme, which will be referred to herein as “Chothia CDR” (see, for example, Chothia and Lesk, 1987, J. Mol. Biol, 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol, 273:927-948; Chothia et al., 1992, J. Mol. Biol, 227:799-817; Tramontano A et al., 1990, J. Mol. Biol, 1990). Biol, 215(1): 175-82; and U.S. Patent No. 7,709,226); or (iii) the ImMunoGeneTics (IMGT) numbering system, as described, for example, in Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res, 27:209-212 (“IMGT CDR”); or (iv) MacCallum et al., 1996, J. Mol. Biol, 262:732-745. See also, for example, Martin, A., “Protein Sequence and Structure Analysis of Antibody Variable Domains”, Antibody Engineering, eds. Kontermann and Diibel, Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0159] Regarding the Kabat numbering system, the CDR within the antibody heavy chain molecule is typically located at amino acid positions 31 to 35, which may optionally contain one or two additional amino acids, followed by 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDR within the antibody light chain molecule is typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). As is well known to those skilled in the art, using the Kabat numbering system, the actual linear amino acid sequence of the antibody variable domain may contain fewer or additional amino acids due to the shortening or lengthening of the FR and / or CDR; therefore, the number of Kabat amino acids is not necessarily the same as the number of linear amino acids.

[0160] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0161] As used herein, the term "human antibody" or "humanized antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, MA and van de Winkel, JG, Curr. Opin. Chem. Biol., 5(2001) 368-374). In some cases, human antibodies are also produced in transgenic animals (e.g., mice) that, upon immunization, are able to produce a full spectrum or selection of human antibodies in the absence of endogenous immunoglobulin production. Transferring human germline immunoglobulin gene arrays in such germline mutant mice results in the production of human antibodies upon antigen challenge (see, for example, Jakobovits, A. et al., Natl. Acad. Sci. USA, 90 (1993) 2551-2555; Jakobovits, A. et al., Nature, 362 (1993) 255-258; Bruggemann, M. et al., Year Immunol., 7 (1993), 33-40). In other cases, human antibodies are also produced in phage display libraries (Hoogenboom, HR and Winter, G., J. Mol. Biol., 227 (1992) 381-388; Marks, JD et al., J. Mol. Biol. 222 (1991), 581-597). The techniques used by Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P. et al., J. Immunol., 147 (1991) 86-95).

[0162] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from host cells (e.g., NSO or CHO cells), antibodies isolated from animals that are transgenic for human immunoglobulin genes (e.g., mice), or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have variable and constant regions in rearranged form. In some cases, recombinant human antibodies have undergone in vivo somatic hypermutation. Therefore, the amino acid sequences of the VH and VL regions of recombinant antibodies are sequences that, while derived from and associated with human germline VH and VL sequences, may not be naturally present within the in vivo human antibody phylogenetic profile.

[0163] As used herein, the terms “individual,” “object,” and “patient” refer to any mammal. In some embodiments, the mammal is human. In some embodiments, the mammal is non-human. None of these terms requires or limits a situation characterized by supervision (e.g., continuous or intermittent) by a healthcare worker (e.g., physician, registered nurse, nurse practitioner, physician assistant, caregiver, or hospice worker).

[0164] As used herein, the sequence-related term "percentage of amino acid sequence identity (%)" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a given sequence after sequence alignment and the introduction of gaps (if necessary) to achieve maximum percentage sequence identity, without considering any conserved substitutions as part of sequence identity. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0165] The following list of embodiments of the invention is intended to disclose various features of the invention, which may be considered specific to the particular embodiments discussed, or may be combined with various other features as listed in other embodiments. Therefore, the use of a feature is not necessarily limited to that embodiment merely because a feature has been discussed in connection with a particular embodiment.

[0166] Implementation scheme A1. A bispecific antibody comprising a CD47 binding domain and an EpCAM binding domain.

[0167] Implementation Scheme A2. The bispecific antibody according to Implementation Scheme A1 further includes an Fc domain.

[0168] Implementation Scheme A3. A bispecific antibody according to Implementation Scheme A1 or A2, wherein the bispecific antibody has a higher affinity for CD47 expressed on the surface of tumor cells than for CD47 expressed on the surface of erythrocytes or platelets.

[0169] Implementation Scheme A4. The bispecific antibody according to Implementation Scheme A3, wherein the tumor cells express EpCAM.

[0170] Implementation Scheme A5. The bispecific antibody according to any one of Implementation Schemes A1 to A4, wherein the concentration of the bispecific antibody required for half-maximal binding to human erythrocytes is greater than 500 nM.

[0171] Implementation Scheme A6. The bispecific antibody according to any one of Implementation Schemes A1 to A5, wherein the bispecific antibody binds to human CD47 with a KD of less than 100 nM.

[0172] Implementation Scheme A7. The bispecific antibody according to Implementation Scheme A6, wherein the bispecific antibody binds to human CD47 with a KD of 1 nM to 100 nM, 1 nM to 50 nM or 5 nM to 50 nM.

[0173] Implementation Scheme A8. The bispecific antibody according to any one of Implementation Schemes A1 to A7, wherein the bispecific antibody binds to EpCAM with a KD of less than 500 nM.

[0174] Implementation Scheme A9. The bispecific antibody according to Implementation Scheme A8, wherein the bispecific antibody is bound to EpCAM with a KD of 0.2 nM to 500 nM, 1 nM to 300 nM, 5 nM to 200 nM or 10 nM to 150 nM.

[0175] Implementation Scheme A10. A bispecific antibody according to any one of Implementation Schemes A6 to A9, wherein KD is determined by surface plasmon resonance.

[0176] Implementation Scheme A11. A bispecific antibody according to any one of Implementation Schemes A1 to A10, wherein the CD47 binding domain is a human or engineered human CD47 binding domain.

[0177] Implementation Scheme A12. The bispecific antibody according to any one of Implementation Schemes A1 to A11, wherein the CD47 binding domain comprises a heavy chain variable domain and a light chain variable domain.

[0178] Implementation Scheme A13. The bispecific antibody according to any one of Implementation Schemes A1 to A11, wherein the CD47 binding domain includes scFv.

[0179] Implementation Scheme A14. The bispecific antibody according to any one of Implementation Schemes A1 to A13, wherein the EpCAM binding domain comprises a heavy chain variable domain and a light chain variable domain.

[0180] Implementation Scheme A15. The bispecific antibody according to any one of Implementation Schemes A1 to A13, wherein the EpCAM binding domain includes scFv.

[0181] Implementation Scheme A16. A bispecific antibody according to any one of Implementation Schemes A1 to A15, wherein the Fc domain is a human Fc domain.

[0182] Implementation Scheme A17. The bispecific antibody according to Implementation Scheme A16, wherein the isotype of the human Fc domain is IgG1 or IgG4.

[0183] Implementation Scheme A18. A bispecific antibody according to any one of Implementation Schemes A1 to A17, wherein the Fc domain is a heterodimeric Fc domain, wherein the heterodimeric Fc region includes a club chain and a mortar chain to form a club-mortar (KiH) structure.

[0184] Implementation Scheme A19. The bispecific antibody according to Implementation Scheme A18, wherein the mortar chain comprises the mutant T366W, and the saliva chain comprises the mutants T366S, L368A, and Y407V, wherein the amino acid position numbers are based on the EU index of Kabat et al.

[0185] Implementation Scheme A20. A bispecific antibody according to any one of Implementation Schemes A1 to A19, wherein the bispecific antibody has an asymmetric triple-chain club-and-mortar structure.

[0186] Implementation Scheme A21. The bispecific antibody according to Implementation Scheme A20, wherein the CD47 binding domain is scFv.

[0187] Implementation Scheme A22. The bispecific antibody according to Implementation Scheme A20, wherein the EpCAM binding domain is scFv.

[0188] Implementation Scheme A23. The bispecific antibody according to any one of Implementation Schemes A1 to A22, wherein the CD47 binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3; and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein HC-CDR1 comprises SEQ ID NO: 15, HC-CDR2 comprises SEQ ID NO: 16, HC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 17, LC-CDR1 comprises SEQ ID NO: 18, LC-CDR2 comprises SEQ ID NO: 19, and LC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 20.

[0189] Implementation Scheme A24. The bispecific antibody according to Implementation Scheme A23, wherein HC-CDR3 includes amino acid substitutions at one or more of K94, E95, G96, S97, F98, G99, V100b, D101, and P102; and LC-CDR3 includes amino acid substitutions at one or more of Y89, S90, T91, D92, I93, S94, G95, N95a, H95b, W96, and V97.

[0190] Implementation Scheme A25. The bispecific antibody according to any one of Implementation Schemes A1 to A24, wherein the heavy chain variable domain of the CD47 binding domain comprises a sequence having at least 90% identity with SEQ ID NO: 5, SEQ ID NO: 10 or SEQ ID NO: 13, and the light chain of the CD47 binding domain comprises a sequence having at least 90% identity with SEQ ID NO: 8, SEQ ID NO: 11 or SEQ ID NO: 14.

[0191] Implementation Scheme A26. A bispecific antibody according to any one of Implementation Schemes A1 to A25, wherein the EpCAM binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein the CDRs are defined by an ordered set of sequences listed as HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3; and wherein the CDRs are selected from sequences having at least 90% identity with the following sequences: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83; SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86; SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89; SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92; SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 95; SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98; SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101; SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104; SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107; SEQ ID NO: 72, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110; SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113; and SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116.

[0192] Implementation Scheme A27. The bispecific antibody according to any one of Implementation Schemes A1 to A26, wherein the EpCAM binding domain comprises: The sequence is at least 90% identical to SEQ ID NO: 21, and A sequence that is at least 90% identical to SEQ ID NO: 33; The sequence is at least 90% identical to SEQ ID NO: 22, and A sequence that is at least 90% identical to SEQ ID NO: 34; or The sequence is at least 90% identical to SEQ ID NO: 24, and The sequence is at least 90% identical to SEQ ID NO: 36.

[0193] Implementation Scheme A28. The bispecific antibody according to any one of Implementation Schemes A1 to A27, wherein the percentage of A431 cells phagocytosed by macrophages is increased by at least 4-fold when the bispecific antibody is less than 1 nM or less than 0.01 nM compared with a nonspecific IgG1 antibody control.

[0194] Implementation Scheme A29. The bispecific antibody according to Implementation Scheme A28, wherein the concentration of the antibody required to mediate antibody-dependent phagocytosis of macrophages against EpCAM-positive and CD47-positive tumor cells is between 0.01 nM and -3 nM.

[0195] Implementation Scheme A30. The bispecific antibody according to Implementation Scheme A29, wherein the EpCAM-positive, CD47-positive tumor cells are OVISE cells or A431 cells.

[0196] Implementation Scheme A31. The bispecific antibody according to Implementation Scheme A29, wherein the EpCAM-positive and CD47-positive tumor cells are selected from pancreatic ductal adenocarcinoma cells, ovarian clear cell adenocarcinoma cells, colon adenocarcinoma cells, lung adenocarcinoma cells, ovarian adenocarcinoma cells, and vulvar squamous cell carcinoma cells.

[0197] Implementation Scheme A32. A bispecific antibody according to any one of Implementation Schemes A1 to A28, wherein 100 nM of the bispecific antibody inhibits the binding of SIRPα to CD47 on the cell surface by at least 30%.

[0198] Implementation Scheme A33. The bispecific antibody according to Implementation Scheme A32, wherein the cells are CD47+EpCAM+ tumor cells.

[0199] Implementation Scheme A34. The bispecific antibody according to Implementation Scheme A33, wherein the cells express at least as much EpCAM protein on their surface as HCC-44 cells.

[0200] Implementation Scheme A35. The bispecific antibody according to Implementation Scheme A33, wherein the cells express at least 100,000, at least 300,000, at least 600,000, or at least 1,500,000 EpCAM proteins on their surface.

[0201] Implementation Scheme A36. A bispecific antibody according to any one of Implementation Schemes A1 to A33, wherein 400 nM of the bispecific antibody does not induce hemolysis of erythrocytes in a hemagglutination assay.

[0202] Implementation Scheme A37. A complex comprising a bispecific antibody according to any one of Implementation Schemes A1 to A36 and CD47+EpCAM+ target cells.

[0203] Implementation Scheme A38. The complex according to Implementation Scheme A37, wherein the target cells express at least as much EpCAM protein on their surface as HCC-44 cells.

[0204] Implementation Scheme A39. The complex according to Implementation Scheme A37, wherein the target cells express at least 100,000, at least 300,000, at least 600,000, or at least 1,500,000 EpCAM proteins on their surface.

[0205] Implementation scheme A40. The complex according to any one of implementation schemes A37 to A39, wherein the CD47+EpCAM+ target cells are cancer cells.

[0206] Implementation Scheme A41. The complex according to Implementation Scheme A40, wherein the cancer cells are pancreatic ductal adenocarcinoma cells, ovarian clear cell adenocarcinoma cells, colonic adenocarcinoma cells, lung adenocarcinoma cells, ovarian adenocarcinoma cells, or vulvar squamous cell carcinoma cells.

[0207] Implementation Scheme A42. A method for inducing phagocytosis in CD47+EpCAM+ target cells, the method comprising administering a bispecific antibody according to any one of Implementation Schemes A1 to A36.

[0208] Implementation Scheme A43. The method according to Implementation Scheme A42, wherein the bispecific antibody is administered at a concentration of less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.1 nM, or less than 0.05 nM.

[0209] Implementation Scheme A44. A method for killing CD47+EpCAM+ target cells, the method comprising administering a bispecific antibody according to any one of Implementation Schemes A1 to A36, wherein the bispecific antibody inhibits the binding of SIRPα to CD47 on the surface of the CD47+EpCAM+ target cells.

[0210] Implementation Scheme A45. A method for killing CD47+EpCAM+ target cells, the method comprising administering a bispecific antibody according to any one of Implementation Schemes A1 to A36, wherein the bispecific antibody induces antibody-dependent cytotoxicity that kills the CD47+EpCAM+ target cells.

[0211] Implementation Scheme A46. The method according to Implementation Scheme A45, wherein the antibody is administered at a concentration of 0.01-1 nM, 0.01-0.5 nM, 0.01-0.25 nM, 0.01-0.1 nM, 0.01-0.05 nM or less than 0.01 nM.

[0212] Implementation Scheme A47. The method according to Implementation Scheme A45 or A46, wherein the CD47+EpCAM+ target cells are selected from A431 cells, HCC-44 cells, SKOV-3 cells, OVISE cells or CFPAC-1 cells.

[0213] Implementation Scheme A48. The method according to Implementation Scheme A45 or A46, wherein the CD47+EpCAM+ target cells are selected from pancreatic ductal adenocarcinoma cells, ovarian clear cell adenocarcinoma cells, colon adenocarcinoma cells, lung adenocarcinoma cells, or vulvar squamous cell carcinoma cells.

[0214] Implementation Scheme A49. A drug comprising a bispecific antibody as described in any one of Implementation Schemes A1 to A36.

[0215] Implementation Scheme A50. A method for treating an individual suffering from cancer, the method comprising administering a pharmaceutical composition according to Implementation Scheme A49.

[0216] Implementation Scheme A51. The method according to Implementation Scheme A50, wherein the cancer is pancreatic ductal adenocarcinoma, ovarian clear cell adenocarcinoma, colon adenocarcinoma, lung adenocarcinoma, or vulvar squamous cell carcinoma.

[0217] Implementation Scheme B1. A bispecific antibody comprising a CD47 binding domain and an EGFR binding domain.

[0218] Implementation Scheme B2. The bispecific antibody according to Implementation Scheme B1 further includes an Fc domain.

[0219] Implementation Scheme B3. A bispecific antibody according to Implementation Scheme B1 or B2, wherein the bispecific antibody has a higher affinity for CD47 expressed on the surface of tumor cells than for CD47 expressed on the surface of erythrocytes or platelets.

[0220] Implementation Scheme B4. The bispecific antibody according to Implementation Scheme B3, wherein the tumor cells express EGFR.

[0221] Implementation Scheme B5. The bispecific antibody according to any one of Implementation Schemes B1 to B4, wherein the concentration of the bispecific antibody required for half-maximal binding to human erythrocytes is greater than 500 nM.

[0222] Implementation Scheme B6. A bispecific antibody according to any one of Implementation Schemes B1 to B5, wherein the bispecific antibody binds to human CD47 with a KD of less than 100 nM.

[0223] Implementation Scheme B7. The bispecific antibody according to Implementation Scheme B6, wherein the bispecific antibody binds to human CD47 with a KD of 1 nM to 100 nM, 1 nM to 50 nM or 5 nM to 50 nM.

[0224] Implementation Scheme B8. The bispecific antibody according to any one of Implementation Schemes B1 to B7, wherein the bispecific antibody binds to EGFR with a KD of less than 25 nM.

[0225] Implementation Scheme B9. The bispecific antibody according to Implementation Scheme B8, wherein the bispecific antibody binds to EGFR with a KD of 0.2 nM to 25 nM, 0.2 nM to 10 nM, 0.2 nM to 2 nM or 2 nM to 10 nM.

[0226] Implementation Scheme B10. A bispecific antibody according to any one of Implementation Schemes B6 to B9, wherein KD is determined by surface plasmon resonance.

[0227] Implementation Scheme B11. A bispecific antibody according to any one of Implementation Schemes B1 to B10, wherein the CD47 binding domain is a human or engineered human CD47 binding domain.

[0228] Implementation Scheme B12. The bispecific antibody according to any one of Implementation Schemes B1 to B11, wherein the CD47 binding domain comprises a heavy chain variable domain and a light chain variable domain.

[0229] Implementation Scheme B13. The bispecific antibody according to any one of Implementation Schemes B1 to B11, wherein the CD47 binding domain includes scFv.

[0230] Implementation Scheme B14. A bispecific antibody according to any one of Implementation Schemes B1 to B13, wherein the EGFR binding domain comprises a heavy chain variable domain and a light chain variable domain.

[0231] Implementation Scheme B15. The bispecific antibody according to any one of Implementation Schemes B1 to B13, wherein the EGFR binding domain includes scFv.

[0232] Implementation Scheme B16. A bispecific antibody according to any one of Implementation Schemes B1 to B15, wherein the Fc domain is a human Fc domain.

[0233] Implementation Scheme B17. The bispecific antibody according to Implementation Scheme B16, wherein the isotype of the human Fc domain is IgG1 or IgG4.

[0234] Implementation Scheme B18. A bispecific antibody according to any one of Implementation Schemes B1 to B17, wherein the Fc domain is a heterodimeric Fc domain, wherein the heterodimeric Fc region includes a club chain and a mortar chain to form a club-mortar (KiH) structure.

[0235] Implementation Scheme B19. The bispecific antibody according to Implementation Scheme B18, wherein the mortar chain comprises the mutant T366W, and the saliva chain comprises the mutants T366S, L368A, and Y407V, wherein the amino acid position numbers are based on the EU index of Kabat et al.

[0236] Implementation Scheme B20. A bispecific antibody according to any one of Implementation Schemes B1 to B19, wherein the bispecific antibody has an asymmetric triple-chain club-and-mortar structure.

[0237] Implementation Scheme B21. The bispecific antibody according to Implementation Scheme B20, wherein the CD47 binding domain is scFv.

[0238] Implementation Scheme B22. The bispecific antibody according to Implementation Scheme B20, wherein the EGFR binding domain is scFv.

[0239] Implementation Scheme B23. The bispecific antibody according to any one of Implementation Schemes B1 to B22, wherein the CD47 binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3; and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein HC-CDR1 comprises SEQ ID NO: 15, HC-CDR2 comprises SEQ ID NO: 16, HC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 17, LC-CDR1 comprises SEQ ID NO: 18, LC-CDR2 comprises SEQ ID NO: 19, and LC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 20.

[0240] Implementation Scheme B24. The bispecific antibody according to Implementation Scheme B23, wherein HC-CDR3 includes amino acid substitutions at one or more of K94, E95, G96, S97, F98, G99, V100b, D101, and P102; and LC-CDR3 includes amino acid substitutions at one or more of Y89, S90, T91, D92, I93, S94, G95, N95a, H95b, W96, and V97.

[0241] Implementation Scheme B25. The bispecific antibody according to any one of Implementation Schemes B1 to B24, wherein the heavy chain variable domain of the CD47 binding domain comprises a sequence having at least 90% identity with SEQ ID NO: 5, SEQ ID NO: 10 or SEQ ID NO: 13, and the light chain of the CD47 binding domain comprises a sequence having at least 90% identity with SEQ ID NO: 8, SEQ ID NO: 11 or SEQ ID NO: 14.

[0242] Implementation Scheme B26. A bispecific antibody according to any one of Implementation Schemes B1 to B25, wherein the EGFR binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein the CDRs are defined by an ordered set of sequences listed as HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3; and wherein the CDRs are selected from sequences having at least 90% identity with the following sequences: SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126.

[0243] Implementation Scheme B27. The bispecific antibody according to any one of Implementation Schemes B1 to B26, wherein the EGFR binding domain comprises: The sequence is at least 90% identical to SEQ ID NO: 127, and The sequence is at least 90% identical to SEQ ID NO: 128.

[0244] Implementation Scheme B28. A bispecific antibody according to any one of Implementation Schemes B1 to B27, wherein the percentage of A431 cells phagocytosed by macrophages is increased by at least 4-fold when the bispecific antibody is less than 1 nM or less than 0.01 nM compared to a nonspecific IgG1 antibody control.

[0245] Implementation Scheme B29. The bispecific antibody according to Implementation Scheme B28, wherein the concentration of the antibody required to mediate antibody-dependent phagocytosis of macrophages against EGFR-positive and CD47-positive tumor cells is between 0.01 nM and -3 nM.

[0246] Implementation Scheme B30. The bispecific antibody according to Implementation Scheme B29, wherein the EGFR-positive, CD47-positive tumor cells are OVISE cells or A431 cells.

[0247] Implementation Scheme B31. The bispecific antibody according to Implementation Scheme B29, wherein the EGFR-positive, CD47-positive tumor cells are selected from epidermal carcinoma cells, colorectal adenocarcinoma cells, pancreatic adenocarcinoma cells, lung squamous cell carcinoma cells, or gastric carcinoma cells.

[0248] Implementation Scheme B32. A bispecific antibody according to any one of Implementation Schemes B1 to B28, wherein 100 nM of the bispecific antibody inhibits the binding of SIRPα to CD47 on the cell surface by at least 30%.

[0249] Implementation Scheme B33. The bispecific antibody according to Implementation Scheme B32, wherein the cells are CD47+EGFR+ tumor cells.

[0250] Implementation Scheme B34. The bispecific antibody according to Implementation Scheme B33, wherein the cells express at least as much EGFR protein on their surface as HCC-44 cells.

[0251] Implementation Scheme B35. The bispecific antibody according to Implementation Scheme B33, wherein the cells express at least 100,000, at least 300,000, at least 600,000, or at least 1,500,000 EGFR proteins on their surface.

[0252] Implementation Scheme B36. A bispecific antibody according to any one of Implementation Schemes B1 to B33, wherein 400 nM of the bispecific antibody does not induce hemolysis of erythrocytes in a hemagglutination assay.

[0253] Implementation Scheme B37. A complex comprising a bispecific antibody according to any one of Implementation Schemes B1 to B36 and CD47+EGFR+ target cells.

[0254] Implementation Scheme B38. The complex according to Implementation Scheme B37, wherein the target cells express at least as much EGFR protein on their surface as HCC-44 cells.

[0255] Implementation Scheme B39. The complex according to Implementation Scheme B37, wherein the target cells express at least 100,000, at least 300,000, at least 600,000, or at least 1,500,000 EGFR proteins on their surface.

[0256] Implementation Scheme B40. The complex according to any one of Implementation Schemes B37 to B39, wherein the CD47+EGFR+ target cells are cancer cells.

[0257] Implementation Scheme B41. The complex according to Implementation Scheme B40, wherein the cancer cells are epidermoid cancer cells, colorectal adenocarcinoma cells, pancreatic adenocarcinoma cells, lung squamous cell carcinoma cells, or gastric cancer cells.

[0258] Implementation Scheme B42. A method for inducing phagocytosis in CD47+EGFR+ target cells, the method comprising administering a bispecific antibody according to any one of Implementation Schemes B1 to B36.

[0259] Implementation Scheme B43. The method according to Implementation Scheme B42, wherein the bispecific antibody is administered at a concentration of less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.1 nM, or less than 0.05 nM.

[0260] Implementation Scheme B44. A method for killing CD47+EGFR+ target cells, the method comprising administering a bispecific antibody according to any one of Implementation Schemes B1 to B36, wherein the bispecific antibody inhibits the binding of SIRPα to CD47 on the surface of CD47+EGFR+ target cells.

[0261] Implementation Scheme B45. A method for killing CD47+EGFR+ target cells, the method comprising administering a bispecific antibody according to any one of Implementation Schemes B1 to B36, wherein the bispecific antibody induces antibody-dependent cytotoxicity that kills CD47+EGFR+ target cells.

[0262] Implementation Scheme B46. The method according to Implementation Scheme B45, wherein the antibody is administered at a concentration of 0.01-1 nM, 0.01-0.5 nM, 0.01-0.25 nM, 0.01-0.1 nM, 0.01-0.05 nM or less than 0.01 nM.

[0263] Implementation Scheme B47. The method according to Implementation Scheme B45 or B46, wherein the CD47+EGFR+ target cells are selected from A431 cells, NCI-H747 cells, ASPC-1 cells, EBC-1 cells or SNU-5 cells.

[0264] Implementation Scheme B48. The method according to Implementation Scheme B45 or B46, wherein the CD47+EGFR+ target cells are selected from epidermal carcinoma cells, colorectal adenocarcinoma cells, pancreatic adenocarcinoma cells, lung squamous cell carcinoma cells, or gastric carcinoma cells.

[0265] Implementation Scheme B49. A drug comprising a bispecific antibody as described in any one of Implementation Schemes B1 to B36.

[0266] Implementation Scheme B50. A method for treating an individual suffering from cancer, the method comprising administering a pharmaceutical composition according to Implementation Scheme B49.

[0267] Implementation Scheme B51. The method according to Implementation Scheme B50, wherein the cancer is epidermoid carcinoma, colorectal adenocarcinoma, pancreatic adenocarcinoma, lung squamous cell carcinoma, or gastric cancer.

[0268] Implementation Scheme C1. A bispecific antibody comprising a CD47 binding domain and a tumor-associated antigen binding domain, wherein the CD47 binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3; and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein HC-CDR1 comprises SEQ ID NO: 15, HC-CDR2 comprises SEQ ID NO: 16, HC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 17, LC-CDR1 comprises SEQ ID NO: 18, LC-CDR2 comprises SEQ ID NO: 19, and LC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 20.

[0269] Implementation Scheme C2. The bispecific antibody according to Implementation Scheme C1, wherein the tumor-associated antigen binding domain binds to a tumor-associated antigen, wherein the tumor-associated antigen includes ACVR2, HER2 / neu, CD20, EGFR, CD3, CD22, CD80, CD23, EpCAM, CD2, CD3, CD19, mesothelin, Mum-1, β -Catalytic protein, CDK4, p53, Ras, CDC27 α - Actin-4, TRP1 / gp75, Wilm, EphA3, prostatic acid phosphatase (PAP), alpha-fetoprotein (AFP), 9D7, cyclin-B1, carcinoembryonic antigen (CEA), gp100 / pmel17, BRCA1 / 2, VEGFR, TGF-β RII, MUC-1, Epithelial Tumor Antigen (ETA), Tyrosinase, Melanoma-associated Antigen (MAGE), Carbonic Anhydrase IX, Cytotoxic T-Lymphocyte Antigen 4, Folate-binding Protein A-33, Prostate-Specific Antigen (PSA), Survivin, EGFRvIII, Melanocyte-derived Peptide, Multiple Melanoma-associated Peptides, Cervical Cancer Antigen HPV-16-E7, PRAME, SSX-2, CA125, MART, CS-1, BING-4, Fibronectin, CML66, MC1R, Calcium-activated Chloride Channel 2, Immature Laminin Receptor, and hTERT.

[0270] Implementation Scheme C3. A bispecific antibody according to Implementation Scheme C1 or C2, wherein the bispecific antibody has a higher affinity for CD47 expressed on the surface of tumor cells than for CD47 expressed on the surface of erythrocytes or platelets.

[0271] Implementation Scheme C4. The bispecific antibody according to Implementation Scheme C3, wherein the bispecific antibody further comprises an Fc domain.

[0272] Implementation Scheme C5. The bispecific antibody according to any one of Implementation Schemes C1 to C4, wherein the concentration of the bispecific antibody required for half-maximal binding to human erythrocytes is greater than 500 nM.

[0273] Implementation Scheme C6. The bispecific antibody according to any one of Implementation Schemes C1 to C5, wherein the bispecific antibody binds to human CD47 with a KD of less than 100 nM.

[0274] Implementation Scheme C7. The bispecific antibody according to Implementation Scheme C6, wherein the bispecific antibody binds to human CD47 with a KD of 1 nM to 100 nM, 1 nM to 50 nM or 5 nM to 50 nM.

[0275] Implementation Scheme C8. A bispecific antibody according to any one of Implementation Schemes C2 to C7, wherein the bispecific antibody binds to the tumor-associated antigen with a KD of less than 500 nM.

[0276] Implementation Scheme C9. The bispecific antibody according to Implementation Scheme C8, wherein the bispecific antibody binds to the tumor-associated antigen at a KD of 0.2 nM to 500 nM, 1 nM to 300 nM, 5 nM to 200 nM or 10 nM to 150 nM.

[0277] Implementation scheme C10. A bispecific antibody according to any one of implementation schemes C6 to C9, wherein KD is determined by surface plasmon resonance.

[0278] Implementation Scheme C11. A bispecific antibody according to any one of Implementation Schemes C1 to C10, wherein the CD47 binding domain is a human or engineered human CD47 binding domain.

[0279] Implementation Scheme C12. The bispecific antibody according to any one of Implementation Schemes C1 to C11, wherein the CD47 binding domain comprises a heavy chain variable domain and a light chain variable domain.

[0280] Implementation Scheme C13. The bispecific antibody according to any one of Implementation Schemes C1 to C11, wherein the CD47 binding domain includes scFv.

[0281] Implementation Scheme C14. A bispecific antibody according to any one of Implementation Schemes C1 to C13, wherein the tumor-associated antigen binding domain includes a heavy chain variable domain and a light chain variable domain.

[0282] Implementation Scheme C15. The bispecific antibody according to any one of Implementation Schemes C1 to C13, wherein the tumor-associated antigen binding domain includes scFv.

[0283] Implementation Scheme C16. The bispecific antibody according to any one of Implementation Schemes C1 to C15, wherein the Fc domain is a human Fc domain.

[0284] Implementation Scheme C17. The bispecific antibody according to Implementation Scheme C16, wherein the isotype of the human Fc domain is IgG1, IgG2 or IgG4.

[0285] Implementation Scheme C18. A bispecific antibody according to any one of Implementation Schemes C1 to C17, wherein the Fc domain is a heterodimeric Fc domain, wherein the heterodimeric Fc region includes a club chain and a mortar chain to form a club-mortar (KiH) structure.

[0286] Implementation Scheme C19. The bispecific antibody according to Implementation Scheme C18, wherein the mortar chain comprises the mutant T366W, and the saliva chain comprises the mutants T366S, L368A, and Y407V, wherein the amino acid position numbers are based on the EU index of Kabat et al.

[0287] Implementation Scheme C20. A bispecific antibody according to any one of Implementation Schemes C1 to C19, wherein the bispecific antibody has an asymmetric triple-chain club-and-mortar structure.

[0288] Implementation Scheme C21. The bispecific antibody according to Implementation Scheme C20, wherein the CD47 binding domain is scFv.

[0289] Implementation Scheme C22. The bispecific antibody according to Implementation Scheme C20, wherein the tumor-associated antigen binding domain is scFv.

[0290] Implementation Scheme C23. A bispecific antibody according to any one of Implementation Schemes C1 to C22, wherein the tumor-associated antigens include: ACVR2, HER2 / neu, CD20, EGFR, CD3, EphA3, and EpCAM.

[0291] Implementation Scheme C24. The bispecific antibody according to any one of Implementation Schemes C1 to C23, wherein HC-CDR3 includes amino acid substitutions at one or more of K94, E95, G96, S97, F98, G99, V100b, D101, and P102; and LC-CDR3 includes amino acid substitutions at one or more of Y89, S90, T91, D92, I93, S94, G95, N95a, H95b, W96, and V97.

[0292] Implementation Scheme C25. The bispecific antibody according to any one of Implementation Schemes C1 to C24, wherein the heavy chain variable domain of the CD47 binding domain comprises a sequence having at least 90% identity with SEQ ID NO: 5, SEQ ID NO: 10 or SEQ ID NO: 13, and the light chain of the CD47 binding domain comprises a sequence having at least 90% identity with SEQ ID NO: 8, SEQ ID NO: 11 or SEQ ID NO: 14.

[0293] Implementation Scheme C26. A bispecific antibody according to any one of Implementation Schemes C23 to C25, wherein the tumor-associated antigen binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein the CDRs are defined by an ordered set of sequences listed as HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3; and wherein the CDRs are selected from sequences having at least 90% identity with the following sequences: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83; SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86; SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89; SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92; SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 95; SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98; SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101; SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104; SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107; SEQ ID NO: 72, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110; SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113; and SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116.

[0294] Implementation Scheme C27. The bispecific antibody according to any one of Implementation Schemes C1 to C26, wherein the tumor-associated antigen binding domain comprises: The sequence is at least 90% identical to SEQ ID NO: 21, and A sequence that is at least 90% identical to SEQ ID NO: 33; The sequence is at least 90% identical to SEQ ID NO: 22, and A sequence that is at least 90% identical to SEQ ID NO: 34; or The sequence is at least 90% identical to SEQ ID NO: 24, and The sequence is at least 90% identical to SEQ ID NO: 36.

[0295] Implementation Scheme C28. A bispecific antibody according to any one of Implementation Schemes C1 to C25, wherein the tumor-associated antigen binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein the CDRs are defined by an ordered set of sequences listed as HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3; and wherein the CDRs are selected from sequences having at least 90% identity with the following sequences: SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126.

[0296] Implementation Scheme C29. The bispecific antibody according to any one of Implementation Schemes C1 to C25, wherein the tumor-associated antigen binding domain comprises: The sequence is at least 90% identical to SEQ ID NO: 127, and The sequence is at least 90% identical to SEQ ID NO: 128.

[0297] Implementation Scheme C30. A bispecific antibody according to any one of Implementation Schemes C1 to C29, wherein the percentage of A431 cells phagocytosed by macrophages is increased by at least 4-fold when the bispecific antibody is less than 1 nM or less than 0.01 nM compared to a nonspecific IgG1 antibody control.

[0298] Implementation Scheme C31. A drug comprising a bispecific antibody as described in any one of Implementation Schemes C1 to C30.

[0299] Implementation Scheme C32. A method for treating an individual suffering from cancer, the method comprising administering a pharmaceutical composition according to Implementation Scheme C31.

[0300] This invention provides, but is not limited to, the following embodiments: 1. A bispecific antibody comprising a CD47-binding domain and an EpCAM-binding domain, wherein the bispecific antibody has a higher affinity for CD47 expressed on the surface of tumor cells than for CD47 expressed on the surface of erythrocytes or platelets.

[0301] 2. The bispecific antibody according to Embodiment 1 further includes an Fc domain.

[0302] 3. The bispecific antibody according to Embodiment 1, wherein the tumor cells express EpCAM.

[0303] 4. The bispecific antibody according to Embodiment 1, wherein the concentration of the bispecific antibody required for half-maximal binding to human erythrocytes is greater than 500 nM.

[0304] 5. The bispecific antibody according to Embodiment 1, wherein the bispecific antibody binds to human CD47 with a KD of less than 100 nM determined by surface plasmon resonance.

[0305] 6. The bispecific antibody according to Embodiment 1, wherein the bispecific antibody binds to human CD47 with a KD of 1 nM to 100 nM, 1 nM to 50 nM or 5 nM to 50 nM as determined by surface plasmon resonance.

[0306] 7. The bispecific antibody according to Embodiment 1, wherein the bispecific antibody binds to EpCAM with a KD of less than 500 nM determined by surface plasmon resonance.

[0307] 8. The bispecific antibody according to Embodiment 1, wherein the bispecific antibody binds to EpCAM with a KD of 0.2 nM to 500 nM, 1 nM to 300 nM, 5 nM to 200 nM or 10 nM to 150 nM as determined by surface plasmon resonance.

[0308] 9. The bispecific antibody according to Embodiment 1, wherein the CD47 binding domain is a human or engineered human CD47 binding domain.

[0309] 10. The bispecific antibody according to Embodiment 1, wherein the CD47 binding domain includes a heavy chain variable domain and a light chain variable domain.

[0310] 11. The bispecific antibody according to Embodiment 1, wherein the CD47 binding domain includes scFv.

[0311] 12. The bispecific antibody according to Embodiment 1, wherein the EpCAM binding domain includes a heavy chain variable domain and a light chain variable domain.

[0312] 13. The bispecific antibody according to Embodiment 1, wherein the EpCAM binding domain includes scFv.

[0313] 14. The bispecific antibody according to Embodiment 2, wherein the Fc domain is a human Fc domain.

[0314] 15. The bispecific antibody according to embodiment 14, wherein the isotype of the human Fc domain is IgG1 or IgG4.

[0315] 16. The bispecific antibody according to embodiment 15, wherein the Fc domain is a heterodimeric Fc domain, wherein the heterodimeric Fc region includes a club chain and a mortar chain to form a club-mortar (KiH) structure.

[0316] 17. The bispecific antibody according to embodiment 16, wherein the mortar chain comprises the mutant T366W, and the saliva chain comprises the mutants T366S, L368A, and Y407V, wherein the amino acid position numbers are based on the EU index of Kabat et al.

[0317] 18. The bispecific antibody according to Embodiment 1, wherein the bispecific antibody has an asymmetric triple-chain club-and-mortar structure.

[0318] 19. The bispecific antibody according to Embodiment 1, wherein the CD47 binding domain is Fab and the EpCAM binding domain is scFv.

[0319] 20. The bispecific antibody according to Embodiment 1, wherein the CD47 binding domain is scFv and the EpCAM binding domain is Fab.

[0320] 21. The bispecific antibody according to Embodiment 1, wherein the CD47 binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3; and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein HC-CDR1 comprises SEQ ID NO: 15, HC-CDR2 comprises SEQ ID NO: 16, HC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 17, LC-CDR1 comprises SEQ ID NO: 18, LC-CDR2 comprises SEQ ID NO: 19, and LC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 20.

[0321] 22. The bispecific antibody according to embodiment 21, wherein HC-CDR3 includes amino acid substitutions at one or more of K94, E95, G96, S97, F98, G99, V100b, D101, and P102; and LC-CDR3 includes amino acid substitutions at one or more of Y89, S90, T91, D92, I93, S94, G95, N95a, H95b, W96, and V97.

[0322] 23. The bispecific antibody according to Embodiment 10, wherein the heavy chain variable domain of the CD47 binding domain includes a sequence having at least 90% identity with SEQ ID NO: 5, SEQ ID NO: 10 or SEQ ID NO: 13, and the light chain of the CD47 binding domain includes a sequence having at least 90% identity with SEQ ID NO: 8, SEQ ID NO: 11 or SEQ ID NO: 14.

[0323] 24. The bispecific antibody according to Embodiment 1, wherein the EpCAM binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein the CDRs are defined by an ordered set of sequences listed as HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3; and wherein the CDRs are selected from sequences having at least 90% identity with the following sequences: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83; SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86; SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89; SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92; SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 95; SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98; SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101; SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104; SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107; SEQ ID NO: 72, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110; SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113; and SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116.

[0324] 25. The bispecific antibody according to Embodiment 1, wherein the EpCAM binding domain comprises: The sequence is at least 90% identical to SEQ ID NO: 21, and A sequence that is at least 90% identical to SEQ ID NO: 33; The sequence is at least 90% identical to SEQ ID NO: 22, and A sequence that is at least 90% identical to SEQ ID NO: 34; or The sequence is at least 90% identical to SEQ ID NO: 24, and The sequence is at least 90% identical to SEQ ID NO: 36.

[0325] 26. The bispecific antibody according to Embodiment 1, wherein the percentage of A431 cells phagocytosed by macrophages is increased by at least 4 times when the bispecific antibody is less than 1 nM, less than 0.1 nM, or less than 0.01 nM compared with a nonspecific IgG1 antibody control.

[0326] 27. The bispecific antibody according to Embodiment 1, wherein the concentration of the antibody required to mediate antibody-dependent phagocytosis of macrophages against EpCAM-positive and CD47-positive tumor cells is between 0.01 nM and -3 nM.

[0327] 28. The bispecific antibody according to embodiment 27, wherein the EpCAM-positive, CD47-positive tumor cells are OVISE cells or A431 cells.

[0328] 29. The bispecific antibody according to Embodiment 27, wherein the EpCAM-positive and CD47-positive tumor cells are selected from pancreatic ductal adenocarcinoma cells, ovarian clear cell adenocarcinoma cells, colonic adenocarcinoma cells, lung adenocarcinoma cells, ovarian adenocarcinoma cells, and vulvar squamous cell carcinoma cells.

[0329] 30. The bispecific antibody according to Embodiment 1, wherein 100 nM of the bispecific antibody inhibits the binding of SIRPα to CD47 on the surface of the tumor cells by at least 30%.

[0330] 31. The bispecific antibody according to Embodiment 1, wherein the tumor cells are CD47+EpCAM+ tumor cells.

[0331] 32. The bispecific antibody according to Embodiment 1, wherein the tumor cells express at least as much EpCAM protein on their surface as HCC-44 cells.

[0332] 33. The bispecific antibody according to Embodiment 1, wherein the tumor cells express at least 100,000, at least 300,000, at least 600,000, or at least 1,500,000 EpCAM proteins on their surface.

[0333] 34. The bispecific antibody according to Embodiment 1, wherein 400 nM of the bispecific antibody does not induce hemolysis of erythrocytes in a hemagglutination assay.

[0334] 35. A bispecific antibody comprising a CD47-binding domain and an EGFR-binding domain, wherein the bispecific antibody has a higher affinity for CD47 expressed on the surface of tumor cells than for CD47 expressed on the surface of erythrocytes or platelets.

[0335] 36. The bispecific antibody according to embodiment 35 further includes an Fc domain.

[0336] 37. The bispecific antibody according to embodiment 35, wherein the tumor cells express EGFR.

[0337] 38. The bispecific antibody according to embodiment 35, wherein the concentration of the bispecific antibody required for half-maximal binding to human erythrocytes is greater than 500 nM.

[0338] 39. The bispecific antibody according to embodiment 35, wherein the bispecific antibody binds to human CD47 with a KD of less than 100 nM determined by surface plasmon resonance.

[0339] 40. The bispecific antibody according to embodiment 35, wherein the bispecific antibody binds to human CD47 with a KD of 1 nM to 100 nM, 1 nM to 50 nM or 5 nM to 50 nM as determined by surface plasmon resonance.

[0340] 41. The bispecific antibody according to embodiment 35, wherein the bispecific antibody binds to EGFR with a KD of less than 500 nM determined by surface plasmon resonance.

[0341] 42. The bispecific antibody according to embodiment 35, wherein the bispecific antibody binds to EGFR with a KD concentration of 0.2 nM to 500 nM, 0.2 nM to 200 nM, 0.2 nM to 20 nM, 0.2 nM to 2 nM, or 2 nM to 10 nM as determined by surface plasmon resonance.

[0342] 43. The bispecific antibody according to embodiment 35, wherein the CD47 binding domain is a human or engineered human CD47 binding domain.

[0343] 44. The bispecific antibody according to embodiment 43, wherein the CD47 binding domain includes a heavy chain variable domain and a light chain variable domain.

[0344] 45. The bispecific antibody according to embodiment 44, wherein the CD47 binding domain includes scFv.

[0345] 46. ​​The bispecific antibody according to embodiment 35, wherein the EGFR binding domain includes a heavy chain variable domain and a light chain variable domain.

[0346] 47. The bispecific antibody according to embodiment 46, wherein the EGFR binding domain includes scFv.

[0347] 48. The bispecific antibody according to embodiment 36, wherein the Fc domain is a human Fc domain.

[0348] 49. The bispecific antibody according to embodiment 36, wherein the isotype of the human Fc domain is IgG1, IgG2 or IgG4.

[0349] 50. The bispecific antibody according to embodiment 36, wherein the Fc domain is a heterodimeric Fc domain, wherein the heterodimeric Fc region includes a club chain and a mortar chain to form a club-mortar (KiH) structure.

[0350] 51. The bispecific antibody according to embodiment 50, wherein the mortar chain comprises the mutant T366W, and the saliva chain comprises the mutants T366S, L368A, and Y407V, wherein the amino acid position numbers are based on the EU index of Kabat et al.

[0351] 52. The bispecific antibody according to embodiment 50, wherein the bispecific antibody has an asymmetric triple-chain club-and-mortar structure.

[0352] 53. The bispecific antibody according to embodiment 35, wherein the CD47 binding domain is scFv.

[0353] 54. The bispecific antibody according to embodiment 35, wherein the EGFR binding domain is scFv.

[0354] 55. The bispecific antibody according to embodiment 35, wherein the CD47 binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3; and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein HC-CDR1 comprises SEQ ID NO: 15, HC-CDR2 comprises SEQ ID NO: 16, HC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 17, LC-CDR1 comprises SEQ ID NO: 18, LC-CDR2 comprises SEQ ID NO: 19, and LC-CDR3 comprises a sequence having at least 50% identity with SEQ ID NO: 20.

[0355] 56. The bispecific antibody according to embodiment 55, wherein HC-CDR3 includes amino acid substitutions at one or more of K94, E95, G96, S97, F98, G99, V100b, D101, and P102; and LC-CDR3 includes amino acid substitutions at one or more of Y89, S90, T91, D92, I93, S94, G95, N95a, H95b, W96, and V97.

[0356] 57. The bispecific antibody according to embodiment 44, wherein the heavy chain variable domain of the CD47 binding domain includes a sequence having at least 90% identity with SEQ ID NO: 5, SEQ ID NO: 10 or SEQ ID NO: 13, and the light chain of the CD47 binding domain includes a sequence having at least 90% identity with SEQ ID NO: 8, SEQ ID NO: 11 or SEQ ID NO: 14.

[0357] 58. The bispecific antibody according to embodiment 35, wherein the EGFR binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and three light chain (LC) CDRs: LC-CDR1, LC-CDR2, and LC-CDR3; wherein the CDRs are defined by an ordered set of sequences listed as HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3; and wherein the CDRs are selected from sequences having at least 90% identity with the following sequences: SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126.

[0358] 59. The bispecific antibody according to embodiment 35, wherein the EGFR binding domain comprises: The sequence is at least 90% identical to SEQ ID NO: 127, and The sequence is at least 90% identical to SEQ ID NO: 128.

[0359] 60. The bispecific antibody according to embodiment 35, wherein the percentage of A431 cells phagocytosed by macrophages is increased by at least 4 times when the bispecific antibody is less than 1 nM or less than 0.01 nM compared with a nonspecific IgG1 antibody control.

[0360] 61. The bispecific antibody according to embodiment 35, wherein the concentration of the bispecific antibody required to mediate antibody-dependent phagocytosis of macrophages against EGFR-positive and CD47-positive tumor cells is between 0.01 nM and -3 nM.

[0361] 62. The bispecific antibody according to embodiment 61, wherein the EGFR-positive, CD47-positive tumor cells are OVISE cells or A431 cells.

[0362] 63. The bispecific antibody according to Embodiment 61, wherein the EGFR-positive, CD47-positive tumor cells are selected from epidermoid carcinoma, colorectal adenocarcinoma, pancreatic adenocarcinoma, lung squamous cell carcinoma, or gastric cancer.

[0363] 64. The bispecific antibody according to embodiment 35, wherein 100 nM of the bispecific antibody inhibits the binding of SIRPα to CD47 on the cell surface by at least 30%.

[0364] 65. The bispecific antibody according to embodiment 64, wherein the cells are CD47+EGFR+ tumor cells.

[0365] 66. The bispecific antibody according to embodiment 65, wherein the cells express at least as much EGFR protein on their surface as LS1034 cells.

[0366] 67. The bispecific antibody according to embodiment 65, wherein the cells express at least 50,000, at least 100,000, at least 300,000, at least 600,000, or at least 1,500,000 EGFR proteins on their surface.

[0367] 68. The bispecific antibody according to embodiment 35, wherein 400 nM of the bispecific antibody does not induce hemolysis of erythrocytes in a hemagglutination assay.

[0368] 5. Examples These embodiments are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0369] 5.1 Example 1. Standard Antibody and Reagents The sequences of the baseline antibodies and reagents used in this study were resolved from patent literature and cloned into the human IgG1 backbone, as shown in Table 1.

[0370] Table 1. Standard reagents. 5.2 Example 2. CD47+EpCAM+ cell line Tumor-derived cell lines expressing CD47 and / or EpCAM were obtained from the American Type Culture Collection (ATCC) and grown in medium containing 10% fetal bovine serum at 37°C in an incubator with 5% CO2. CFPAC-1 cells were cultured in IMDM medium, Raji cells in RPMI-1640 medium, and A431 cells in DMEM medium.

[0371] The expression of CD47 and EpCAM on the cell surface was measured by flow cytometry. Tumor cells were harvested, centrifuged, and measured at 1×10⁻⁶. 6 Resuspend the cells in FACS buffer (PBS + 2% FBS) at a concentration of 100 μL / mL. Dispense 100 μL of the cell suspension into each well of a 96-well plate, add 100 μL of 10 μg / mL CD47 BMK-1 or EpCAM BMK-6 to each well, and incubate at 4°C for 1 hour. Wash the cells three times with FACS buffer. After the third wash, resuspend the cells in 100 μL of Alexa Fluor-488 mouse anti-human IgG1 Fc secondary antibody (Invitrogen, catalog number A10631) diluted 1:500 and incubate at 4°C in the dark for 1 hour. Wash the cells three times with 200 μL of PBS by centrifugation at 2000 RPM for 5 minutes. After the final wash, resuspend the cells in 300 μL of cold PBS and incubate in FACS buffer. TM Analyzed using a flow cytometer from BD Biosciences. Table 2 shows the relative cell surface expression levels of CD47 and EpCAM in these cell lines.

[0372] Table 2. Cancer cell lines used in this study and their CD47 and EpCAM expression levels. N / A: Not available Puro et al., Molecular Cancer Therapeutics, published online on December 26, 2016.

[0373] ^Casaletto et al., PNAS 116(15) 7533-7542, March 2019.

[0374] Stable human CD47 cell lines were generated by transfecting CHOK1 cells with the pLVX-IRES-Puro lentiviral expression vector (Clontech, catalog number 632183) encoding the full-length human CD47 gene.

[0375] 5.3 Example 3. Bispecific CD47×EpCAM antibody induces phagocytosis in CD47+EpCAM+ cells A bispecific antibody was constructed using a CD47-binding domain derived from CD47 BMK-2, an EpCAM-binding scFv domain derived from EpCAM BMK-5, and an IgG1 Fc domain. This bispecific antibody possesses an asymmetric club-and-mortar structure that inhibits the formation of heavy chain dimers (Carter, J. Immunol. Protein Engineering, Vol. 9, No. 7, pp. 617-621, 1996). This antibody form is referred to as triple club-and-mortar (3C-KIH) CD47×EpCAM scFv (…). Figure 1 Additional bispecific antibody design is described in Klein et al., mAbs 4(6) 653-663, 2012. The amino acid sequences of the polypeptide components of this antibody are shown in Table 3.

[0376] Table 3. Peptide chain sequences of the proof-of-concept CD47×EpCAM bispecific antibody. Bispecific antibodies were tested in an antibody-dependent cytophagy (ADCP) assay. Peripheral blood mononuclear cells (PBMCs) were isolated from human donors. PBMCs were enriched using a human mononuclear cell enrichment kit without CD16 depletion (STEMCELL, catalog 19058). The isolated PBMCs were differentiated into macrophages by culturing them in complete medium (RPMI 1640 + 10% FBS) containing 20 ng / ml human macrophage colony-stimulating factor (M-CSF, Peprotech, catalog 3-25-10). The medium was changed every three days. After culturing in M-CSF-containing medium for 7 days, macrophages were collected and counted. Target tumor cells were collected and washed twice with D-PBS to remove residual FBS. The washed tumor cells were cultured at 5-10 × 10⁶ cells / mL. 6 Cells were resuspended in PBS at a concentration of 1 / mL. Cancer cells were stained with CFSE (ebiosciences, catalog number 65-0580-84) to a final concentration of 3 μM and mixed immediately. Cells were stained in the dark at room temperature for 10 minutes. Staining was terminated by adding 4–5 volumes of cold complete culture medium and incubating on ice for 5 minutes. The stained cells were washed three times with RPMI 1640 + 10% FBS. Cells were resuspended in 1 ml of RPMI 1640 + 10% FBS, counted, and then diluted or concentrated to 3 × 10⁶ cells / mL. 5Cells / mL. 50 μl of cells were seeded into 96-well deep U-shaped plates (Axygen, catalog number P-DW-20-C), with each well containing 1.5 × 10⁶ cells / mL. 4 Add 50 μl of diluted antibody to each well. Add 100 μl of macrophages (1.5 × 10⁶ cells) to each well. 4 Cells were incubated at 37°C and 5% CO2 for 1.5 hours. After incubation, the cells were washed once with 2 ml of 2% FBS in D-PBS. 100 μl of diluted Fc blocking agent (human TruStain FcX (Fc receptor blocking solution), Biolegend catalog number 422302) was added, and the cells were incubated at room temperature for 10 minutes. 20 μl of diluted anti-human CD11b antibody was added to each well, and the cells were incubated in the dark at 4°C for 30 minutes. The cells were washed once with 2% FBS-D-PBS. Phagocytosis was detected by flow cytometry by the presence of CFSE / CD11b double-positive cells, which indicated macrophages phagocytosing tumor cells.

[0377] Bispecific antibodies promoted phagocytosis in CFPAC-1 pancreatic cancer cells, OVISE ovarian cancer cells, CW-2 colon cancer cells, and HCC44 lung cancer cells. Exemplary results for CFPAC-1 cells are shown in [the original text]. Figure 2 The results are presented in Table 4. A summary of the four cell lines with additional controls is presented in Table 4.

[0378] Table 4. Antibody-dependent phagocytosis induced by bispecific CD47×EpCAM antibody in CD47+ and EpCAM+ cancer cell lines. nd: Not detected In all four cell lines, the bispecific antibody promoted phagocytosis more effectively than the bivalent anti-CD47 antibody with the same anti-CD47 binding domain. The nonspecific IgG1 isotype control antibody did not promote phagocytosis in any of the tested CD47+ cells.

[0379] EpCAM BMK-5 (both monovalent and bivalent) also promotes phagocytosis by directly facilitating it through its IgG1 domain interacting with Fcγ receptors on phagocytes. This effect is more pronounced in OVISE and CW-2 cells with high EpCAM expression.

[0380] 5.4 Example 4. Engineering Modification and Testing of CD47-Resistant Binding Domains VIR47 is an anti-CD47 antibody identified in OmniRat® rats. Compared to the sequence of natural human IgG4, the variable domain of VIR47 was transplanted into a sequence with S... 228 P-substituted human IgG4 framework (IgG4.SPL). VIR47 effectively blocks the interaction between CD47 and SIRPα. VIR47 mAb has a relatively weak binding to RBCs (with little ability to induce reduced phagocytosis of RBCs) and does not induce hemagglutination of human RBCs. VIR47 is described in PCT / CN2019 / 113296 (which is incorporated herein by reference in its entirety).

[0381] To enhance Fcγ receptor interaction, the IgG4 Fc domain of VIR47 was replaced with the IgG1 Fc domain. Furthermore, the framework region of VIR47 was further engineered by replacing rat-derived amino acids with amino acids found at corresponding positions in the sequence of the closest human germline ortholog. According to Kabat numbering (EU index of Sequences of Proteins of Immunological Interest, Kabat et al., 1991), two of these optimized VIR47 variants are known as VIR47.V7 (HC:F...). 79 Y, G 82b S;LC:E 103 K) and VIR47.V8 (HC:I) 67 F, F 79 Y, G 82b S; LC: M4L, E 103 K). The heavy and light chain sequences of VIR47 and related mAbs are shown in Table 5. The complementarity-determining region (CDR) of VIR47 is presented in Table 6.

[0382] Table 5. Peptide sequences of the anti-CD47 binding domain derived from VIR47. CDRs are underlined and bolded. Table 6. CDR sequence of VIR47 IgG1 forms and engineered variants were characterized by ELISA-based binding assays, flow cytometry quantification of binding to CD47-expressing cells, and binding kinetics measurements via surface plasmon resonance using Biacore instruments.

[0383] 5.4.1 Production of human CD47 DNA encoding the extracellular domain (ECD) of human CD47 was cloned into pRK5 (ATCC catalog number 209784). The resulting construct with a C-terminal 6xHis tag (SEQ ID NO: 129) was transfected into Expi293F cells. After 72 hours, CD47-expressing cells were harvested by centrifugation at 2000 rpm for 5 minutes at 4°C. The supernatant was collected. Ni-NTA (Qiagen, catalog number 30410) resin was pre-equilibrated with buffer A (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and incubated with the supernatant at 4°C for 2 hours on a vortex mixer. The resin was packed into a NiSepharose excel (General Electric (GE), catalog GE17371201) and washed with buffer A until no signal was observed through Coomassie-Brilliant Blue G-250 (OD595, approximately 20–30 column volumes (CV)). The target protein was eluted using 3 column volumes (CV) of buffer B (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, 250 mM imidazole). A SuperdexTX 200 increase column (General Electric, catalog GE28-9909-44) was pre-equilibrated with buffer A, and then the eluent was loaded onto the column. The column was washed with buffer A, and the fraction was collected. Different fractions were separated on 12% SDS-PAGE, and the desired fractions were combined and neutralized with buffer C (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). The target protein was concentrated using ultrafiltration tubes with a molecular cutoff of 30 kDa (Amicon, catalog number 42409), then aliquoted and rapidly frozen with liquid N2 and stored at -80°C.

[0384] 5.4.2 Antibody-antigen binding measured by ELISA A 96-well plate was coated overnight at 4°C with 1 μg / ml recombinant CD47. After washing three times, the plate was blocked for 1 hour at 37°C with 300 μl of 1% BSA in PBST. Serially diluted antibody was added and incubated at 37°C for 1 hour. The plate was then washed four times with PBST and incubated at 37°C for 1 hour with a 1:5000 dilution of peroxidase-labeled goat anti-human IgG (Fab-specific) secondary antibody (Sigma, catalog number A0293). The plate was washed four times again with PBST, incubated at room temperature with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate for 15 minutes, terminated with 1N HCl, and read at 450 nM. By ELISA, VIR47 and its IgG1, V7, and V8 variants showed comparable binding to human CD47.

[0385] 5.4.3 Antibody binding to cells as measured by flow cytometry Antibodies binding to the CD47+ cell line were quantified by flow cytometry. On the cells, CD47 ECDs exist in their native orientation and are naturally glycosylated. Harvested cells were centrifuged at 2000 rpm for 5 minutes, resuspended in 10-15 ml of ice-cold culture medium, and then counted. Cells were resuspended in a 3×10⁻⁶ medium solution. 6 Cells / mL of blocking buffer (PBS plus 2% FBS). Dispense 100 μL of cell suspension into each well of a 96-well plate. Dilute the purified antibody to the desired concentration with blocking buffer and add 100 μL of the diluted antibody to each well, incubating at 4°C for 1 hour. Then wash the cells three times with PBS plus 2% FBS. After the third wash, resuspend the cells in 100 μL of 1:500 diluted Alexa Fluor488-labeled mouse anti-human IgG1 Fc secondary antibody (Invitrogen, catalog number A10631) and incubate at 4°C in the dark for 1 hour. Then wash the cells three times with 200 μL PBS by centrifugation at 2000 rpm for 5 minutes. After the final wash, resuspend the cells in 300 μL of cold PBS and incubate in a FACSVerse... TM Analyzed on a flow cytometer (BD Biosciences).

[0386] In flow cytometry assays, VIR47 and its IgG1, V7 and V8 variants bind to CHO cells expressing human CD47 with comparable affinity.

[0387] 5.4.4 Surface Plasmon Resonance Binding Measurement Binding kinetics of the anti-CD47 antibody were evaluated on a Biacore 8K instrument (GE Healthcare). Biacore Series SCM5 sensor chips were immobilized with monoclonal mouse anti-human IgG (Fc) antibody (from GE Healthcare's Human Antibody Capture Kit). Antibodies were captured in each flow cell. Serial 3-fold dilutions of each antigen were injected at a flow rate of 30 μL / min. Each sample was analyzed at room temperature (25°C) for 1 min of binding and 10 min of dissociation. After each injection, the chip was regenerated with 3M MgCl2. Simultaneous fitting of k... 开 and k 关 The 1:1 Langmuir model was used for kinetic analysis. The monomeric antigen flowing through immobilized antibody produces binding results unaffected by antibody titer.

[0388] Through surface plasmon resonance, VIR47 and its IgG1, V7, and V8 variants exhibit similar binding affinities to human CD47, with KD values ​​ranging from 5 to 50 nM. CD47 BMK-1 binds to human CD47 with a KD value ranging from 1 to 5 nM.

[0389] 5.4.5 Antibody-dependent phagocytosis ADCP activity of the IgG1 form of VIR47 and its engineered variants was tested on OVISE ovarian cancer cells and compared with the benchmark αCD47 antibody. The three forms of VIR47 exhibited similar ADCP activity to each other, but their activity was lower than the BMK-1 benchmark.

[0390] 5.4.6 Antibody-dependent cytotoxicity The ability of anti-CD47 antibody to mediate antibody-dependent cytotoxicity (ADCC) was tested on CFPAC-1 pancreatic cancer cells expressing high levels of CD47. Cells were washed once with balanced salt solution or culture medium and the cell number was adjusted to 1 × 10⁻⁶. 6 Cells / ml. Then, 2 μL of BATDA fluorescence-enhancing ligand (PerkinElmer, catalog number C136-100) was added to each milliliter of cells, and the cells were incubated at 37°C for 20 minutes. After incubation, the cells were centrifuged and the culture medium was aspirated. The labeled cells were washed four times with PBS. After the final wash, the cells were resuspended in the culture medium and adjusted to 5 × 10⁶ cells / ml. 4 Cells / ml. Then 200 μL of cell suspension was added to each well of the 96-well plate to achieve a cell count of 1 × 10⁶ cells / ml per well. 4Background release was determined by removing aliquots of labeled target cells, centrifuging, and transferring the supernatant to empty wells. The readings represent background release. (1×10⁻⁶) 4 Transfer labeled target cells to a sterile 96-well assay plate. Serially dilute the antibody with RPMI-1640 containing 10% FBS. Add 50 μL of serially diluted antibody to the assay plate containing target cells and incubate at 37°C and 5% CO2 for 5–10 minutes. Harvest effector cells NK92 / CD16a176V and resuspend them in RPMI-1640 containing 10% FBS. Add 50 μL / well of effector cells to each well of the assay plate at different effector-to-target ratios. Set up controls: spontaneous target (target cells + 100 μL medium); maximum target (target cells + 100 μL medium + 10 μL lysis buffer); background (100 μL labeled target cell supernatant and 100 μL diluted medium). Incubate the plate at 37°C in a humid 5% CO2 atmosphere for 2 hours. At the end of the incubation, 10 μL of lysis buffer (Perkin Elmer, catalog number 4005-0010) was added to the largest release well. The plate was centrifuged at 500 g for 5 minutes. 20 μL of supernatant from each well was transferred to a flat-bottomed detection plate. Then, 200 μL of europium solution (Perkin Elmer, catalog number C135-100) was added to each well of the detection plate. The plate was shaken at 250 rpm for 15 minutes at room temperature, and fluorescence was measured using a time-resolved fluorometer over 5 hours.

[0391] VIR47 and its IgG1, V7, and V8 variants exhibit considerable ADCC activity. As expected, the IgG4 form is essentially inactive.

[0392] 5.4.7 Binding with red blood cells and hemagglutination It is desirable to have a CD47 binding domain that binds to the form of CD47 expressed on tumor cells, rather than the form expressed on red blood cells or platelets, to enhance the safety profile of the therapeutic agent.

[0393] RBC binding was determined by centrifuging fresh human whole blood at 200 g for 10 minutes. Collected RBCs were washed twice with PBS and counted using flow cytometry. 1 × 10⁻⁶ RBCs were... 6Cells were aliquoted into each well of a 96-well plate. Serially diluted anti-CD47 antibody was added, and the plates were incubated at 4°C for 1 hour. Cells were washed twice with FACS buffer (PBS + 2% FBS). Secondary antibody (Alexa Fluor® 488 goat anti-human IgG (H+L)) was added, and the plates were incubated at 4°C for 1 hour. Cells were washed twice, resuspended in 200 μL of FACS buffer, and analyzed by flow cytometry.

[0394] Hemagglutination assays were performed by diluting human erythrocytes (RBCs) and titrating them with CD47 antibody (starting at 100 μg / ml) in round-bottom 96-well plates, followed by incubation at 37°C for 2 hours. Hemagglutination was demonstrated by the presence of cross-linked RBCs, which appeared as a mist because they did not settle to the bottom of the wells, compared to non-hemagglutinated RBCs.

[0395] VIR47 was chosen for its low RBC binding and lack of hemagglutination activity. These properties are shared by the IgG1 form and engineered variants. In particular, >20-fold higher concentrations of VIR47 and its variants are required for RBC binding compared to the CD47 BMK-1 benchmark. Furthermore, hemagglutination of any VIR47 variant was not detected even at the highest tested concentration (666.7 nM).

[0396] 5.5 Example 5. Alanine scan of VIR47 To optimize the binding affinity of VIR47 anti-CD47 antibody, a variant of VIR47 in the IgG1 form was generated by scanning mutagenesis of the alanine residues in the heavy and light chain CDR3 domains, such as... Figure 3 A and Figure 3 As shown in B. The binding of these variants to human CD47 was measured by ELISA and compared with the parental VIR47 IgG1 antibody, as shown in Table 7.

[0397] Table 7. Determination of binding of the CDR3 alanine scanning variant of αCD47 mAb VIR47 to human CD47 by ELISA. EC50 range for huCD47: A: 0.01 nM–0.15 nM; B: 0.15 nM–0.4 nM; C: 0.4 nM–5 nM; D: ≥5 nM. 5.6 Example 6. Engineering Modification and Testing of the EpCAM-Resistant Bonded Domain Anti-EpCAM antibodies were generated by immunizing mice with a human EpCAM protein (Novoprotein, catalog number CM50) with a C-terminal Fc tag and a cyno-EpCAM protein (Sino Biological, catalog number 90299-C02H) with a C-terminal Fc tag. Mouse hybridomas were generated using B cells using the method described in U.S. Patent No. 5,597,725. Initial screening was performed using human EpCAM with a C-terminal His tag by ELISA. Secondary screening was performed by ELISA for human EpCAM with a C-terminal His tag (Novoprotein, catalog number C339). Antibody binding was also tested by flow cytometry analysis of CHO-K1 cells expressing or not expressing human EpCAM. Hybridomas expressing antibodies specific to human EpCAM were used to generate chimeric antibodies.

[0398] 5.6.1 Sequence of the anti-EpCAM binding domain Chimeric antibodies were generated by fusing the EpCAM-specific mouse VH region with the human IgG1 constant region. Binding was assessed by ELISA and by flow cytometry. The baseline antibody from these studies was EpCAM BMK-6, and Tables 8-11 summarize the polypeptide sequences of the binding domains of the heavy and light chains and their corresponding CDRs.

[0399] Table 8: Peptide sequences against the EpCAM heavy chain binding domain. CDRs are underlined and bolded. Table 9: Peptide sequences against the EpCAM light chain binding domain. CDRs are underlined and bolded. Table 10: CDR sequence anti-EpCAM heavy chain binding domain. Table 11: CDR sequence anti-EpCAM light chain binding domain. 5.6.2 Binding affinity against EpCAM binding domains The chimeric anti-EpCAM antibody binds to human EpCAM, with EC50 values ​​ranging from 0.1 nM to 1 nM as determined by ELISA.

[0400] The binding of chimeric antibodies to EpCAM on the cell surface was tested by flow cytometry using CHO-K1 cells transfected with human EpCAM. mAb-1, mAb-2, mAb-8, mAb-8, mAb-28, mAb-30, mAb-33, mAb-35, mAb-36, mAb-39, and EpCAMBMK-6 bound to CHO-K1-huEpCAM cells, with EC50 values ​​ranging from 0.5 nM to 10 nM.

[0401] The ability of selected chimeric antibodies to bind to the surface of tumor cell lines expressing EpCAM was also tested. In flow cytometry assays, mAb-1, mAb-2, mAb-8, mAb-21, and EpCAM BMK-6 bound to MCF-7, Caco-2, and A431 cells with EC50 less than 20 nM.

[0402] The binding affinity of chimeric antibodies to the extracellular domain (ECD) of human EpCAM (Novoprotein #C339) was measured using surface plasmon resonance. In this assay, mAb-1, mAb-2, mAb-4, mAb-8, and EpCAM BMK-6 exhibited binding philosophies ranging from 2 nM to 10 nM, while mAb-28, mAb33, and mAb35 showed binding philosophies ranging from 0.2 nM to 2 nM.

[0403] 5.7 Example 7. Bispecific CD47×EpCAM antibody with engineered binding domain 5.7.1 Bispecific antibody structure Bispecific antibodies in a three-chain mortar and pestle form were constructed using an engineered scFv CD47-binding domain derived from VIR47.V8 and an EpCAM-binding domain including Mab-1, Mab-2, or Mab-8. The sequences of the heavy chain peptides of these antibodies are shown in Table 12, and the peptide composition of the 3C-KIH EpCAM×CD47 scFv bispecific antibody is shown in Table 13.

[0404] Table 12. Heavy chain polypeptide sequence of 3C-KIH EpCAM-CD47 scFv bispecific antibody. Table 13. Peptide composition of the 3C-KIH EpCAM-CD47 scFv bispecific antibody. 5.7.2 Binding Activity The binding of these bispecific antibodies to human CD47 and human EpCAM was compared with the binding of bivalent antibodies with the same binding domain by ELISA. As expected, the Bi-1, Bi-2, and Bi-8 CD47×EpCAM bispecific antibodies bound to both CD47 and EpCAM. Figure 4 shows the binding of Bi-1 to CD47 (… Figure 4A ) and EpCAM Figure 4B The combination of ).

[0405] 5.7.3 Inhibition of CD47 binding to immobilized SIRPα The inhibition of CD47 binding to immobilized SIRPα protein was tested by ELISA. 96-well plates were coated with 100 μl of 1 μg / ml CD47 BMK-4 in PBS per well and incubated overnight at 4°C. The next day, the plates were blocked for 1.5 hours at 37°C with 300 μl of blocking buffer (1% BSA in PBST) per well. The plates were then washed three times with PBST. Serial 1:5 dilutions of mAb were prepared starting at 30 μg / ml and mixed with huCD47-His (0.11 μg / ml) in a total of 50 μl of blocking buffer, and added to the coated plates. After incubation at 37°C for 1 hour, the plates were washed three times with PBST. 100 μl of THETM His antibody [HRP] (Genscript, catalog number A00612) diluted 1:5000 was added to each well, and the plates were incubated at 37°C for 45 minutes. 100 μl of TMB (InnoReagents, catalog number TMB-S-003) was added to each well, and the reaction was developed at room temperature for 5 minutes. The reaction was quenched using 50 μl of 1N HCl. The reaction products on the plate were quantified at 450 nM.

[0406] The ability of the CD47×EpCAM bispecific antibody to inhibit the binding of CD47 to SIRPα was measured by ELISA and compared with that of CD47 BMK-1. Bi-1, Bi-2, and Bi-8 bispecific antibodies inhibited the binding of SIRPα to CD47 protein. The CD47 BMK-1 antibody, possessing two CD47-binding domains, inhibited the binding of SIRPα to CD47 protein at concentrations 5 to 50 times lower than that of the bispecific antibodies.

[0407] 5.7.4 Inhibition of SIRPA binding to CD47+ cells Harvest tumor cells, centrifuge, and then at 2×10⁻⁶. 6Resuspend the cells at a concentration of 100 cells / mL in FACS buffer (PBS plus 2% FBS). Dispense 100 μL of the cell suspension into each well of a 96-well plate. Centrifuge the plate at 300 g for 5 min and discard the supernatant. Incubate the cells in FACS buffer at 4°C for 1 h with 50 μL per well of serially diluted bispecific or bivalent anti-CD47 antibody and a constant amount of SIRPα-mIgG2a fusion protein (0.2 μg / mL for Raji cells; 2.5 µg / mL for OVISE cells). Then, wash the plate twice with FACS buffer and incubate in the dark at 4°C for 1 h with 100 µL of Alexa Fluor488 donkey anti-mouse IgG (H+L) secondary antibody (Invitrogen, catalog number A21202, 1:1000). After washing twice with FACS buffer, resuspend the plate in 300 μL of FACS buffer and analyze by flow cytometry.

[0408] The bispecific antibody Bi-2 inhibited the binding of SIRPα to CD47+ / EpCAM+ OVISE cells, with an EC50 lower than that of αCD47 VIR47.V8 (a bivalent antibody with the same CD47 binding domain). Figure 5A In contrast, Bi-2 and Bi-8 were much less effective at inhibiting the binding of SIRPα to Raji cells, which are CD47+ but express >40-fold less EpCAM than OVISE cells. Figure 5B (See Table 2). These results demonstrate the importance of EpCAM expression for the specific targeting of CD47+ / EpCAM+ cells by the bispecific CD47×EpCAM antibody, and the ability of the bispecific CD47×EpCAM antibody to inhibit CD47 / SIRPa interaction on CD47+ / EpCAM+ cells.

[0409] 5.7.5 Phagocytosis of CD47+EpCAM+ cancer cell lines The ability of Bi-1, Bi-2, and Bi-8 to promote phagocytosis of A431 vulvar cancer cells was tested in an ADCP assay and compared with bivalent IgG1 antibodies having the same CD47 or EpCAM binding domain. All three bispecific antibodies promoted phagocytosis of A431 cells. Phagocytosis was increased >10-fold compared with the nonspecific IgG1 control. The Bi-1, Bi-2, and Bi-8 bispecific antibodies had lower EC50 values ​​(0.01–3 nM) than bivalent αCD47 antibodies (>10 nM) with the same anti-CD47 binding domain. The results for Bi-2, as an example, were... Figure 6As shown in the figure, anti-EpCAM mAb may function independently of the CD47-SIRPα do-not-eat-me signaling and promote phagocytosis by directly interacting with Fc receptors on phagocytes.

[0410] 5.7.6 Off-target binding to the reduction of non-tumor cells The extratumor effects of the Bi-1, Bi-2, and Bi-8 CD47×EpCAM bispecific antibodies were tested in hemagglutination and flow cytometry assays. Figure 7 As shown, compared with the CD47 BMK-1 benchmark antibody, the bivalent and bispecific antibody with a CD47 binding domain derived from VIR47 does not induce hemagglutination of RBCs.

[0411] In flow cytometry assays, no binding of Bi-1, Bi-2, or Bi-8 to RBCs was detected at concentrations up to 500 nM. Significant binding of the CD47 BMK-1 benchmark antibody was detected at approximately 1 nM EC50. The parental bivalent anti-CD47 antibody VIR47.V8 mAb exhibited significantly lower binding than CD47 BMK-1 and even lower binding than the CD47 BMK-3 benchmark antibody at lower concentrations. As an example, Bi-2 showed similar results. Figure 8A As shown in the image.

[0412] On platelets, adequate amounts of Bi-1, Bi-2, and Bi-8 binding were detected at concentrations of 100 nM and higher, such as Figure 8B As shown, Bi-1, Bi-2, and Bi-8 have less platelet binding than any of the anti-CD47 benchmarks (including CD47 BMK-1 and CD47 BMK-3 mAb). Furthermore, the parental anti-CD47 VIR47.V8 antibody has a lower affinity than both benchmark anti-CD47 antibodies.

[0413] 5.7.7 Antibody-dependent cytotoxicity CD47×EpCAM bispecific antibodies can also mediate their therapeutic effects through antibody-dependent cytotoxicity (ADCC). ADCC activity was measured using cancer cell lines with different CD47 and EpCAM expression levels, as shown in Table 14. Bi-1, Bi-2, and Bi-8 exhibited ADCC activity in all tested CD47+EpCAM+ cell lines. Bi-8, with the highest EpCAM affinity, showed the highest ADCC activity. The parental anti-CD47 antibody VIR47.V8 had lower ADCC than Bi-8 in all cell lines and lower ADCC activity than Bi-2 in all cell lines except SKOV-3 ovarian cancer cells, exhibiting moderate EpCAM affinity. SKOV-3 ovarian cancer cells showed relatively higher CD47 expression and lower EpCAM expression.

[0414] Table 14. Antibody-dependent cytotoxic activity of bispecific and bivalent antibodies against cancer cell lines. Results are expressed as EC50 (nM) values. nd: Undetermined; N / A: Not applicable (not tested) 5.8 Example 8: Generation of anti-CD47 / anti-EGFR bispecific antibody Using sequences derived from the anti-CD47 antibody VIR47.V8 and the anti-EGFR antibody cetuximab, V H and V LSequences were derived, and anti-CD47 / EGFR bispecific antibodies with different binding stoichiometry and geometries were designed and generated. The corresponding heavy chain (HC) and light chain (LC) DNAs were synthesized and cloned into the pRK5 mammalian expression vector (ATCC). Each HC and LC pair was then co-transfected into CHO cells. Conditioned medium was harvested by centrifugation (4°C, 4000 rpm for 40 min) followed by filtration to remove cell debris. The clarified medium was loaded onto a MabSelect SuRe column (GE, 17-5438), which was pre-equilibrated with buffer A (25 mM Tris, 150 mM NaCl, pH 8.0). The column was washed sequentially with 5 column volumes of buffer A, then with 30 column volumes of buffer B (buffer A + 0.1% Triton X100 + 0.1% Triton X114), and finally with 15 column volumes of buffer A. The antibody was eluted with buffer C (100 mM sodium citrate, 150 mM NaCl, pH 3.0) and immediately neutralized with buffer D (200 mM arginine, 137 mM succinate, pH 5.0). The final product was dialyzed against PBS buffer at pH 7.4, concentrated, and filtered through a Millex-MP 0.22 μm (MILLIPORE) filter.

[0415] Figures 9A-9B The bispecific formats generated for evaluation and referred to as cetuximab.Fab / VIR47.V8.scFv. bispecific (9A) and VIR47.V8.Fab / cetuximab. bispecific (9B) are shown, and the amino acid and nucleotide sequences are provided in Tables 15-17.

[0416] Table 15. CDR sequences of cetuximab (anti-EGFR) as defined by Kabat. Table 16. Cetuximab.Fab / VIR47.V8 scFv. Bispecific Table 17. VIR47.V8.Fab / Cetuximab.scFv.Bispecific 5.9 Example 9: Antibody-antigen binding measured by ELISA 96-well plates were coated overnight at 4°C with 1 µg / ml recombinant huCD47 or huEGFR. After washing three times, the plates were blocked for 1 hour at 37°C with 300 µl of 1% BSA in PBST. Serially diluted antibodies were added and incubated at 37°C for 1 hour. The plates were then washed four times with PBST and incubated for 1 hour at 37°C with 1:5000 diluted peroxidase-labeled goat anti-human IgG (Fab-specific) secondary antibody (Sigma, catalog number A0293). The plates were washed four times again with PBST, incubated for 15 minutes at room temperature with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate, terminated with 1N HCl, and read at 450 nM. Figures 10A-10B The ELISA binding results of the anti-CD47 / EGFR bispecific antibody on human CD47 and human EGFR are shown respectively.

[0417] 5.10 Example 10: Inhibition of SIRPα binding to CD47+ cells Harvest tumor cells, centrifuge, and then at 2×10⁻⁶. 6 Resuspend the cells at a concentration of 100 cells / mL in FACS buffer (PBS plus 2% FBS). Dispense 100 µL of the cell suspension into each well of a 96-well plate. Centrifuge the plate at 300 g for 5 min and discard the supernatant. Incubate the cells with 50 µL per well of serially diluted bispecific or bivalent anti-CD47 antibody and a constant amount of SIRPα-mIgG2a fusion protein (1 µg / mL for A431 cells) in FACS buffer at 4 °C for 1 h. Then, wash the plate twice with FACS buffer and incubate with 100 µL of Alexa Fluor 488 donkey anti-mouse IgG (H+L) secondary antibody (Invitrogen, catalog number A21202, 1:1000) in the dark at 4 °C for 1 h. After washing twice with FACS buffer, resuspend the plate in 300 µL of FACS buffer and analyze by flow cytometry.

[0418] Figure 11 The results of SIRPα blockade by the anti-CD47 / EGFR bispecific antibody on A431 cells are shown, demonstrating high expression of both CD47 and EGFR, indicating EGFR-directed inhibition. The bispecific VIR47.V8 / cetuximab showed superior SIRPα blocking activity compared to VIR47.V8. 5.11 Example 11: Antibody-dependent phagocytosis Bispecific antibodies were tested in an antibody-dependent cytophagy (ADCP) assay. Peripheral blood mononuclear cells (PBMCs) were isolated from human donors. PBMCs were enriched using a human mononuclear cell enrichment kit without CD16 depletion (STEMCELL, catalog number 19058). The isolated PBMCs were differentiated into macrophages by culturing them in complete medium (RPMI 1640 + 10% FBS) containing 20 ng / ml human macrophage colony-stimulating factor (M-CSF, Peprotech, catalog number 3-25-10). The medium was changed every three days. After culturing in M-CSF-containing medium for 7 days, macrophages were collected and counted. Target tumor cells were collected and washed twice with D-PBS to remove residual FBS. The washed tumor cells were then cultured at 5-10 × 10⁶ cells / mL. 6 Cells were resuspended in PBS at a concentration of 3 µM / mL. Cancer cells were stained with CFSE (ebiosciences, catalog number 65-0580-84) to a final concentration of 3 µM and immediately mixed. Cells were stained in the dark at room temperature for 10 minutes. Staining was terminated by adding 4–5 volumes of cold complete culture medium and incubating on ice for 5 minutes. The stained cells were washed three times with RPMI 1640 + 10% FBS. Cells were resuspended in 1 ml of RPMI 1640 + 10% FBS and counted, then the cell number was adjusted to 3 × 10⁶ cells / mL. 5 Cells / mL. 50 μl of cells were seeded into 96-well deep U-shaped plates (Axygen, catalog number P-DW-20-C), with each well containing 1.5 × 10⁶ cells / mL. 4 Add 50 μl of diluted antibody to each well. Add 100 μl of macrophages (1.5 × 10⁶ cells) to each well. 4 Cells were incubated at 37°C and 5% CO2 for 1.5 hours. After incubation, the cells were washed once with 2 ml of 2% FBS in D-PBS. 100 μl of diluted Fc blocking agent (human TruStain FcX (Fc receptor blocking solution), Biolegend catalog number 422302) was added, and the cells were incubated at room temperature for 10 minutes. 20 μl of diluted anti-human CD11b antibody was added to each well, and the cells were incubated in the dark at 4°C for 30 minutes. The cells were washed once with 2% FBS-D-PBS. Phagocytosis was detected by flow cytometry by the presence of CFSE / CD11b double-positive cells, which indicated macrophages phagocytosing tumor cells.

[0419] exist Figure 12In comparison with the VIR47.V8 bivalent antibody and BMK-1, the bispecific antibody cetuximab / VIR47.V8 showed better ADCP activity.

[0420] 5.12 Example 12: Antibody binding to red blood cells / platelets and hemagglutination RBC binding was determined by centrifuging fresh human whole blood at 200 g for 10 minutes. Collected RBCs were washed twice with PBS and counted using flow cytometry. 1 × 10⁻⁶ RBCs were... 6 Cells were aliquoted into each well of a 96-well plate. Serially diluted anti-CD47 antibody was added, and the plates were incubated at 4°C for 1 hour. Cells were washed twice with FACS buffer (PBS + 2% FBS). Secondary antibody (Alexa Fluor® 488 goat anti-human IgG (H+L)) was added, and the plates were incubated at 4°C for 1 hour. Cells were washed twice, resuspended in 200 μL of FACS buffer, and analyzed by flow cytometry.

[0421] Hemagglutination assays were performed by diluting human erythrocytes (RBCs) and titrating them with CD47 antibody (starting at 100 µg / ml) in round-bottom 96-well plates, followed by incubation at 37°C for 2 hours. Hemagglutination was demonstrated by the presence of cross-linked RBCs, which appeared as a mist because they did not settle to the bottom of the wells, compared to non-hemagglutinating RBCs.

[0422] exist Figures 13A-13B In comparison with the CD47 BMK-1 baseline antibody and its parental VIR47.V8 bivalent antibody, the bispecific antibody cetuximab / VIR47.V8 showed less RBC and platelet binding. Furthermore, no hemagglutination of VIR47.V8 / cetuximab was detected even at the highest tested concentration (666.7 nM; data not shown).

[0423] 5.13 Example 13: The in vivo antitumor activity of CD47×EGFR bispecific antibody was studied in the SNU-5 gastric cancer xenograft model.

[0424] SNU-5 tumor cells were cultured at 37°C in air under a 5% CO2 atmosphere in Iscove's modified Dulbecco medium supplemented with 20% heat-inactivated fetal bovine serum. Tumor cells in the exponential growth phase were harvested for tumor inoculation. Six- to seven-week-old female CB17 / SCID mice (Shanghai Lingchang Biotechnology Co., Ltd.) were subcutaneously inoculated with 0.2 mL of PBS supplemented with Matrigel (1:1). 7Each cell is used for tumor development. Tumor volume is measured using calipers and calculated using the following formula: Tumor volume = (Length × Width) / (Length × Width) 2 ) / 2. On day 6 post-tumor inoculation, when the average tumor volume reached approximately 175 mm... 3 Animals were randomly divided into groups of six based on tumor volume. The test antibody was administered via bolus injection into the tail vein at a dose of 10 mg / kg, once weekly for five weeks. Tumor volume was measured twice weekly. If an animal's tumor volume exceeded 3000 mm², the animal was considered a candidate for tumor screening. 3 Alternatively, if they experience a weight loss of more than 20%, they should be euthanized.

[0425] The results are summarized in Figure 14 Treatment with the bispecific antibody CD47×EGFR and the bivalent CD47 antibody resulted in complete tumor regression, while treatment with cetuximab only inhibited tumor growth. Furthermore, all mice in both the CD47×EGFR bispecific antibody group and the CD47 antibody group remained tumor-free until the end of the study. These data demonstrate that the CD47×EGFR bispecific antibody exhibits superior in vivo antitumor activity against its monospecific parental cetuximab, which expresses both EGFR and CD47 in gastric cancer.

[0426] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. This is not to imply that the invention is limited to the specific examples provided in the specification. Although the invention has been described with reference to the foregoing description, the description and illustration of embodiments herein are not intended to be construed as limiting. Many variations, alterations, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, and depend on a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in carrying out the invention. Therefore, the invention is also intended to cover any such substitutions, modifications, variations, or equivalents. The appended claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. Use of a bispecific antibody comprising a CD47-binding domain and an EpCAM-binding domain in the manufacture of a medicament for treating cancer, wherein the CD47-binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs): HC-CDR1 as shown in SEQ ID NO: 15, HC-CDR2 as shown in SEQ ID NO: 16, and HC-CDR3 as shown in SEQ ID NO: 17; and three light chain (LC) CDRs: LC-CDR1 as shown in SEQ ID NO: 18, LC-CDR2 as shown in SEQ ID NO: 19, and LC-CDR3 as shown in SEQ ID NO:

20.

2. The use according to claim 1, wherein the CD47 binding domain includes a heavy chain variable region as shown in SEQ ID NO: 5, SEQ ID NO: 10 or SEQ ID NO: 13, and a light chain variable region as shown in SEQ ID NO: 8, SEQ ID NO: 11 or SEQ ID NO:

14.

3. The use according to claim 1 or 2, wherein the CD47 binding domain is scFv or Fab.

4. The use according to any one of claims 1-3, wherein the EpCAM-bonded structural domain is scFv or Fab.

5. The use according to any one of claims 1-4, wherein the CD47 binding domain is Fab and the EpCAM binding domain is scFv.

6. The use according to any one of claims 1-4, wherein the CD47 binding domain is scFv and the EpCAM binding domain is Fab.

7. The use according to any one of claims 1-6, wherein the bispecific antibody further comprises an Fc domain.

8. The use according to claim 7, wherein the Fc domain is a human Fc domain.

9. The use according to claim 8, wherein the human Fc domain is IgG1 or IgG4.

10. The use according to any one of claims 1-9, wherein the bispecific antibody has an asymmetric triple-chain club-and-mortar structure.

11. The use according to claim 10, wherein the mortar chain comprises the mutant T366W, and the mortar chain comprises the mutants T366S, L368A, and Y407V, wherein the amino acid position numbers are based on the EU index of Kabat et al.

12. The use according to any one of claims 1-11, wherein the EpCAM binding domain comprises three heavy chain (HC) complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 and three light chain (LC) CDRs LC-CDR1, LC-CDR2, and LC-CDR3; wherein HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3 are defined by an ordered set of the sequences listed below: (i) SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83; (ii) SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86; or (iii) SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO:

92.

13. The use according to any one of claims 1-12, wherein the EpCAM binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are defined by an ordered set of the sequences listed below: (i) The sequences of SEQ ID NO: 21 and SEQ ID NO: 33; (ii) The sequence of SEQ ID NO: 22 and the sequence of SEQ ID NO: 34; or (iii) The sequences of SEQ ID NO: 24 and SEQ ID NO:

36.

14. The use according to any one of claims 1-13, wherein the bispecific antibody inhibits the binding of SIRPα to CD47 on the surface of tumor cells.

15. The use according to claim 14, wherein the tumor cells express CD47 and EpCAM.

16. The use according to any one of claims 1-15, wherein the bispecific antibody does not induce hemolysis of erythrocytes.

17. The use according to any one of claims 1-16, wherein the cancer is pancreatic cancer, ovarian cancer, colorectal cancer, lung cancer, stomach cancer, liver cancer, head and neck cancer, or vulvar cancer.

18. The use according to any one of claims 1-17, wherein the cancer is pancreatic ductal adenocarcinoma, ovarian clear cell adenocarcinoma, colorectal adenocarcinoma, small cell lung cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, non-small cell lung cancer, or vulvar squamous cell carcinoma.

19. Use of a bispecific antibody comprising a CD47-binding domain and an EGFR-binding domain in the manufacture of a medicament for treating cancer, wherein the bispecific antibody has a higher affinity for CD47 expressed on the surface of tumor cells than for CD47 expressed on the surface of erythrocytes or platelets.

20. The use according to claim 19, wherein the cancer is epidermoid carcinoma, colorectal adenocarcinoma, pancreatic adenocarcinoma, squamous cell carcinoma of the lung, or gastric cancer.

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