Anti-HER2 / anti-CD47 molecules and uses thereof

Abstract

Provided herein are anti-CD47 / anti-HER2 polypeptide complexes, nucleic acid molecules encoding variable domains of the polypeptide complexes, expression vectors and host cells for expressing the polypeptide complexes. The present disclosure further provides methods for producing polypeptide complexes and uses thereof.

Description

[0001] Cross-references

[0002] This application claims priority to International Patent Application No. PCT / CN2022 / 082951 filed on March 25, 2022. The entire contents of this application are incorporated herein by reference. Technical Field

[0003] The present application generally relates to antibodies. More specifically, the present application relates to bispecific antibodies that specifically bind to HER2 and CD47, methods for preparing the same, and uses thereof. Background Art

[0004] Cluster of differentiation 47 (CD47) is an immunoglobulin superfamily membrane protein of approximately 50 kDa, consisting of a single extracellular V-setIgSF domain, a presenilin domain with five transmembrane segments, and a short cytoplasmic domain. CD47 interacts with its ligand, signal regulatory protein alpha (SIRPα), expressed on myeloid cells such as macrophages, and then sends out antiphagocytic (“don’t eat me”) signals to evade immune surveillance [1-2]. CD47 is a ubiquitous cell surface glycoprotein expressed on most normal cell types and is overexpressed in a variety of malignancies, including acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), non-small cell lung cancer (NSCC), breast cancer (BC), and gastric cancer (GC). Therefore, CD47 can be used as an innate immune checkpoint target for cancer therapy to shut down the “don’t eat me” signal by blocking the CD47-SIRPα interaction [3].

[0005] HER2 (also known as erb-b2 receptor tyrosine kinase 2 or ERBB2) is a member of the epidermal growth factor receptor (EGFR) family, along with HER1 (also known as EGFR), HER3, and HER4. These receptors function as homodimers or heterodimers to activate multiple cellular pathways, such as the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol-3-kinase (PI3K) pathway, which then stimulate cell growth, survival, and differentiation [4].

[0006] High expression of CD47 is associated with poor prognosis in patients with various cancers, and co-expression of CD47 and Her2 may lead to +Disease progression of cancer (e.g., BC, GC) after Her2-targeted therapy [5-6]. Specially designed CD47xHer2 bispecific antibodies (BsAbs) may preferentially target Her2+ / CD47+ double-positive tumor cells and minimize the effects on CD47 single-positive normal cells to reduce systemic CD47 antigen-mediated sinking effects and hematotoxicity. Blocking CD47 / SIRPα signaling can enhance the anti-tumor efficacy of Her2-targeted therapy by increasing antibody-dependent cellular phagocytosis (ADCP) of tumor cells and further stimulating adaptive immunity. In addition, the IgG1 Fc of BsAb can maintain the anti-her2 + Antibody-dependent cellular cytotoxicity (ADCC) of tumor cells.

[0007] Although patients with Her2-overexpressing metastatic breast and gastric cancer initially respond to Her2-targeted therapy, most patients with advanced Her2-positive solid tumors (e.g., breast cancer) who initially respond to trastuzumab eventually acquire treatment resistance and relapse (despite persistent Her2 gene amplification or overexpression), and there is a high unmet medical need for patients with Her2-positive relapsed / refractory cancer. Therefore, CD47xHer2 BsAb may provide a new treatment option for Her2-positive BC, GC, and other solid tumors.

[0008] The present disclosure provides bispecific antibodies that dual-target CD47 and HER2 and block the dual functions of CD47 and HER2. SUMMARY OF THE INVENTION

[0010] The present disclosure provides these and other purposes, which broadly relate to providing compounds, methods, compositions and articles of manufacture of antibodies with improved efficacy. The benefits provided by the present disclosure are broadly applicable to the field of antibody therapy and diagnosis, and can be used in combination with other therapeutic and diagnostic agents (e.g., antibodies reactive with a variety of targets).

[0011] The present disclosure provides bispecific polypeptide complexes or bispecific antibodies against CD47 and HER2. It also provides nucleic acid molecules encoding anti-CD47 / anti-HER2 antibodies, expression vectors, and host cells for expressing bispecific antibodies. The present disclosure also provides methods for preparing anti-CD47 / anti-HER2 antibodies (CD47xHer2 BsAbs) and verifying their functions in vivo and in vitro. The bispecific antibodies of the present disclosure provide very effective agents for preventing or treating diseases including proliferative disorders and immune disorders.

[0012] In one aspect, the present disclosure provides a bispecific polypeptide complex, or antigen binding portion thereof, comprising a first antigen binding portion that specifically binds HER2 (ie, a HER2 binding portion) and a second antigen binding portion that specifically binds CD47 (ie, a CD47 binding portion).

[0013] In some embodiments, the present disclosure provides a bispecific polypeptide complex, or an antigen binding portion thereof, comprising a first antigen binding moiety that specifically binds HER2 (i.e., a HER2 binding moiety) and a second antigen binding moiety that specifically binds CD47 (i.e., a CD47 binding moiety), wherein the first antigen binding moiety comprises:

[0014] a heavy chain complementary determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, a light chain complementary determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and

[0015] The second antigen binding moiety comprises:

[0016] HCDR1 comprising the amino acid sequence of SEQ ID NO:7, HCDR2 comprising the amino acid sequence of SEQ ID NO:8, HCDR3 comprising the amino acid sequence of SEQ ID NO:9, LCDR1 comprising the amino acid sequence of SEQ ID NO:10, LCDR2 comprising the amino acid sequence of SEQ ID NO:11, and LCDR3 comprising the amino acid sequence of SEQ ID NO:12.

[0017] In some embodiments, the first antigen binding moiety and the second antigen binding moiety are in Fab format.

[0018] In some embodiments, the first antigen binding portion comprises a first heavy chain variable domain (VH1) operably linked to a first T cell receptor (TCR) constant region (referred to as C1 or CBeta) and a first light chain variable domain (VL1) operably linked to a second TCR constant region (referred to as C2 or CA1pha); and the second antigen binding portion comprises a second VH (VH2) operably linked to an antibody heavy chain CH1 domain and a second VL (VL2) operably linked to an antibody light chain constant (CL) domain, wherein C1 and C2 are capable of forming one or more non-natural interchain disulfide bonds. The positions of the C1 and C2 domains in the bispecific polypeptide complex or antigen binding portion thereof can be interchanged.

[0019] In some other embodiments, the first antigen binding portion comprises a first heavy chain variable domain (VH1) operably connected to an antibody heavy chain CH1 domain and a first light chain variable domain (VL1) operably connected to an antibody light chain constant region (CL); and the second antigen binding portion comprises a second VH (VH2) operably connected to a first T cell receptor (TCR) constant region (C1) and a second VL (VL2) operably connected to a second TCR constant region (C2), wherein C1 and C2 are capable of forming one or more non-natural interchain disulfide bonds. The positions of the C1 and C2 domains in the bispecific polypeptide complex or antigen binding portion thereof can be interchanged.

[0020] In some embodiments, the first VH comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 13, and the first VL comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 14; and / or

[0021] The second VH comprises the amino acid sequence of SEQ ID NO:15, or an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO:15, and the second VL comprises the amino acid sequence of SEQ ID NO:16, or an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO:16.

[0022] The bispecific polypeptide complex may further comprise an Fc region, such as a human IgG (IgG1, IgG2, IgG3 or IgG4) Fc region, such as a human IgG1 Fc region, an IgG2 Fc region or an IgG4 Fc region. The Fc region may be a native Fc region or an engineered Fc region. For example, a human IgG1 Fc region may be engineered to include a "knob into hole" structure or other modifications conventionally known in the art. Optionally, the Fc region may be selected from one of the following: (a) a human IgG1 Fc region engineered to include a "knob into hole" structure; (b) a human IgG4 Fc region engineered to include a "knob into hole" structure and an S228P mutation; and (c) a human IgG4 Fc region engineered to include an S228P mutation and an M252Y / S254T / T256E mutation.

[0023] In some embodiments, the bispecific polypeptide complex comprises a CD47 binding portion and a HER2 binding portion. For example, the bispecific polypeptide complex comprises two heavy chains and two light chains, wherein the first heavy chain comprises a domain operably linked from the N-terminus to the C-terminus such as VH1-C1-hinge-Fc, the second heavy chain comprises a domain operably linked from the N-terminus to the C-terminus such as VH2-CH1-hinge-Fc, the first light chain comprises a domain operably linked from the N-terminus to the C-terminus such as VL1-C2, and the second light chain comprises a domain operably linked from the N-terminus to the C-terminus such as VL2-C1. As an alternative example of a bispecific polypeptide complex, from N-terminus to C-terminus, the first heavy chain comprises an operably linked domain such as VH1-CH1-hinge-Fc, the second heavy chain comprises an operably linked domain such as VH2-C1-hinge-Fc, the first light chain comprises an operably linked domain such as VL1-CL, and the second light chain comprises an operably linked domain such as VL2-C2. The positions of the C1 and C2 domains in the bispecific polypeptide complex or antigen binding portion thereof can be interchanged.

[0024] In some embodiments, the bispecific polypeptide complex disclosed herein comprises:

[0025] The first and second heavy chains comprise SEQ ID NOs: 19 and 20, respectively, and the first and second light chains comprise SEQ ID NOs: 21 and 22, respectively.

[0026] In some embodiments, the bispecific polypeptide complex comprises two CD47 binding moieties and two HER2 binding moieties. For example, the bispecific polypeptide complex comprises two heavy chains and four light chains, wherein from N-terminus to C-terminus: the first and second heavy chains each comprise an operably linked domain as in VH1-C1-VH2-CH1-hinge-Fc, VH2-CH1-VH1-C1-hinge-Fc, VH1-C1-hinge-Fc-VH2-CH1 or VH2-CH1-hinge-Fc-VH1-C1, the first and second light chains each comprise an operably linked domain as in VL1-C2, and the third and fourth light chains each comprise an operably linked domain as in VL2-C1. Alternatively, the bispecific polypeptide complex comprises two heavy chains and four light chains, wherein from N-terminus to C-terminus: the first and second heavy chains each comprise an operably linked domain as in VH1-CH1-VH2-C1-hinge-Fc, VH2-C1-VH1-CH1-hinge-Fc, VH1-CH1-hinge-Fc-VH2-C1 or VH2-C1-hinge-Fc-VH1-CH1, the first and second light chains each comprise an operably linked domain as in VL1-CL, and the third and fourth light chains each comprise an operably linked domain as in VL2-C2. The positions of the C1 and C2 domains in the bispecific polypeptide complex or antigen binding portion thereof may be interchanged.

[0027] In some embodiments, the bispecific polypeptide complex comprises one CD47 binding moiety and two HER2 binding moieties. For example, the bispecific polypeptide complex comprises two heavy chains and three light chains, wherein from N-terminus to C-terminus: the first heavy chain comprises an operably linked domain as in VH2-CH1-VH1-C1-hinge-Fc or VH1-C1-hinge-Fc-VH2-CH1, the second heavy chain comprises an operably linked domain as in VH1-C1-hinge-Fc, the first and second light chains each comprise an operably linked domain as in VL1-C2, and the third light chain comprises an operably linked domain as in VL2-C1. Alternatively, the first heavy chain comprises an operably linked domain as in VH2-C1-VH1-CH1-hinge-Fc or VH1-CH1-hinge-Fc-VH2-C1, the second heavy chain comprises an operably linked domain as in VH1-CH1-hinge-Fc, the first and second light chains each comprise an operably linked domain as in VL1-CL, and the third light chain comprises an operably linked domain as in VL2-C2. The positions of the C1 and C2 domains in the bispecific polypeptide complex or antigen binding portion thereof can be interchanged.

[0028] In some embodiments, the bispecific polypeptide complex comprises two CD47 binding moieties and one HER2 binding moiety. For example, the bispecific polypeptide complex comprises two heavy chains and three light chains, wherein from N-terminus to C-terminus: the first heavy chain comprises an operably linked domain as in VH1-C1-VH2-CH1-hinge-Fc or VH2-CH1-hinge-Fc-VH1-C1, the second heavy chain comprises an operably linked domain as in VH2-CH1-hinge-Fc, the first light chain comprises an operably linked domain as in VL1-C2, and the second and third light chains each comprise an operably linked domain as in VL2-CL. Alternatively, the first heavy chain comprises an operably linked domain as in VH1-CH1-VH2-C1-hinge-Fc or VH2-C1-hinge-Fc-VH1-CH1, the second heavy chain comprises an operably linked domain as in VH2-C1-hinge-Fc, the first light chain comprises an operably linked domain as in VL1-CL, and the second and third light chains each comprise an operably linked domain as in VL2-C2. The positions of the C1 and C2 domains in the bispecific polypeptide complex or antigen binding portion thereof can be interchanged.

[0029] Operably linked or "-" can be via direct linkage or via a peptide linker, such as a GS linker, e.g., (GS)n, (GGS)n, (GGGS)n, (GGGS)n, (GGGGS)n, (GGGGS)n, (GGGS)n, (GGGS)n, where n is an integer from 1 to 9. The bispecific polypeptide complex can be a humanized antibody or a fully human antibody. In some embodiments, the bispecific polypeptide complex is a fully human antibody.

[0030] In one aspect, disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a bispecific polypeptide complex or an antigen binding portion thereof.

[0031] In one aspect, disclosed herein is a vector comprising a nucleic acid molecule as described above. In one aspect, disclosed herein is a host cell comprising a nucleic acid molecule or a vector as described above.

[0032] In one aspect, disclosed herein is a pharmaceutical composition comprising a bispecific polypeptide complex or an antigen binding portion thereof and a pharmaceutically acceptable carrier.

[0033] In one aspect, disclosed herein is a method for producing a bispecific polypeptide complex comprising the steps of:

[0034] - culturing a host cell comprising a nucleic acid sequence encoding a polypeptide complex under suitable conditions; and

[0035] - Isolation of the polypeptide complex from the host cell.

[0036] In one aspect, disclosed herein are methods for modulating a HER2 and / or CD47-related immune response in a subject, comprising administering to the subject a bispecific polypeptide complex, or an antigen binding portion thereof, or a pharmaceutical composition as disclosed herein.

[0037] In one aspect, disclosed herein is a method for inhibiting the growth of tumor cells (such as CD47 and / or HER2-positive tumor cells) in a subject, comprising administering to the subject an effective amount of a bispecific polypeptide complex or antigen binding portion thereof or a pharmaceutical composition as disclosed herein.

[0038] In one aspect, disclosed herein is a method for inducing macrophage-mediated phagocytosis of tumor cells in a subject, comprising administering to the subject an effective amount of a bispecific polypeptide complex or an antigen binding portion thereof or a pharmaceutical composition as disclosed herein.

[0039] In one aspect, disclosed herein is a method for inducing natural killer cell-mediated cytotoxicity against (or directed against) tumor cells in a subject, comprising administering to the subject an effective amount of a bispecific polypeptide complex or antigen binding portion thereof or a pharmaceutical composition as disclosed herein.

[0040] In one aspect, disclosed herein are methods for diagnosing, preventing or treating cancer in a subject, comprising administering to the subject an effective amount of a bispecific polypeptide complex or an antigen binding portion thereof or a pharmaceutical composition. In some embodiments, the cancer is a HER2 and / or CD47 positive cancer and is selected from colon cancer, colorectal cancer, breast cancer, lung cancer (including NSCLC and small cell lung cancer), cervical cancer, kidney cancer, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, gastric cancer, melanoma, liver cancer, head and neck cancer, skin cancer, bladder cancer, brain cancer, bronchial cancer, bile duct cancer, endometrial cancer, ependymoma, glioma, cancer of unknown primary origin, medulloblastoma, nasopharyngeal cancer, neuroblastoma, squamous cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thymoma, thyroid cancer, urachal cancer, uterine cancer, vaginal cancer, astrocytoma, basal cell carcinoma and combinations thereof.

[0041] In some further embodiments, the cancer is breast cancer, gastric cancer, lung cancer, skin cancer, or colorectal cancer.

[0042] In one aspect, disclosed herein is a composition for diagnosing, preventing or treating cancer in a subject, comprising an effective amount of a bispecific polypeptide complex disclosed herein, or an antigen binding portion thereof.

[0043] In one aspect, disclosed herein is a use of a bispecific polypeptide complex, or an antigen binding portion thereof, for:

[0044] i) modulating HER2 and / or CD47 related immune responses; and / or

[0045] ii) inducing macrophage-mediated phagocytosis of tumor cells; and / or

[0046] iii) inducing natural killer cell-mediated tumor cell cytotoxicity; and / or

[0047] iv) inhibiting the growth of tumor cells, such as CD47 and / or HER2 positive tumor cells.

[0048] In one aspect, disclosed herein is the use of a bispecific polypeptide complex, or an antigen binding portion thereof, for diagnosing, treating or preventing cancer.

[0049] In one aspect, disclosed herein is the use of a bispecific polypeptide complex or an antigen binding portion thereof in the preparation of a medicament for:

[0050] i) Modulate HER2 and / or CD47-related immune responses;

[0051] ii) inducing macrophage-mediated phagocytosis of tumor cells;

[0052] iii) inducing natural killer cell-mediated tumor cell cytotoxicity; and / or

[0053] iv) inhibiting the growth of tumor cells, such as CD47 and / or HER2 positive tumor cells.

[0054] In one aspect, disclosed herein is the use of a bispecific polypeptide complex or an antigen binding portion thereof in the preparation of a medicament for diagnosing, treating or preventing cancer.

[0055] The bispecific polypeptide complexes, or antigen binding portions thereof, as disclosed herein can be administered in combination with chemotherapeutic agents, radiation, and / or other agents for cancer immunotherapy, and can be used in combination therapy with chemotherapeutic agents, radiation, and / or other agents for cancer immunotherapy.

[0056] In one aspect, disclosed herein is a kit, wherein the kit comprises a container comprising a bispecific polypeptide complex as described above, or an antigen binding portion thereof.

[0057] The foregoing is a summary and necessarily contains simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that this summary is illustrative only and is not intended to be in any way limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1A schematic diagram of the W308032 bispecific antibody in the WuXiBody E17 format is shown. From N-terminus to C-terminus, the first heavy chain, VH1-Cbeta-hinge-Fc; the second heavy chain, VH2-CH1-hinge-Fc; the first light chain, VL1-CA1pha; and the second light chain, VL2-CL.

[0059] Figure 2-3 SDS-PAGE and SEC-HPLC characterization of W308032-U5T6.E17-57.uIgG1 are shown, respectively.

[0060] Figure 4-6 DSF, DLS and HIC-HPLC characterization of W308032-U5T6.E17-57.uIgG1 are shown, respectively.

[0061] Figures 7A-7F The binding affinities of W308032-U5T6.E17-57.uIgG1 to human Her2 protein (A: 1st round, B: 2nd round, C: 3rd round) and human CD47 protein (D: 1st round, E: 2nd round, F: 3rd round) determined by SPR are shown.

[0062] Figures 8A-8B It shows that W308032-U5T6.E17-57.uIgG1 has no antigen sinking ( Fig. 8A ) or after being sunk by human blood cells ( Figure 8B ) combined with SK-BR-3.

[0063] Figures 9A-9B It shows that W308032-U5T6.E17-57.uIgG1 has no antigen sinking ( Fig. 9A ) or after being submerged by Jurkat cells ( Fig. 9B ) combined with SK-BR-3.

[0064] Figures 10A-10B It shows that W308032-U5T6.E17-57.uIgG1 has no antigen sinking ( Fig. 10A ) or after being sunk by Jurkat cells ( Fig. 10B ) combined with HCC1954.

[0065] Fig.11 Binding of W308032-U5T6.E17-57.uIgG1 to human blood cells is shown.

[0066] Fig.12 Binding of W308032-U5T6.E17-57.uIgG1 to Jurkat cells is shown.

[0067] Figures 13A-13B showed that in the absence of antigen sinking ( Fig.13A ) or after being sunk by human blood cells ( Fig. 13B ), W308032-U5T6.E17-57.uIgG1 blocks CD47 ligand on SK-BR-3.

[0068] Figures 14A-14B showed that in the absence of antigen sinking ( Fig.14A ) or after being sunk by Jurkat cells ( Fig. 14B ), W308032-U5T6.E17-57.uIgG1 blocks CD47 ligand on SK-BR-3.

[0069] Fig.15 Blockade of CD47 ligand on Jurkat cells by W308032-U5T6.E17-57.uIgG1 is shown.

[0070] Figures 16A-16B HER2 and CD47 occupancy of W308032-U5T6.E17-57.uIgG1 on SK-BR-3 are shown, respectively.

[0071] Figures 17A-17B Shown are the ADCP of W308032-U5T6.E17-57.uIgG1 against SK-BR-3 from two independent experiments.

[0072] Figure 18-19 The results of W308032-U5T6.E17-57.uIgG1 on HCC1954 ( Fig.18 ) and NCI-N87( Fig.19 )’s ADCP.

[0073] Figure 20-21 The effects of W308032-U5T6.E17-57.uIgG1 on human blood cells ( Fig. 20 ) and Jurkat cells ( Fig.21 )’s ADCP.

[0074] Figure 22-24 The results of W308032-U5T6.E17-57.uIgG1 on SK-BR-3 ( Fig. 22 )、HCC1954( Fig.23 ) and NCI-N87( Fig.24 )’s ADCC.

[0075] Fig.25 ADCC of W308032-U5T6.E17-57.uIgG1 on Jurkat cells is shown.

[0076] Figure 26-27 The results of W308032-U5T6.E17-57.uIgG1 on SK-BR-3 ( Fig.26 ) and NCI-N87( Fig. 27 ) proliferation inhibition.

[0077] Fig.28 The hemagglutination effect of W308032-U5T6.E17-57.uIgG1 on human blood cells is shown.

[0078] Fig.29 The thermal stability results of W308032-U5T6.E17-57.uIgG1 are shown.

[0079] Fig.30 The combined antitumor effect of anti-HER2 and anti-CD47 therapy in the HCC1954 xenograft model was shown.

[0080] Fig.31 The anti-tumor effects of different dose levels of W308032-U5T6.E17-57.uIgG1 in the HCC1954 xenograft model were shown.

[0081] Fig.32 The antitumor effect of combination therapy with Pertuzumab in the HCC1954 xenograft model was shown.

[0082] Fig.33 A summary of tumor weights on day 36 in the HCC1954 xenograft model is shown. Note: Two-way ANOVA Bonferroni post-test, *p<0.05; **p<0.01.

[0083] Fig.34 The combined anti-tumor effect of anti-Her2 and anti-CD47 therapy in the NCI-N87 xenograft model was shown.

[0084] Fig.35 A summary of the anti-tumor effects of W308032-U5T6.E17-57.uIgG1 in the NCI-N87 xenograft model is shown.

[0085] Fig.36 A summary of the antitumor effects of combination therapy with paclitaxel in the NCI-N87 xenograft model is shown.

[0086] Fig.37 A summary of the antitumor effects of combination therapy with paclitaxel in the JIMT-1 xenograft model is shown.

[0087] Fig.38A summary of tumor rebound after the last dose is shown. The dashed line indicates the mean initial tumor volume at grouping.

[0088] Fig.39 A summary of the rodent pharmacokinetics of W308032-U5T6.E17-57.uIgG1 and trastuzumab is shown. DETAILED DESCRIPTION OF THE INVENTION

[0090] While the present disclosure can be implemented in many different forms, disclosed herein are specific illustrative embodiments that illustrate the principles of the present disclosure. It should be emphasized that the present disclosure is not limited to the specific embodiments shown. In addition, any section headings used herein are only for organizational purposes and should not be interpreted as limiting the subject matter described.

[0091] Unless otherwise defined herein, scientific and technical terms related to the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include plural terms and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural indicators unless the context clearly indicates otherwise. Thus, for example, reference to "a protein" includes multiple proteins; reference to "a cell" includes a mixture of cells, etc. In this application, "or" is used to mean "and / or" unless otherwise indicated. In addition, the use of the term "comprising" and other forms, such as "comprises" and "comprised", is not restrictive. In addition, the ranges provided in the specification and the appended claims include both endpoints and all points between the endpoints.

[0092] Generally, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present disclosure are generally described in accordance with conventional methods well known in the art and in various general and more specific references cited and discussed throughout the present specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6 thed., WB Saunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coliganet al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Other aspects, features and advantages of the methods, compositions and / or devices and / or other subjects described herein will become apparent in the teachings set forth herein. In addition, the contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference in their entirety.

[0093] definition

[0094] In order to better understand the present disclosure, definitions and explanations of relevant terms are provided below.

[0095] As used herein, the term "antibody" or "Ab" is used in the broadest sense and covers any form of antibody that exhibits the desired biological or binding activity. It covers, but is not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single domain antibodies. The bispecific polypeptide complexes disclosed herein also belong to antibodies. Common antibodies generally contain heavy chains and light chains. The heavy chains can be divided into μ, δ, γ, α, and ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a heavy chain variable region (V H ) and the heavy chain constant region (C H The heavy chain constant region consists of three domains (CH 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L As demonstrated herein, various modifications may be made to the constant regions or they may be replaced with other immunoglobulin derived constant regions. H and V L The regions can be further divided into regions of hypervariability, termed complementarity determining regions (CDRs), separated by regions that are relatively conserved, termed framework regions (FRs). H and V L It consists of 3 CDRs and 4 FRs in the following order: from N-terminus to C-terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L) respectively form an antigen binding site. The scope of the framework region and CDR can be accurately identified using methods known in the art, such as by Kabat definition, Chothia definition, AbM definition, EU definition and / or contact definition, all of which are well known in the art. See, for example: Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. al. (1987) J. Mol. Biol. 196: 901-917, Al-lazikani et al (1997) J. Molec. Biol. 273: 927-948; Edelman et al., Proc Natl Acad Sci US A. 1969 May, 63(1):78-85; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. The correspondence or comparison between the numbers according to different definitions can be found, for example, at www.imgt.org / (see also Giudicelli V et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. (1997) 25: 206–11; and Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Igsuperfamily V-like domains. Dev Comp Immunol. (2003) 27: 55–77). The antibodies can be of different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0096] As used herein, the term "antigen binding portion" refers to an antibody fragment formed by a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain a complete native antibody structure. Examples of antigen binding portions include, but are not limited to, variable domains, variable regions, diabodies, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv fragments (scFv), disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), multispecific antibodies, camelized single domain antibodies, single variable domains (i.e., VHH), nanobodies, domain antibodies, and bivalent domain antibodies. The antigen binding portion is capable of binding to the same antigen bound by the parent antibody. In certain embodiments, the antigen binding portion may include one or more CDRs from a specific human antibody that are transplanted to a framework region from one or more different human antibodies. More detailed formats for antigen binding portions are described in Spiess et al, (2015) Molecular Immunology 67:95-106, and Brinkman et al., mAbs, 9(2), pp. 182–212 (2017), which are incorporated herein in their entireties.

[0097] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody are used interchangeably in the context of this application and refer to a polypeptide comprising a fragment of a full-length antibody or polypeptide complex disclosed herein that retains the ability to specifically bind to an antigen to which the full-length antibody specifically binds, and / or the ability to compete with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., the second edition, Raven Press, NY (1989), which is incorporated herein by reference for all purposes.

[0098] With respect to antibodies, "Fab" refers to the portion of an antibody consisting of a single light chain (both variable and constant regions) associated by disulfide bonds with the variable region of a single heavy chain and a first constant region. In certain embodiments, the constant regions of both the light and heavy chains are replaced by TCR constant regions.

[0099] With respect to antibodies, "Fc" (abbreviation for crystallizable fragment) refers to a portion of an antibody comprising the following: the second and third constant regions of the first heavy chain bound to the second (CH2) and third (CH3) constant regions of the second heavy chain via disulfide bonds. The Fc region as used herein may also include a portion or all of the hinge region. The term "hinge" or "hinge region" refers to a flexible stretch of amino acids in the central portion of the heavy chains of the IgG and IgA immunoglobulin classes, which connects the two chains via disulfide bonds, and as used herein, may include a natural hinge region as a whole, a part, a homologue or a functional equivalent thereof. The Fc region of an antibody is responsible for a variety of effector functions such as ADCC and CDC, but does not usually function in antigen binding. The ability of an antibody to initiate and regulate effector functions through its Fc domain is a key component of its in vivo protective activity. Although the neutralizing activity of antibodies was previously thought to be solely the result of Fab-antigen interactions, it is clear that their in vivo activity is highly dependent on the interaction of the IgG Fc domain with its cognate receptor, the Fcγ receptor (FcγR) expressed on the surface of effector leukocytes.

[0100] As used herein, the term "humanized antibody" refers to antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0101] As used herein, the term "human antibody" or "fully human antibody" is intended to include antibodies with variable regions, wherein the framework region and CDR region are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from another mammalian species (e.g., mouse) germline have been transplanted to human framework sequences.

[0102] The terms "operably linked" and "operably linked" refer to two or more biological sequences of interest, with or without spacers or linkers, juxtaposed in a manner such that they are in a relationship that allows them to function in the intended manner. When used in relation to polypeptides, it is intended to mean that the polypeptide sequences are linked in a manner that allows the linked product to have the intended biological function. For example, an antibody variable region can be operably linked to a constant region to provide a stable product with antigen binding activity. The term can also be used for polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, an enhancer, a silencer sequence, etc.), it is intended to mean that the polynucleotide sequences are linked in a manner that allows regulated expression of the polypeptide from the polynucleotide. The term operably linked, when used herein to describe domains that are linked to form a polypeptide, can be represented by "-", and can refer to a direct connection between domains or to a connection via a linker, the linker length comprising 1-30 amino acids, such as a single amino acid or a series of (G4S)n linkers, where n=1-5 (1, 2, 3, 4 or 5).

[0103] As used herein, the term "Ka" is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "Kd" as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. D " is intended to refer to the dissociation constant for a specific antibody-antigen interaction, which is obtained by the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed in molar concentration (M). The K D A preferred method is by using surface plasmon resonance, preferably using a biosensor system, such as system.

[0104] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as a non-random binding reaction between an antibody and an antigen. As used herein, the term "high affinity" of an IgG antibody refers to an antibody that has a K of less than 1.0 for a target antigen. D :1×10 -7 M or less, more preferably 5×10 -8 M or less, even more preferably 1×10 -8 M or less, even more preferably 5×10 -9 M or less, even more preferably 1×10 -9 M or less, and even more preferably 5×10 -10 M or less, such as measured by SPR.

[0105] As used herein, the term “EC 50 EC, also known as the “half-maximal effective concentration”, is the concentration of a drug, antibody, or toxicant that elicits a response halfway between baseline and maximum after a specified exposure time. In the context of this application, EC 50 The unit is "nM".

[0106] As used herein, the term “IC 50 IC, also known as the "half maximal inhibitory concentration", refers to the half maximal inhibitory concentration of a drug, antibody or other substance. It is a measure of the effectiveness of a drug, antibody or other substance in inhibiting a biological or biochemical function. In the context of this application, IC 50 The unit is "nM".

[0107] As used herein, the ability to "inhibit binding" or "block binding" refers to the ability of an antibody to inhibit the binding interaction between two molecules (e.g., human CD47 and CD47 ligand SIRPα) to any detectable extent. In some embodiments, an antibody as disclosed herein has an IC of 50 Blocking the binding between CD47 and SIRPα: no more than 1 nM, no more than 0.8 nM, no more than 0.6 nM, no more than 0.4 nM or no more than 0.3 nM.

[0108] As used herein, the term "epitope" refers to the part on the antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to as "antigenic determinant". An epitope or antigenic determinant is generally composed of chemically active surface groups of molecules such as amino acids, carbohydrates or sugar side chains, and generally has a specific three-dimensional structure and a specific charge characteristic. For example, an epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or discontinuous amino acids in a unique spatial conformation, which may be "linear" or "conformational". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0109] As used herein, the term "isolated" refers to a state obtained from a natural state by artificial means. If a certain "isolated" substance or component exists in nature, it may be because its natural environment has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a certain living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances, nor does it exclude other impure substances that do not affect the activity of the isolated substance.

[0110] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated bispecific antibody that specifically binds to CD47 and HER2 proteins is substantially free of antibodies with different targeting antigens or epitopes). However, isolated antibodies that specifically bind to human CD47 proteins and HER2 proteins may have cross-reactivity with other antigens (e.g., CD47 or HER2 proteins from other species). In addition, the isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0111] As used herein, the term "vector" refers to a nucleic acid vector that may have a polynucleotide inserted therein. When a vector allows expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. A vector may have genetic material elements carried and expressed in a host cell by transformation, transduction or transfection into a host cell. Vectors are well known to those skilled in the art, including but not limited to plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phages or M13 phages and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40). A vector may include multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, screening elements, and reporter genes. In addition, a vector may include an origin of replication.

[0112] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0113] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by alignment and comparison of sequences. "Identity percentage" represents the percentage of identical residues between the amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule compared. For these calculations, gaps in the alignment, if any, are preferably resolved by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAM J. Applied Math. 48:1073.

[0114] As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It not only refers to the property of antigens to stimulate the activation, proliferation, and differentiation of specific immune cells to ultimately generate immune effectors such as antibodies or sensitized lymphocytes, but also refers to the specific immune response of antibodies or sensitized T lymphocytes that can be formed after the body's immune system is stimulated by antigens. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce an immune response in the host depends on three factors: the characteristics of the antigen, the host's reactivity, and the means of immunity.

[0115] As used herein, the term "transfection" or "transfect" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Transfection protocols and techniques include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197.

[0116] As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes an optical phenomenon that allows real-time biospecific interactions to be analyzed by detecting changes in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see Examples 5 and U.,et al.(1993)Ann.Biol.Clin.51:19-26; U., et al. (1991) Biotechniques 11: 620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnson, B., et al. (1991) Anal. Biochem. 198: 268-277.

[0117] As used herein, the term "fluorescence activated cell sorting" or "FACS" refers to a special type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers (one cell at a time) based on the specific light scattering and fluorescence characteristics of each cell (Flow Metric. "Sorting Out Fluorescence Activated Cell Sorting". Retrieved 2017-11-09.). Instruments for performing FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, Calif.), Epics C from Coulter Epics Division (Hialeah, Fla.), and MoFlo from Cytomation (Colorado Springs, Colo.).

[0118] The term "subject" includes any human or non-human animal, preferably a human.

[0119] As used herein, the terms "associated with HER2" or "related to HER2" in relation to a disease or condition refer to any disease or condition caused by, exacerbated by, or otherwise associated with an increase or decrease in the expression or activity of HER2 (e.g., human HER2).

[0120] As used herein, the term "cancer" refers to any tumor or malignant cell growth, proliferation or metastasis mediated by solid tumors and non-solid tumors such as leukemia and resulting in a medical condition.

[0121] As used herein in the context of treating a condition, the terms "treatment," "treating," or "treated," generally relate to treatment and therapy, whether for humans or animals, in which some desired therapeutic effect is achieved, such as inhibition of progression of the condition, including a reduction in the rate of progression, cessation of the rate of progression, regression of the condition, amelioration of the condition, and cure of the condition. For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, or some combination thereof. For a tumor, "treatment" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, reducing the number of tumors, preventing or delaying tumor development, or some combination thereof.

[0122] As used herein in the context of preventing a condition, the terms "prevent," "preventing," or "prevention" generally refer to preventing or delaying the onset of a disease, or preventing the appearance of clinical or subclinical symptoms thereof in a subject, whether human or animal, e.g., preventing a disease from occurring in a subject susceptible to the condition or disease but not yet diagnosed with the condition or disease.

[0123] As used herein, the term "therapeutically effective amount" relates to an amount of an active compound or a material, composition or dosage form containing an active compound, which, when administered according to a desired treatment regimen, is effective in producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. Specifically, "therapeutically effective amount" refers to an amount or concentration of an antibody that is effective in treating a human CD47 / HER2-related disease or condition.

[0124] As used herein, a "host cell" in the present disclosure refers to a cell into which an exogenous polynucleotide has been introduced.

[0125] As used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0126] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, the pH regulator includes, but is not limited to, a phosphate buffer; the surfactant includes, but is not limited to, a cationic, anionic, or nonionic surfactant, such as Tween-80; and the ionic strength enhancer includes, but is not limited to, sodium chloride.

[0127] As used herein, the term "adjuvant" refers to a non-specific immunopotentiator that, when delivered to an organism together with an antigen or delivered to an organism in advance, can enhance the immune response to the antigen in the organism or change the type of immune response. There are many types of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvants are widely used in clinical trials.

[0128] Bispecific peptide complexes

[0129] The bispecific polypeptide complexes provided herein include bispecific antibodies and antigen-binding portions thereof. As used herein, the term "polypeptide complex" can be used interchangeably with "antibody". In some embodiments, the bispecific antibody and its antigen-binding portion have a first specificity for HER2 (e.g., human, cynomolgus monkey and mouse HER2), and a second specificity for CD47 (e.g., human, cynomolgus monkey and mouse CD47). Such antibodies may be referred to herein as, for example, "anti-HER2 / anti-CD47" or "anti-CD47 / HER2" or "anti-CD47xHER2" or "CD47xHER2" bispecific antibodies or other similar terms.

[0130] In some embodiments, the bispecific antibodies herein comprise a first antigen binding portion (CD47 binding portion) that specifically binds to CD47 and a second antigen binding portion (HER2 binding portion) that specifically binds to HER2. Taking into account the stability, expression level, binding ability and other functions of the assembled antibody, the first and second antigen binding portions can be in the form of Fab, scFv or VHH, etc. For example, the HER2 binding portion is in the form of Fab, scFv or VHH, and the CD47 binding portion is in the form of Fab; or, the CD47 binding portion is in the form of Fab, scFv or VHH, and the HER2 binding portion is in the form of Fab. In some embodiments, both the HER2 binding portion and the CD47 binding portion are in the form of Fab, forming the two arms of the bispecific antibody.

[0131] In some embodiments, the bispecific antibodies disclosed herein comprise more than one antigen binding portion that specifically binds to CD47 and / or more than one antigen binding portion that specifically binds to HER2. Typically, for bispecific antibodies, the more than one antigen binding portion has the same variable region (thereby targeting the same antigen / epitope), or is completely identical in the variable region and constant region (if present). For example, the antibody may comprise two identical CD47 binding portions and one HER2 binding portion, or one CD47 binding portion and two identical HER2 binding portions, or two identical CD47 binding portions and two identical HER2 binding portions. In addition, when there are two CD47 binding portions or two HER2 binding portions, preferably, the two CD47 binding portions and / or the two HER2 binding portions are not on the same chain.

[0132] In some specific embodiments, the CD47 binding portion is in Fab form, comprising a first VH operably linked to an antibody heavy chain CH1 domain and a first VL operably linked to an antibody light chain constant (CL) domain, and the HER2 binding portion is also in Fab form, but comprises a second heavy chain variable domain (VH) operably linked to a first T cell receptor (TCR) constant region (C1) and a second light chain variable domain (VL) operably linked to a second TCR constant region (C2). In other words, in the second antigen binding portion, the commonly used CH1 domain and CL domain are replaced by a pair of TCR constant regions. The positions of C1 and C2 can be interchanged.

[0133] Alternatively, the CD47 binding portion may comprise a first heavy chain variable domain (VH) operably linked to a first T cell receptor (TCR) constant region (C1) and a first light chain variable domain (VL) operably linked to a second TCR constant region (C2), wherein the positions of C1 and C2 may be interchanged, and the HER2 binding portion may comprise a second VH operably linked to an antibody heavy chain CH1 domain and a second VL operably linked to an antibody light chain constant (CL) domain.

[0134] Introducing TCR constant regions to replace commonly used CH1 and CL domains has been shown to increase the stability and expression levels of the generated antibody forms. A detailed description of the use of TCR constant regions in assembling two parent antibodies into bispecific molecules with desired valence and functionality has been disclosed in WO2019 / 057122, the entire contents of which are incorporated herein by reference. Replacement of TCR constant regions (Cbeta / CA1pha) produces chimeric Fabs with unique light-heavy chain interfaces orthogonal to conventional antibody Fabs. The assembly of different forms of chimeric Fabs and conventional Fabs can produce various bispecific molecules with different structures and valences.

[0135] The first TCR constant region and the second TCR constant region are associated via non-natural interchain disulfide bonds. A pair of TCR constant regions in the antigen binding portion includes TCR alpha and beta constant regions (wild type or preferably engineered) in the light chain and heavy chain, respectively. The TCR constant regions in the bispecific antibody can associate with each other to form dimers through non-natural disulfide bonds.

[0136] TCR, or T cell receptor, is a heterodimeric T cell surface protein that belongs to the immunoglobulin superfamily and is similar to a half antibody with a single heavy chain and a single light chain. The natural TCR has an extracellular part, a transmembrane part, and an intracellular part. The extracellular domain of TCR has a membrane-proximal constant region and a membrane-distal variable region.

[0137] The sequences of the wild-type human TCR beta and alpha chain constant regions are available at NCBI under accession number A0A5B9 ( www.uniprot.org / uniprot / A0A5B9 ) and NCBI accession number P01848 ( www.uniprot.org / uniprot / P01848 The pair of TCR constant regions used to construct the bispecific antibodies herein are derived from wild-type TCR constant regions, with one or more substitutions, additions or deletions of one or more amino acids.

[0138] As shown in the present application, the bispecific antibody comprises an engineered TCR beta chain constant region, whose sequence is shown in SEQ ID NO:17; and an engineered TCR alpha chain constant region, whose sequence is shown in SEQ ID NO:18.

[0139] It should be understood that variants of the TCR constant region are not limited to the above sequences, as long as they can stabilize the VH and VL regions to form an antigen binding portion. Multiple C1 and C2 variants for constructing WuXiBody antibody formats have been disclosed in PCT / CN2021 / 072601, the entire contents of which are incorporated herein by reference.

[0140] In some embodiments, the TCR beta chain constant region replaces the CH1 domain and the TCR alpha chain constant region replaces the CL domain. Alternatively, they can be interchanged with the TCR beta chain constant region in the light chain and the TCR alpha chain constant region in the heavy chain.

[0141] The benefits of replacing the CH1 and CL domains with TCR constant regions are significant. In bispecific antibodies, C1 and C2 containing antigen-binding portions with at least one non-natural disulfide bond can be recombinantly expressed and assembled into the desired conformation, which stabilizes the TCR constant region dimer while providing good antigen-binding activity of the antibody variable region. In addition, it was found that C1 and C2 containing antigen-binding portions can tolerate conventional antibody engineering well, such as modification of glycosylation sites and removal of some natural sequences. In addition, due to the presence of the TCR constant region in the antigen-binding portion, this form of bispecific antibody can be easily expressed and assembled, and the mismatch of the antigen-binding sequence is minimal or substantially free of mismatch.

[0142] In some specific embodiments, the bispecific antibodies disclosed herein comprise one HER2 binding moiety and one CD47 binding moiety in each arm, i.e., each antibody comprises two HER2 binding moieties and two CD47 binding moieties. The Fc region can be located at the C-terminus of the antibody, operably linked to the CD47 binding moiety or the HER2 binding moiety at the N-terminus of the Fc region. Alternatively, the Fc region can be located between the CD47 binding moiety and the HER2 binding moiety. Such bispecific antibodies can be constructed as homodimers with two identical heavy chains.

[0143] According to some exemplary embodiments, the bispecific polypeptide complex herein comprises two heavy chains and four light chains, wherein from N-terminus to C-terminus:

[0144] (a) the first and second heavy chains each comprise an operably linked domain as in VH1-C1-VH2-CH1-hinge-Fc (VH1-C1 is from the HER2 binding portion, VH2-CH1 is from the CD47 binding portion); the two light chains comprise an operably linked domain as in VL1-C2 (VL1-C2 is from the HER2 binding portion); and the other two light chains comprise an operably linked domain as in VL2-CL (VL2-CL is from the CD47 binding portion);

[0145] (b) the first and second heavy chains each comprise an operably linked domain as in VH2-CH1-VH1-C1-hinge-Fc (VH1-C1 is from the HER2 binding portion, VH2-CH1 is from the CD47 binding portion); the two light chains comprise an operably linked domain as in VL1-C2 (VL1-C2 is from the HER2 binding portion); and the other two light chains comprise an operably linked domain as in VL2-CL (VL2-CL is from the CD47 binding portion);

[0146] (c) the first and second heavy chains each comprise an operably linked domain as in VH1-C1-hinge-Fc-VH2-CH1 (VH1-C1 is from the HER2 binding portion, VH2-CH1 is from the CD47 binding portion); the two light chains comprise an operably linked domain as in VL1-C2 (VL1-C2 is from the HER2 binding portion); and the other two light chains comprise an operably linked domain as in VL2-CL (VL2-CL is from the CD47 binding portion);

[0147] (d) the first and second heavy chains each comprise an operably linked domain as in VH2-CH1-hinge-Fc-VH1-C1 (VH1-C1 is from the HER2 binding portion, VH2-CH1 is from the CD47 binding portion); the two light chains comprise an operably linked domain as in VL1-C2 (VL1-C2 is from the HER2 binding portion); and the other two light chains comprise an operably linked domain as in VL2-CL (VL2-CL is from the CD47 binding portion);

[0148] (e) the first and second heavy chains each comprise an operably linked domain as in VH1-CH1-VH2-C1-hinge-Fc (VH1-CH1 is from the HER2 binding portion, VH2-C1 is from the CD47 binding portion); the two light chains each comprise an operably linked domain as in VL1-CL (VL1-CL is from the HER2 binding portion); and the other two light chains each comprise an operably linked domain as in VL2-C2 (VL2-C2 is from the CD47 binding portion);

[0149] (f) the first and second heavy chains each comprise an operably linked domain as in VH2-C1-VH1-CH1-hinge-Fc (VH1-CH1 is from the HER2 binding portion, VH2-C1 is from the CD47 binding portion); the two light chains each comprise an operably linked domain as in VL1-CL (VL1-CL is from the HER2 binding portion); and the other two light chains each comprise an operably linked domain as in VL2-C2 (VL2-C2 is from the CD47 binding portion);

[0150] (g) the first and second heavy chains each comprise an operably linked domain as in VH1-CH1-hinge-Fc-VH2-C1 (VH1-CH1 is from the HER2 binding portion, VH2-C1 is from the CD47 binding portion); the two light chains each comprise an operably linked domain as in VL1-CL (VL1-CL is from the HER2 binding portion); and the other two light chains each comprise an operably linked domain as in VL2-C2 (VL2-C2 is from the CD47 binding portion); or

[0151] (h) the first and second heavy chains each comprise an operably linked domain as in VH2-C1-hinge-Fc-VH1-CH1 (VH1-CH1 is from the HER2 binding portion, VH2-C1 is from the CD47 binding portion); the two light chains each comprise an operably linked domain as in VL1-CL (VL1-CL is from the HER2 binding portion); and the other two light chains each comprise an operably linked domain as in VL2-C2 (VL2-C2 is from the CD47 binding portion).

[0152] In some specific embodiments, the bispecific antibodies disclosed herein comprise one HER2 binding moiety and two CD47 binding moieties per antibody, or two HER2 binding moieties and one CD47 binding moiety per antibody.

[0153] According to some exemplary embodiments, the bispecific polypeptide complex herein comprises two heavy chains and three light chains, wherein from N-terminus to C-terminus:

[0154] (a) the first heavy chain comprises domains operably linked as in VH1-C1-VH2-CH1-hinge-Fc, the second heavy chain comprises domains operably linked as in VH2-CH1-hinge-Fc; the first light chain comprises domains operably linked as in VL1-C2, and the second and third light chains comprise domains operably linked as in VL2-CL;

[0155] (b) the first heavy chain comprises domains operably linked as in VH2-CH1-hinge-Fc-VH1-C1, the second heavy chain comprises domains operably linked as in VH2-CH1-hinge-Fc; the first light chain comprises domains operably linked as in VL1-C2, and the second and third light chains comprise domains operably linked as in VL2-CL;

[0156] (c) the first heavy chain comprises domains operably linked as in VH2-CH1-VH1-C1-hinge-Fc, the second heavy chain comprises domains operably linked as in VH1-C1-hinge-Fc; the first and second light chains each comprise a domain operably linked as in VL1-C2, and the third light chain comprises a domain operably linked as in VL2-CL;

[0157] (d) the first heavy chain comprises domains operably linked as in VH1-C1-hinge-Fc-VH2-CH1, the second heavy chain comprises domains operably linked as in VH1-C1-hinge-Fc; the first and second light chains each comprise a domain operably linked as in VL1-C2, and the third light chain comprises a domain operably linked as in VL2-CL;

[0158] (e) the first heavy chain comprises domains operably linked as in VH1-CH1-VH2-C1-hinge-Fc, the second heavy chain comprises domains operably linked as in VH2-C1-hinge-Fc; the first light chain comprises domains operably linked as in VL1-CL, and the second and third light chains comprise domains operably linked as in VL2-C2;

[0159] (f) the first heavy chain comprises domains operably linked as in VH2-C1-hinge-Fc-VH1-CH1, the second heavy chain comprises domains operably linked as in VH2-C1-hinge-Fc; the first light chain comprises domains operably linked as in VL1-CL, and the second and third light chains comprise domains operably linked as in VL2-C2;

[0160] (g) the first heavy chain comprises an operably linked domain as in VH2-C1-VH1-CH1-hinge-Fc, the second heavy chain comprises an operably linked domain as in VH1-CH1-hinge-Fc, the first and second light chains each comprise an operably linked domain as in VL1-CL, and the third light chain comprises an operably linked domain as in VL2-C2; or

[0161] (h) the first heavy chain comprises domains operably linked as in VH1-CH1-hinge-Fc-VH2-C1, the second heavy chain comprises domains operably linked as in VH1-CH1-hinge-Fc, the first and second light chains each comprise a domain operably linked as in VL1-CL, and the third light chain comprises a domain operably linked as in VL2-C2.

[0162] In some specific embodiments, the bispecific antibodies disclosed herein comprise a HER2 binding moiety in one arm and a CD47 binding moiety in the other arm. Figure 1 Shown is a bispecific antibody comprising the following structure (E17): from N-terminus to C-terminus, in the first heavy chain, VH1-C1-hinge-Fc; in the second heavy chain, VH2-CH1-hinge-Fc; in the first light chain, VL1-C2; and in the second light chain, VL2-CL. VH1 and VL1 refer to the first VH and VL that form the HER2 binding site, respectively, and VH2 and VL2 refer to the second VH and VL that form the CD47 binding site, respectively. "-" indicates an operably linked, usually via a linker comprising a peptide bond or a peptide linker having about 1-30 amino acids. The peptide linker can be a series of (G4S)n, where n=1-5.

[0163] Depending on the bispecific format and / or numbering convenience, the numbering sequence disclosed herein, such as first or second, may be different. For example, the first VH may be numbered as the second VH, and the first VL may be numbered as the second VL. As another example, depending on preference and / or convenience, the second antigen binding moiety may be numbered as the first antigen binding moiety, and the first antigen binding moiety may be numbered as the second antigen binding moiety.

[0164] CDRs and variable regions of bispecific peptide complexes

[0165] In some embodiments, the bispecific polypeptide complex or antigen binding portion thereof comprises a first antigen binding portion that specifically binds to HER2 (preferably human HER2) and a second antigen binding portion that specifically binds to CD47 (preferably human CD47), wherein the first and second antigen binding portions are derived from anti-CD47 antibodies and anti-HER2 antibodies, respectively. The parent antibody may have been developed and known to the public, or developed de novo. "Derived from" herein generally means that the antigen binding portion comprises the CDR sequence of the parent antibody or a highly homologous CDR sequence, preferably, the variable region of the parent antibody. The antigen binding portion may also comprise a variant of the CDR sequence of the parent antibody that retains the antigen binding specificity. For example, one or two amino acids in one or more CDR regions may be modified to reduce the risk of glycosylation and deamidation compared to the original CDR sequence of the parent antibody.

[0166] Specifically, in the bispecific antibodies exemplified herein, the HER2 binding portion comprises:

[0167] A) one or more heavy chain CDRs (HCDRs) selected from the group consisting of:

[0168] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO: 1;

[0169] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO: 2; and

[0170] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO: 3;

[0171] B) one or more light chain CDRs (LCDRs) selected from the group consisting of:

[0172] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO:4;

[0173] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO:5; and

[0174] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO:6; or

[0175] C) one or more HCDRs of A) and one or more LCDRs of B); and

[0176] The CD47 binding moiety contains:

[0177] A') one or more HCDRs selected from the group consisting of:

[0178] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO: 7;

[0179] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO: 8; and

[0180] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO: 9;

[0181] B') one or more LCDRs selected from the group consisting of:

[0182] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO: 10;

[0183] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO: 11; and

[0184] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence in which no more than 1, 2 or 3 amino acids are added, deleted and / or substituted compared to SEQ ID NO: 12; or

[0185] C') one or more HCDRs of A') and one or more LCDRs of B').

[0186] The variable region and CDR in the antibody sequence can be identified according to the general rules developed in the art or by comparing the sequence with a database of known variable regions. CDR is described in Kabat et al., J.Biol.Chem.252:6609-6616 (1977); Kabat et al., USDept.of Health and Human Services, "Sequences of proteins of immunological interest" (1991); described in Chothia et al., J.Mol.Biol.196:901-917 (1987); and MacCallum et al., J.Mol.Biol.262:732-745 (1996), wherein the definition includes overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to the CDR of the antibodies disclosed herein is intended to fall within the scope of the present application. The CDRs shown in Table 2 below are defined by the Kabat and IMGT numbering systems. However, Chothia, MacCallum and other methods known in the art can also be used to define CDRs. Methods for identifying these regions are described, for example, in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described below and can be evaluated by the following: "Abysis" website www.bioinf.org.uk / abs (maintained by AC Martin, Department of Biochemistry and Molecular Biology, University College London, London, UK), and the VBASE2 website www.vbase2.org, as described by Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). Preferably, the sequence is analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT and protein database (PDB) with structural data from PDB. See the protein sequence and structural analysis of antibody variable domains in the Antibody Engineering Lab Manual (Antibody Engineering Lab Manual) book by Dr. Andrew CR Martin (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg.uk / abs). The Abysis database website also includes general rules developed for identifying CDRs that can be used according to the teachings of this article.

[0187] In some embodiments, the HER2 binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0188] (a) VH comprises: HCDR1 as shown in SEQ ID NO: 1; HCDR2 as shown in SEQ ID NO: 2; and HCDR3 as shown in SEQ ID NO: 3; and

[0189] (b) VL comprises: LCDR1 as shown in SEQ ID NO:4; LCDR2 as shown in SEQ ID NO:5; and LCDR3 as shown in SEQ ID NO:6.

[0190] In some embodiments, the CD47 binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0191] (a) VH comprises: HCDR1 as shown in SEQ ID NO:7; HCDR2 as shown in SEQ ID NO:8; and HCDR3 as shown in SEQ ID NO:9; and

[0192] (b) VL comprises: LCDR1 as shown in SEQ ID NO:10; LCDR2 as shown in SEQ ID NO:11; and LCDR3 as shown in SEQ ID NO:12.

[0193] In some embodiments, the bispecific antibody, or antigen binding portion thereof, comprises a first antigen binding portion that specifically binds HER2, wherein the first antigen binding portion comprises:

[0194] (A) Heavy chain variable region:

[0195] (i) an amino acid sequence comprising SEQ ID NO: 13;

[0196] (ii) comprises an amino acid sequence that is at least 85%, 90% or 95% identical (preferably, at least 90%, more preferably, at least 95% (e.g., 95%, 96%, 97%, 98% or 99%)) to SEQ ID NO: 13; or

[0197] (iii) comprising an amino acid sequence having one or more (e.g., one, two, three or more, preferably one, two or three, more preferably one or two) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 13; and / or

[0198] (B) Light chain variable region:

[0199] (i) an amino acid sequence comprising SEQ ID NO: 14;

[0200] (ii) comprises an amino acid sequence that is at least 85%, at least 90% or at least 95% identical (preferably, at least 90%, more preferably, at least 95% (e.g., 95%, 96%, 97%, 98% or 99%)) to SEQ ID NO: 14; or

[0201] (iii) an amino acid sequence comprising one or more (e.g., one, two, three or more, preferably one, two or three, more preferably one or two) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 14.

[0202] In some further embodiments, the bispecific antibody, or antigen binding portion thereof, comprises a second antigen binding portion that specifically binds CD47, wherein the second antigen binding portion comprises:

[0203] (A) Heavy chain variable region:

[0204] (i) an amino acid sequence comprising SEQ ID NO: 15;

[0205] (ii) comprises an amino acid sequence that is at least 85%, 90% or 95% identical (preferably, at least 90%, more preferably, at least 95% (e.g., 95%, 96%, 97%, 98% or 99%)) to SEQ ID NO: 15; or

[0206] (iii) comprising an amino acid sequence having one or more (e.g., one, two, three or more, preferably one, two or three, more preferably one or two) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 15; and / or

[0207] (B) Light chain variable region:

[0208] (i) an amino acid sequence comprising SEQ ID NO: 16;

[0209] (ii) comprises an amino acid sequence that is at least 85%, at least 90% or at least 95% identical (preferably, at least 90%, more preferably, at least 95% (e.g., 95%, 96%, 97%, 98% or 99%)) to SEQ ID NO: 16; or

[0210] (iii) comprises an amino acid sequence having one or more (e.g., one, two, three or more, preferably one, two or three, more preferably one or two) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 16.

[0211] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0212] Additionally or alternatively, the protein sequences of the present disclosure may also be used as a "query sequence" to search against public databases to, for example, identify related sequences. Such searches may be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215: 403-10. BLAST protein searches may be performed using the XBLAST program with score = 50 and word length = 3 to obtain amino acid sequences homologous to the antibody molecules of the present disclosure. To obtain gapped alignments for comparison purposes, gapped BLAST may be used as described in Altschul et al, (1997) Nucleic Acids Res. 25 (17): 3389-3402. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) may be used. See www.ncbi.nlm.nih.gov.

[0213] In some specific embodiments, the heavy chain variable region of the HER2 binding portion consists of the amino acid sequence of SEQ ID NO:13, the light chain variable region of the HER2 binding portion consists of the amino acid sequence of SEQ ID NO:14, and / or the heavy chain variable region of the CD47 binding portion consists of the amino acid sequence of SEQ ID NO:15, and the light chain variable region of the CD47 binding portion consists of the amino acid sequence of SEQ ID NO:16.

[0214] In other embodiments, the amino acid sequences of the heavy chain variable region and / or the light chain variable region may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably at least 95%, 96%, 97%, 98% or 99% identical to the corresponding sequences described above.

[0215] In some further embodiments, the bispecific antibodies or antigen-binding portions thereof may contain conservative substitutions or modifications of amino acids in the heavy and / or light chain variable regions. It is understood in the art that certain conservative sequence modifications may be made that will not eliminate antigen binding. See, e.g., Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0216] As mentioned above, the term "conservative substitution" used herein refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which one amino acid residue is replaced by another amino acid residue with a similar side chain, for example, physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0217] Antigen binding of bispecific peptide complexes

[0218] Preferably, the bispecific polypeptide complexes of the present disclosure are capable of binding to human HER2 and CD47. The binding of the antibody to HER2 and CD47 can be assessed using one or more techniques recognized in the art, such as ELISA or FACS, which measure the binding of the antibody to soluble HER2 / CD47 protein or HER2 / CD47 protein expressed on the cell surface, respectively. For example, the antibody can be tested by flow cytometry assay, wherein the antibody reacts with a cell line expressing human HER2 or human CD47, such as CHO cells that have been transfected to express HER2 or CD47 on their cell surface, or a HER2 or CD47 positive cell line, or a HER2 and CD47 double positive cell line.

[0219] Additionally or alternatively, antibodies can be tested for binding in BIAcore binding assays, including binding kinetics (e.g., K D For example, the antibodies disclosed herein are expressed as 1×10 -9 M or smaller K D Bind to human HER2 protein; 8×10 -10 M or smaller K D Bind to human HER2 protein; 6×10 -10 M or smaller K D Bind to human HER2 protein; 4×10 -10 M or smaller K D Bind to human HER2 protein; 2×10 -10 M or smaller K D Bind to human HER2 protein; or at 1.5×10 -10 M or smaller K D Binding to human HER2 protein as measured by surface plasmon resonance.

[0220] Functions of BsAbs

[0221] The bispecific antibodies disclosed herein are characterized by specific functional characteristics or properties. In some embodiments, the antibodies have one or more of the following properties:

[0222] (a) specifically binds to HER2 with high affinity and to CD47 with relatively low affinity compared to a control antibody;

[0223] (b) Bind to target cells without being affected by antigen sinking effect (e.g., human blood cells or Jurkat cells);

[0224] (c) Negligible binding to red blood cells (RBCs), avoiding induction of hemagglutination and phagocytosis of human RBCs;

[0225] (d) binding to Jurkat cells is greatly reduced (e.g., reduced by ∼200-fold) compared to a control antibody;

[0226] (e) effectively blocks the binding of CD47 to SIRPα on CD47 / HER2 double-positive cells without being affected by the antigen sinking effect (e.g., human blood cells or Jurkat cells);

[0227] (f) There is almost no CD47 ligand blocking effect on CD47 single-positive cells;

[0228] (g) has significantly higher CD47 occupancy on CD47 / HER2 double-positive cells than control antibodies;

[0229] (h) Compared with the combination of anti-CD47 and anti-HER2 monotherapy, it has significantly increased ADCP efficacy against CD47 / HER2 double-positive cells, while showing almost no ADCP efficacy against human blood cells;

[0230] (i) It has effective ADCC efficacy against CD47 / HER2 double-positive cells, but shows weak ADCC efficacy against Jurkat cells;

[0231] (j) It has an effective inhibitory effect on the proliferation of CD47 / HER2 double-positive cells;

[0232] (k) significantly inhibits tumor cell growth in HER2-positive cancer models; and

[0233] (l) Having better or comparable rodent pharmacokinetics than a control antibody.

[0234] In some embodiments, the control antibody is a monoclonal antibody. In some embodiments, the control antibody is an anti-CD47 antibody. In some embodiments, the control antibody is a monoclonal anti-CD47 antibody, such as mololimab. In some embodiments, the control antibody is an anti-HER2 antibody. In some embodiments, the control antibody is a monoclonal anti-HER2 antibody, such as trastuzumab or panitumumab.

[0235] In some embodiments, the bispecific polypeptide complexes disclosed herein have a higher binding affinity for HER2 than monospecific anti-HER2 antibodies or other anti-HER2 / CD47 bispecific antibodies. In some embodiments, the bispecific polypeptide complexes disclosed herein have a binding affinity for HER2 that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than monospecific anti-HER2 antibodies or other anti-HER2 / CD47 bispecific antibodies, such as K D Measured in .

[0236] In some embodiments, the bispecific polypeptide complexes disclosed herein have a lower binding affinity for CD47 than a monospecific anti-CD47 antibody or other anti-HER2 / CD47 bispecific antibody. In some embodiments, the bispecific polypeptide complexes disclosed herein have a binding affinity for CD47 that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than a monospecific anti-CD47 antibody or other anti-HER2 / CD47 bispecific antibody, such as K D Measured in .

[0237] In some embodiments, the bispecific polypeptide complexes disclosed herein have a higher binding affinity to HER2 than a monospecific anti-HER2 antibody, and / or a lower binding affinity to CD47 than a monospecific anti-CD47 antibody. In some embodiments, the bispecific polypeptide complexes disclosed herein have a higher or comparable binding affinity to HER2 than trastuzumab, and a lower binding affinity to CD47 than mololimab.

[0238] In some embodiments, the bispecific polypeptide complexes disclosed herein have a higher binding affinity for HER2 than other anti-HER2 / CD47 bispecific antibodies, and / or have a lower binding affinity for CD47 than other anti-HER2 / CD47 bispecific antibodies.

[0239] Not affected by antigen sinking effect

[0240] Antibodies directed against cell membrane-associated antigens often undergo target-mediated clearance, known as the antigen sinking effect. The ubiquitous expression of CD47 is known to reduce the bioavailability of anti-CD47 mAbs at tumors due to the antigen sinking effect, and there is also a risk of off-target toxicity, most notably from cross-linking of erythrocytes that display high CD47 expression.

[0241] The expression of CD47 on normal tissues may produce "antigen sinking" that prevents anti-CD47 therapeutic antibodies from reaching their intended tumor cell targets in vivo. As demonstrated herein, one strategy that may circumvent this problem is to employ BsAbs with reduced affinity for CD47. These BsAbs retain the ability to block the CD47-SIRPα interaction but require binding to a second tumor antigen to achieve high-affinity binding.

[0242] The BsAb disclosed herein shows that targeting CD47 and HER2 can specifically direct CD47-SIRPα blockade to cells co-expressing HER2, and the BsAb exhibits therapeutic synergy not observed with monospecific anti-CD47 antibodies or monospecific anti-HER2 antibodies. The BsAbs specific for CD47 (with reduced affinity) and HER2 (with high affinity) herein show negligible RBC antigen sinking, and exhibit binding specificity and potent functional effects on CD47 and HER2 double-positive cancer cells.

[0243] Blocking the binding of CD47 to SIRPα

[0244] CD47 is normally expressed on the surface of normal healthy cells and migrates hematopoietic stem cells to prevent phagocytosis, and is upregulated in nearly all hematological and solid tumors to evade immune surveillance and escape phagocytosis. Disrupting the interaction between CD47 and SIRPa enables phagocytes to "eat" and destroy cancer cells. CD47 blockade repolarizes tumor-associated macrophages to a pro-inflammatory, anti-tumor state, and clearance of malignant cells by phagocytes provides an additional pathway for presentation of new antigens to the adaptive immune system.

[0245] Signal regulatory protein alpha (SIRPα, also known as CD172a) is a receptor for CD47, which is primarily expressed on the surface of macrophages. CD47 is known to interact with SIRPα and can therefore escape immune surveillance. The antibodies disclosed herein can modulate, such as block, inhibit, reduce, antagonize, neutralize or otherwise interfere with the binding of CD47 to SIRPα. Blocking the CD47-SIRPα interaction using the antibodies described herein can improve or overcome immune escape, thereby generating potential clinical benefits.

[0246] As demonstrated in the examples, the bispecific antibodies described herein can effectively block the binding of CD47 ligands to CD47 / HER2 double-positive cell lines, i.e., block the interaction of CD47 with SIRPα, while CD47 ligand blocking is not shown on CD47 single-positive cell lines (e.g., Jurkat cells). In addition, the blocking effect on CD47 / HER2 double-positive cell lines is not affected by the antigen sinking effect caused by, for example, human blood cells or Jurkat cells.

[0247] Weakly binds to human red blood cells (RBC) to prevent hemagglutination

[0248] The ubiquitous expression of CD47, especially on RBCs, limits the use of anti-CD47 antibody therapy. Many anti-CD47 antibodies have been reported to cause hemagglutination of human erythrocytes. In preclinical studies, transient hemolytic anemia has been associated with anti-CD47 therapy due to increased erythrocyte clearance.

[0249] The antibodies disclosed herein show negligible binding to human red blood cells and avoid the undesirable effects of hemagglutination. Compared with the control anti-CD47 antibody, the bispecific antibodies herein also show about 200-fold reduced binding to Jurkat cells. Consistently, the phagocytosis of human RBCs induced by the bispecific antibodies herein will be much milder than the control antibodies.

[0250] Mediation of ADCP or ADCC activity

[0251] The term "antibody-dependent cellular phagocytosis" or "ADCP" is a cellular process in which effector cells with phagocytic potential (such as monocytes and macrophages) can internalize target cells. Once engulfed, the target cell resides in a phagosome, which fuses with a lysosome and begins to degrade the target cell via an oxygen-dependent or -independent mechanism. This function relies on opsonization, or the identification of target cells with antibodies, which then also act as a bridge between target cells and phagocytes. Mechanistically, antibodies bind to cognate antigens on target cells through their antigen recognition domains, and then recruit phagocytes to the target with their Fc regions. Once bound to the Fc receptors of phagocytes, the target cell is engulfed and degraded. This process also leads to the production of soluble factors by effector cells that help initiate and drive immune responses.

[0252] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are absolutely necessary for this killing. NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). In order to assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0253] As disclosed herein, ADCP can be the mechanism of action of anti-CD47 therapeutic antibodies. The bispecific antibodies herein show effective ADCP efficacy to CD47 / HER2 double positive cells. In addition, these antibodies disclosed herein can induce effective ADCC activity against CD47 / HER2 double positive tumor cells. On the other hand, BsAb has little or very weak ADCP or ADCC efficacy to CD47 single positive cells, indicating that binding to HER2 is essential for the effector function of these antibodies herein.

[0254] Synergy

[0255] The bispecific antibodies disclosed herein combine the dual binding activities of CD47 and HER2 and exhibit a synergistic effect compared to monotherapy such as trastuzumab, panitumumab or mololimab alone.

[0256] The present disclosure has demonstrated that in the HCC1954 breast cancer model or the NCI-N87 xenograft model in mice, administration of the bispecific antibodies disclosed herein achieved significantly improved tumor growth inhibition compared to anti-HER2 mAb or anti-CD47 mAb alone. The combination of the bispecific antibodies disclosed herein with another anti-HER2 mAb further improved the anti-tumor effect at the same dose level.

[0257] Fc region

[0258] The Fc region of the bispecific antibodies disclosed herein is preferably a human IgG Fc region. The IgG Fc region can be of any isotype, including but not limited to IgG1, IgG2, IgG3 or IgG4. In certain embodiments, the Fc region is of the IgG1 isotype.

[0259] In the context of the bispecific antibodies of the present disclosure, the Fc region may comprise one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to the wild-type Fc region. The present disclosure encompasses bispecific antigen-binding molecules comprising one or more modifications in the Fc region to obtain the desired function, such as a "knob-into-hole" structure that promotes heterodimerization, or a modified Fc region that alters the binding interaction between the Fc and FcRn or FcγR.

[0260] As used herein, the term "knob into hole" refers to engineering the CH3 domain of an antibody Fc region to create a "knob" or "hole" in each heavy chain to promote heterodimerization. The knob can be obtained by replacing a small amino acid residue in the first CH2 / CH3 polypeptide with a larger amino acid residue, and the hole can be obtained by replacing a large residue with a smaller amino acid residue. For detailed information on the mutation sites for the knob into the hole, see Spiess et al., 2015, supra and Brinkmann et al., 2017, supra; U.S. Patent Application US2003078385A1. In general, according to the EU numbering of Kabat et al., the "knob" is constructed by replacing T366 with a large residue W on one heavy chain, while the corresponding "hole" is formed by a triple mutation of T366S, L368A and Y407V on the other heavy chain. In some embodiments, the "hole" mutation is Y349C, T366S, L368A and / or Y407V, and the "knob" mutation is S354C and / or T366W. In some embodiments, the heavy chain of the bispecific antibody comprising C1 has a "hole" structure, while the heavy chain comprising C2 has a "knob" structure. Alternatively, the heavy chain of the bispecific antibody comprising C1 has a "knob" structure, while the heavy chain comprising C2 has a "hole" structure.

[0261] In certain embodiments, the first heavy chain of the bispecific antibody comprises an Fc region comprising S354C and T366W substitutions (knob), and the second heavy chain of the bispecific antibody comprises an Fc region of the IgG1 isotype comprising Y349C, T366S, L368A, and Y407V substitutions (hole). In some other embodiments, the first heavy chain of the bispecific antibody comprises an Fc region of the IgG4 isotype, wherein the Fc region comprises S354C and T366W substitutions (knob), and the second heavy chain of the bispecific antibody comprises an Fc region of the IgG4 isotype, wherein the Fc region comprises Y349C, T366S, L368A, and Y407V substitutions (hole).

[0262] In addition, the Fc region may include one or more amino acid modifications (e.g., Leu234Ala / Leu235Ala or LALA) that alter antibody-dependent cellular cytotoxicity (ADCC) or other effector functions. In certain embodiments, the Fc modification includes a LALA mutation, i.e., a mutation of L234A and L235A (according to EU numbering in Kabat et al.). The LALA mutation may be the most commonly used mutation to disrupt antibody effector functions, for example, eliminating the binding of Fc to specific FcγRs and reducing ADCC activity mediated by PBMCs and monocytes. In addition, it has been found that the therapeutic potential can be enhanced by introducing YTE (M252Y / S254T / T256E) and LS (M428L / N434S) in the Fc region, thereby increasing the half-life and prolonging the duration of protection. The S228P mutation was also found to prevent IgG4 Fab arm exchange in vivo and in vitro, as demonstrated using a combination of a novel quantitative immunoassay and physiological matrix preparation (J Biol Chem 2015 Feb 27; 290(9): 5462-9).

[0263] The bispecific antibodies disclosed herein may comprise an Fc region selected from one of the following:

[0264] (a) Human IgG1 Fc region engineered to contain a “knob-into-hole” structure;

[0265] (b) a human IgG4 Fc region engineered to contain a "knob-into-hole" structure and optionally an S228P mutation; and

[0266] (c) human IgG4 Fc region engineered to contain the S228P mutation and optionally the M252Y / S254T / T256E mutations.

[0267] When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" (e.g., the EU index reported by Kabat et al., supra) is generally used. "EU numbering in Kabat" or "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise indicated herein, references to residue numbers in an antibody constant domain refer to residue numbering by the EU numbering system.

[0268] Nucleic acid molecules encoding BsAb

[0269] In some aspects, the disclosure relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding one or more chains of a bispecific antibody, or antigen-binding portion thereof.

[0270] Nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared by transgenic mice carrying human immunoglobulin genes as further described below), cDNAs encoding the light and heavy chains of antibodies prepared by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from gene libraries.

[0271] The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably linking the nucleic acid encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3 or TCR beta constant region). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al. (1991), supra), and DNA fragments covering these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably an IgG1 or IgG4 constant region.

[0272] By operably linking the DNA encoding VL to another DNA molecule encoding the constant region of the light chain CL or TCR alpha constant region, the isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequence of the human light chain constant region gene is known in the art (see, e.g., Kabat et al., supra), and the DNA fragments covering these regions can be obtained by standard PCR amplification. In some embodiments, the light chain constant region can be a kappa or lambda constant region.

[0273] Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" is intended to mean that the two DNA fragments are connected so that the amino acid sequences encoded by the two DNA fragments remain in the reading frame.

[0274] In some embodiments, the isolated nucleic acid molecule comprises one or more nucleic acid sequences selected from the group consisting of:

[0275] (A) a nucleic acid sequence encoding a heavy chain sequence of a CD47 binding portion or a heavy chain sequence of a HER2 binding portion;

[0276] (B) a nucleic acid sequence encoding a heavy chain sequence of a CD47 binding portion operably linked to a heavy chain sequence of a HER2 binding portion;

[0277] (C) a nucleic acid sequence encoding a light chain sequence of a CD47 binding portion;

[0278] (D) a nucleic acid sequence encoding a light chain sequence of a HER2 binding portion;

[0279] (E) Any combination of (A)-(D); and

[0280] (F) Nucleic acid sequence that hybridizes to the complementary strand of the nucleic acid sequence of (A) to (E) under high stringency conditions.

[0281] In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding SEQ ID NO: 19. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding SEQ ID NO: 20. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding SEQ ID NO: 21. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding SEQ ID NO: 22.

[0282] Exemplary high stringency conditions include hybridization at 45°C in 5X SSPE and 45% formamide, and final washing in 0.1X SSC at 65°C. It is understood in the art that conditions of equal stringency can be achieved by changing the temperature and buffer or salt concentration, as described in Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications to hybridization conditions can be determined empirically or accurately calculated based on the length and percentage of guanosine / cytosine (GC) base pairing of the probe. Hybridization conditions can be calculated as described in Sambrook, et al, (Eds.), Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.

[0283] Vectors and host cells

[0284] Nucleic acid molecules encoding bispecific polypeptide complexes can be inserted into vectors for further cloning (amplification of DNA) or for expression using recombinant techniques known in the art. In some embodiments, antibodies can be produced by homologous recombination known in the art. DNA encoding monoclonal antibodies is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding antibody heavy chains). Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence. Selectable marker genes help select host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically, the selection marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Selectable marker genes can include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0285] In some embodiments, the vector system includes mammals, bacteria, yeast systems, etc., and includes plasmids, such as but not limited to pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc. and other laboratories and commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). In one embodiment of the invention, the vector may be pET, such as pETbac containing a hexahistidine tag and a c-Myc tag gene.

[0286] Vectors containing nucleic acid sequences encoding the bispecific polypeptide complexes can be introduced into host cells for cloning or gene expression. Therefore, the present disclosure also relates to recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the bispecific polypeptide complexes of the present disclosure.

[0287] Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast or higher eukaryotic cells, such as mammalian cells. Mammalian host cells for expressing the antibodies of the present disclosure include Chinese hamster ovary (CHO cells) (including dhfr CHO cells, which are described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. L. A. 77: 4216-4220, used with a DHFR selective marker, for example, as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159: 601-621), COS cells and SP2 cells. Specifically, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036 and EP 338,841.Also included are monkey kidney CV1 lines transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 or 293 cells subcloned for suspension culture growth, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216); mouse supporting cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; mouse myeloma cells, such as NSO (e.g., RCB0213, 1992, Bio / Technology 10:16 9) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC CRL 1581); rat myeloma cells, such as YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16 AG.20 cells, ATCC CRL 1662); PER.C 6 cells; and human hepatoma cell line (Hep G2). CHO cells are one of the cell lines that can be used herein, with CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555 (1986)) and Lec13 being exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44 or CHO-DP12 host cells, these can be altered so that they lack the ability to fucosylate proteins expressed therein.

[0288] Suitable prokaryotes for this purpose include eubacteria, e.g., Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, marcescans and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

[0289] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for bispecific antibody encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among the lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and can be used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. Wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0290] Other suitable host cells for expressing the bispecific polypeptide complexes provided herein are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. Many baculovirus strains and variants and corresponding licensed insect host cells have been identified from hosts such as Spodopterafrugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori (Bombyx mori). A variety of virus strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses according to the present disclosure, particularly for transfection of Spodopterafrugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.

[0291] Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

[0292] Host cells used to produce the bispecific polypeptide complexes provided herein can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the culture media described in the following can be used as culture media for host cells: Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. Re. 30,985. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drugs), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., are those previously used with the host cells selected for expression and are apparent to those of ordinary skill in the art.

[0293] When using recombinant technology, antibodies can be produced in cells, periplasmic space, or directly secreted into culture medium. If the antibody is produced in cells, as a first step, particle debris (host cells or cleavage fragments) is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describes a method for separating antibodies secreted into the periplasmic space of Escherichia coli. In short, cell paste is thawed for about 30min in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When the antibody is secreted into culture medium, the supernatant from such expression system is usually first concentrated using a commercially available protein concentration filter (such as Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0294] Antibodies produced from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.

[0295] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably at low salt concentration (e.g., from about 0-0.25 M salt).

[0296] Pharmaceutical composition

[0297] In some aspects, the present disclosure relates to a pharmaceutical composition comprising a bispecific polypeptide complex disclosed herein and a pharmaceutically acceptable carrier.

[0298] Components of the composition

[0299] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present disclosure may also be administered in combination therapy with, for example, another immunostimulant, anticancer agent, antiviral agent or vaccine, so that the bispecific polypeptide complex enhances the immune response to the vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobials, isotonic agents, buffers, antioxidants, anesthetics, suspending agents / dispersants, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, various component combinations or more known in the art.

[0300] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propylgalactate. As disclosed herein, the composition containing the antibody includes one or more antioxidants such as methionine to reduce the oxidation of the antibody. Redox can prevent or reduce the reduction of binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the present disclosure provides a composition comprising one or more antibodies and one or more antioxidants such as methionine. The present invention also provides a variety of methods, wherein the antibody is mixed with one or more antioxidants such as methionine, so that the antibody can be prevented from being oxidized, to extend its shelf life and / or increase activity.

[0301] To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antibacterial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (Tween-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multidose containers including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, dissolution enhancers or, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0302] Administration, dosage form and dose

[0303] The pharmaceutical compositions of the present disclosure can be administered to subjects in need in vivo by a variety of routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, external, transdermal and intrathecal, or otherwise by implantation or inhalation. The compositions of the present subject can be formulated into preparations in solid, semisolid, liquid or gaseous form; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. Appropriate preparations and routes of administration can be selected according to the intended application and treatment regimen.

[0304] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalants and controlled release forms thereof.

[0305] Preparations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the preparation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohol, polyols, glycerol, vegetable oils, etc. Examples of suitable isotonic carriers for such preparations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosing regimens, i.e., dosage, time, and repetition, will depend on the specific individual and the individual's medical history, as well as empirical considerations, such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0306] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferative or tumorigenic cells, maintaining the reduction of such tumor cells, reducing the proliferation of neoplastic cells, or delaying the development of metastasis. In some embodiments, the dosage administered can be adjusted or weakened to control potential side effects and / or toxicity. Alternatively, a sustained continuous release formulation of the subject therapeutic composition may be suitable.

[0307] It will be appreciated by those skilled in the art that suitable dosages may vary from patient to patient. Determining the optimal dosage generally involves a balance between the level of therapeutic benefit and any risk or harmful side effects. The selected dosage level will depend on a variety of factors, including but not limited to the activity of a particular compound, route of administration, time of administration, excretion rate of the compound, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, sex, age, weight, illness, general health and previous medical history of the patient. The amount of the compound and route of administration will ultimately be determined by a physician, veterinarian or clinician, but in general, the choice of dosage will reach a local concentration at the site of action, thereby achieving the desired effect, and will not cause substantial harmful or toxic side effects.

[0308] In general, the bispecific polypeptide complexes can be administered in various ranges. In some embodiments, the bispecific polypeptide complexes provided herein can be administered in a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In some of these embodiments, the antibody is administered at a dosage of about 50 mg / kg or less, and in some of these embodiments, the dosage is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the dosage can be changed during treatment. For example, in certain embodiments, the initial dosage can be higher than the subsequent dosage. In certain embodiments, the dosage can change during treatment according to the reaction of the subject.

[0309] In any case, the antibodies of the present disclosure are preferably administered to subjects in need as needed. The frequency of administration can be determined by those skilled in the art, such as the attending physician based on considerations of the condition being treated, the age of the subject being treated, the severity of the condition being treated, the overall health status of the subject being treated, etc.

[0310] In certain preferred embodiments, the course of treatment involving the antibodies of the present disclosure will include multiple doses of the selected drug product over a period of weeks or months. More specifically, the antibodies of the present disclosure may be administered once a day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks, or every three months. In this regard, it will be appreciated that the dosage may be varied or the interval adjusted based on patient response and clinical practice.

[0311] The dosage and regimen of the disclosed therapeutic composition in an individual to which one or more administrations have been given can also be determined empirically. For example, an increasing dose of the therapeutic composition produced as described herein can be given to an individual. In a selected embodiment, the dosage can be gradually increased or decreased or weakened based on the side effects or toxicity determined or observed empirically, respectively. In order to evaluate the efficacy of the selected composition, the markers of a specific disease, disorder or condition can be tracked as described above. For cancer, including direct measurement of tumor size via palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; assessment of improvement by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor markers (such as PSA for prostate cancer) or tumorigenic antigens, pain or paralysis identified according to the methods described herein; improvement of speech, vision, breathing or other tumor-related disabilities; increased appetite; or improvement of quality of life or extension of survival measured by recognized tests. It is obvious to those skilled in the art that the dosage will vary according to the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other locations in the individual, and the treatment used in the past and at the same time.

[0312] Compatible formulations for parenteral administration (e.g., intravenous injection or infusion) may include a concentration of about 10 μg / ml to about 100 mg / ml of the bispecific polypeptide complex provided herein. In some embodiments, the concentration of the bispecific antigen binding molecule may include 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml or 1 mg / ml. In other preferred embodiments, the concentration of the bispecific antigen binding molecule comprises 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml or 100 mg / ml.

[0313] Applications / Indications

[0314] The bispecific polypeptide complexes disclosed herein have a variety of in vitro and in vivo uses. For example, these molecules can be administered to cells in culture in vitro or ex vivo, or, for example, administered to human subjects in vivo, to enhance immunity in various situations. The immune response can be enhanced, stimulated or upregulated.

[0315] Preferred subjects include human patients who need to enhance immune response. The method is particularly suitable for treating human patients with diseases that can be treated by enhancing immune response (e.g., T cell-mediated immune response, phagocytosis of tumor cells). The method is particularly suitable for the treatment of cancer in vivo. In order to achieve antigen-specific enhancement of immunity, bispecific antibodies can be administered together with an antigen of interest, or the antigen may already be present in a subject to be treated (e.g., a subject carrying a tumor or carrying a virus). When a bispecific antibody is administered together with another agent, the two can be administered in either order or simultaneously.

[0316] Treating medical conditions including cancer

[0317] In some aspects, the disclosure provides a method of treating a disorder in a subject, comprising administering a therapeutically effective amount of an antibody or antigen-binding portion thereof disclosed herein to a subject (e.g., a human) in need of treatment. For example, the disorder is cancer.

[0318] A variety of cancers involving CD47 and / or HER2, whether malignant or benign and whether primary or secondary, can be treated or prevented using the methods provided by the present disclosure. The cancer can be a solid cancer or a hematological malignancy. Examples of such cancers include lung cancers such as bronchogenic cancers (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (non-cancerous) and sarcoma (cancerous); cardiac cancers such as myxoma, fibroma and rhabdomyoma; bone cancers such as osteochondroma, enchondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, especially Ewing's tumor (Ewing's sarcoma) and reticulum cell sarcoma; brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytomas, astrocytomas, oligodendrogliomas, medulloblastomas, chordomas, schwannomas, ependymomas, meningiomas, pituitary adenomas, pinealomas, osteomas, hemangioblastomas, craniopharyngiomas, chordomas, germ cell tumors, teratomas, dermoid cysts, and hemangiomas; digestive system cancers such as colon cancer, leiomyoma, epidermoid carcinoma, adenocarcinoma, Leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colorectal polyps and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, bile duct cancer, hepatoblastoma and angiosarcoma; kidney cancers such as renal adenocarcinoma, renal cell carcinoma, adrenal tumor and renal pelvic transitional cell carcinoma; bladder cancer; skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma and Paget's disease; head and neck cancer; eye-related cancers such as retinoblastoma and intraocular melanoma Cancer; male reproductive system cancers such as benign prostatic hyperplasia, prostate cancer and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma and choriocarcinoma); breast cancer; female reproductive system cancers such as uterine cancer (endometrial cancer), cervical cancer (cervical cancer), ovarian cancer (ovarian cancer), vulvar cancer, vaginal cancer, fallopian tube cancer and hydatidiform mole; thyroid cancer (including papillary, follicular, anaplastic or medullary cancer); pheochromocytoma (adrenal gland); non-cancerous growths of the parathyroid glands; pancreatic cancer. In some specific embodiments, the cancer is skin cancer (e.g., squamous cell carcinoma of the skin), colon cancer, colorectal cancer (e.g., colorectal adenocarcinoma), lung cancer (e.g., lung adenocarcinoma) or breast cancer (e.g., triple-negative breast adenocarcinoma).

[0319] In some other embodiments, the condition is an autoimmune disease. Examples of autoimmune diseases that can be treated with the antibody or its antigen-binding portion include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. The antibody or its antigen-binding portion can also be used to treat or prevent infectious diseases, inflammatory diseases (e.g., allergic asthma), and chronic graft-versus-host disease.

[0320] Used in combination with chemotherapy

[0321] The antibodies can be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0322] The term "anticancer agent" or "antiproliferative agent" refers to any agent that can be used to treat cell proliferative disorders such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, tumor reduction agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents and immunotherapeutic agents. It should be understood that in selected embodiments as described above, such anticancer agents may comprise conjugates and may be associated with the disclosed site-specific antibodies prior to administration. More specifically, in some embodiments, the selected anticancer agent will be connected to the unpaired cysteine ​​of the engineered antibody to provide an engineered conjugate as described herein. Therefore, such engineered conjugates are clearly intended to be within the scope of the present disclosure. In other embodiments, the disclosed anticancer agent will be administered in combination with a site-specific conjugate comprising a different therapeutic agent as described above.

[0323] As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell destruction. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins that are bacterial (e.g., diphtheria toxin, Pseudomonas endotoxins and exotoxins, Staphylococcus enterotoxin A), fungal (e.g., α-sarcin, restrictocin), plant (e.g., abrin, ricin, capsularin, quercetin, pokeweed antiviral protein, saporin, gelonin, momordica charantia, trichosanthin, hordeoside toxin, Aleurites fordii protein, dianthin protein, pokeweed protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, Curcin, crotonin, saponin inhibitor, gelonin, mitegrin toxin, restrictocin, phenomycin, neomycin and trichothecenes), or animal (e.g., cytotoxic RNases, such as extracellular pancreatic RNase; DNase I, including fragments and / or variants thereof).

[0324] For the purposes of this disclosure, "chemotherapeutic agents" include chemical compounds (e.g., cytotoxic agents or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation and / or survival of cancer cells. Such chemical agents are generally directed to the intracellular processes necessary for cell growth or division, and are therefore particularly effective for cancer cells that usually grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. In general, chemotherapeutic agents may include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become cancerous or produce tumorigenic progeny (e.g., TICs). Such agents are generally administered in combination, and the combination is generally the most effective, for example in regimens such as CHOP or FOLFIRI.

[0325] Examples of anticancer agents that can be used in combination with the site-specific constructs of the present disclosure (either as a component of a site-specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethyleneimine and methylmelamine, polyacetyl, camptothecin, bryostatin, callystatin, CC-1065, cryptophycin, dolestacin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongestatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne antibiotic chromophores, aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin, carabicin, carminomicin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogamycin, olivomycin, peplomycin, porfibrinocin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, tuberculocidin, ubenimex, netastatin, daunorubicin; antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folate analogs, purine analogs, androgens, antiadreners, folic acid supplements (eg, frolinic acid, acetylglucosylceramide , aldehyde phosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, amustine, bisantrene, edatraxate, defofamine, colcemid, serquinone, elfornithine, elliptonium acetate, epothilone, etoglu, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoid alkaloids, mitoguanidine, mitoxantrone, mopidarol, diamine nitracrine, pentostatin, mustard, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, methylbenzalazine, polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane; rhizoxin; sizofuran; spirogermaniun; tenuisporic acid; triazoquinone; 2,2',2"-trichloroethylamine; trichothecenes (especially T-2 toxin, verracurin A, baculozolin A, and serpentin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; gacytosine; cytarabine "(Ara-C"); cyclophosphamide; sulfotep; taxanes, chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; mitoxantrone; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (CPT-11), topoisomerase inhibitor RFS2000; difluorometlhylornithine; retinoic acid; capecitabine; combretin; folinic acid; oxaliplatin; PKC-alpha, Raf, H-Ras, HER2 and VEGF-A inhibitors that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the above. The definition also includes anti-hormonal agents used to regulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase that regulates the production of estrogen in the adrenal glands, and antiandrogens; as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, rIL-2; Topoisomerase 1 inhibitors; rmRH; Vinorelbine and Esperamicins and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0326] Used in combination with radiation therapy

[0327] The present disclosure also provides a combination of antibodies and radiotherapy (i.e., any mechanism for inducing DNA damage locally in tumor cells, such as gamma-radiation, X-rays, UV-radiation, microwaves, electron emission, etc.). Combination therapy using radioisotopes for directed delivery to tumor cells is also contemplated, and the disclosed conjugates can be used in combination with targeted anticancer agents or other targeted means. Typically, radiotherapy is administered in pulses over a period of about 1 to about 2 weeks. Radiotherapy can be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple continuous doses.

[0328] Pharmaceutical packaging and kits

[0329] Also provided are pharmaceutical packaging and kits comprising one or more containers, which contain one or more doses of antibodies. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition, the composition comprising, for example, an antibody, with or without one or more additional agents. For other embodiments, such a unit dose is supplied in a disposable prefilled syringe for injection. In yet other embodiments, the composition contained in the unit dose may include saline, sucrose, etc.; buffers such as phosphates, etc.; and / or formulated within a stable and effective pH range. Alternatively, in some embodiments, the conjugate composition may be provided in the form of a lyophilized powder, which may be reconstituted when an appropriate liquid (e.g., sterile water or saline solution) is added. In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the enclosed conjugate composition is used to treat a selected tumor disease condition.

[0330] The present disclosure also provides a kit for producing a single dose or multiple dose administration unit of a site-specific conjugate and optionally one or more anticancer agents. The kit includes a container and a label or package associated with or inserted into the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of a variety of materials such as glass or plastic, and contains a pharmaceutically effective amount of the disclosed conjugate in a conjugated or non-conjugated form. In other preferred embodiments, the container includes a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). Such kits will generally include a pharmaceutically acceptable formulation of an engineered conjugate in a suitable container, and optionally one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnosis or combined therapy. For example, in addition to antibodies, such a kit may include any one or more of a series of anticancer agents, such as chemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents.

[0331] More specifically, the kit can have a single container containing the disclosed antibodies, with or without additional components, or they can have different containers for each desired agent. When providing a combination therapeutic agent for conjugation, a single solution can be combined in molar equivalents or premixed with one component more than another component. Alternatively, the conjugate of the kit and any optional anticancer agent can be stored alone in different containers before being administered to the patient. The kit can also include a second / third container device for accommodating sterile pharmaceutically acceptable buffers or other diluents, such as antibacterial water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and dextrose solution.

[0332] When the components of the kit are provided in the form of one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit may be provided as dry powders. When the reagents or components are provided as dry powders, the powders may be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in another container.

[0333] As briefly noted above, the kit may also include a device for administering the antibody and any optional components to a patient, such as one or more needles, IV bags or syringes, or even a dropper, pipette or other similar device from which the formulation can be injected or introduced into an animal or applied to a diseased area of ​​the body. The kits of the present disclosure will also typically include a device for containing vials and the like, as well as other components that are strictly limited for commercial sale, such as an injection or blow molded plastic container in which the desired vials and other equipment are placed and retained.

[0334] SEQUENCE LISTING SUMMARY

[0335] Attached to this application is a sequence listing containing a number of amino acid sequences. Table A below provides a summary of the sequences contained.

[0336] Table A

[0337]

[0338]

[0339] Example

[0340] The present disclosure thus generally described will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present disclosure.The examples are not intended to represent that the following experiments are all or the only experiments performed.

[0341] Example 1

[0342] Material preparation

[0343] Table 1 provides information on commercially available materials used in the examples.

[0344] Table 1: Commercial Materials

[0345]

[0346]

[0347] Example 2

[0348] Generation of W308032 bispecific antibody

[0349] To generate BsAbs, a CD47 binding portion derived from an anti-CD47 antibody (CD47:T6) and a HER2 binding portion derived from an anti-HER2 antibody (HER2:U5) were combined and used to construct a bispecific antibody.

[0350] Bispecific antibodies E17 form construction ( Figure 1 ). CA1pha and CBeta genes were synthesized by Genewiz Inc. Light chain 1 is the VL sequence of HER2:U5 inserted into a linearized vector containing CA1pha, and light chain 2 is the VL sequence of CD47:T6 inserted into a linearized vector containing the lambda light chain constant region. Heavy chain 1 is a VH(HER2:U5)-CBeta DNA fragment inserted into a linearized vector containing the human IgG1 constant region CH2-CH3 with S354C-T366W mutations, and heavy chain 2 is a VH(CD47:T6) DNA fragment inserted into a linearized vector containing the human IgG1 constant region CH1-CH2-CH3 with Y349C-T366S-L368A-Y407V mutations. All sequences were cloned into a modified pcDNA3.4 expression vector.

[0351] According to the manufacturer's instructions, the heavy chain and light chain expression plasmids were co-transfected into Expi293 cells using the Expi293 expression system kit. After transfection for five days, the supernatant of the Expi293 cells expressing the target protein was collected and purified by filtration using a Protein A column. The fractions eluted from Protein A were collected, and the pH was adjusted to 5.0 to perform IEX purification using a CEX column. Before and after loading, the CEX column was balanced with 50mM NaAc (pH5.0). The peak fractions collected were detected by UA with a linear step using 50mM NaAc, 500mM NaCl pH5.0, and then dialyzed in PBS buffer. The concentration of the purified protein was determined by the absorbance at 280nm. The size and purity of the purified protein were tested by SDS-PAGE and SEC-HPLC, respectively.

[0352] The obtained antibody was named W308032-U5T6.E17-57.uIgG1 (or abbreviated as "W308032-U5T6.E17").

[0353] Table 2: CDR sequences of W308032-U5T6.E17 antibody

[0354]

[0355]

[0356] Example 3

[0357] In vitro characterization of the W308032 antibody

[0358] 3.1 Protein analysis

[0359] The size and purity of the W308032 antibody were assessed by SDS-PAGE and size exclusion chromatography (SEC-HPLC). Figure 2 and Figure 3 And Table 3 shows the SDS-PAGE and SEC-HPLC characterization of W308032-U5T6.E17-57.uIgG1 after purification. Figure 2 The apparent molecular weights were about 150 kDa under non-reducing conditions and about 50 kDa and 25 kDa under reducing conditions. The results indicated that the bispecific molecules could be assembled as expected.

[0360] Table 3: Purification summary

[0361]

[0362] 3.2 Differential Scanning Fluorescence (DSF)

[0363] The melting temperature (Tm) of the studied antibodies was determined by DSF using the 7500 Fast Real-Time PCR System (Applied Biosystems). Briefly, 19 μL of antibody solution was mixed with 1 μL of 62.5x SYPRO Orange solution (TheromFisher-S6650) and added to a 96-well plate. The plate was heated from 26°C to 95°C at a rate of 2°C / min, and the resulting fluorescence data were collected. Data collection and Tm calculations were performed by the operating software ( Real-time PCR software v1.3) was used for automatic execution.

[0364] DSF results showed that Tm1 reached 63.1℃ and Tm2 reached 70.1℃, indicating that W308032-U5T6.E17-57.uIgG1 has good thermal stability ( Figure 4 ).

[0365] Table 4: Summary of DSF characterization

[0366] Antibody Name Tm1(℃) Tm2(℃) evaluate W308032-U5T6.E17-57.uIgG1 63.1 70.1 good

[0367] 3.3 Measurement of diffusion interaction parameter (kD) by dynamic light scattering (DLS)

[0368] kD measurements were studied using DynaPro Plate Reader III (Wyatt DynaproTM). The kD parameter is a first-order diffusion interaction parameter obtained in DLS assays as an indicator of molecular colloidal stability and thermal stability. The sample was first filtered with a 0.02 μm filter and concentrated to more than 20 mg / mL. The sample was diluted with PBS buffer to a final concentration of 2.5, 5, 10, 15 and 20 mg / mL. Then 7.5 μL of sample solution was added to a 1536-well microplate. The plate was sealed with ClearSealFilm and centrifuged at 3,000 rpm for 5 min to allow the sample to sink to the bottom of the well. Each sample was tested in two wells. The plate was placed in the corresponding position and data was collected by DYNAMICS operating software (v7.8.1.3). Five acquisitions were collected for each protein sample, and each acquisition time was 5 s. For each measurement, the diffusion coefficient was determined and plotted against the protein concentration. The kD value is automatically calculated by the software.

[0369] The measured kD was -13.0 mL / g, indicating that W308032-U5T6.E17-57.uIgG1 had good colloidal stability ( Figure 5 ).

[0370] Table 5: Summary of DLS characterization

[0371] Antibody Name kD(mL / g) R^2 Size distribution evaluate W308032-U5T6.E17-57.uIgG1 -13.0 98.6 Monodisperse size good

[0372] 3.4 Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC)

[0373] The HPLC was carried out on a 1260 Infinity II system (Agilent Technologies) with a TSKgel Butyl-NPR column (Tosoh-0042168). TM ) to detect the hydrophobicity of the antibody. 20 μL of sample was injected into the column and separated at a flow rate of 0.5 ml / min for 61 min. The running buffer was 25 mM sodium phosphate, pH 7.0 (buffer A) and 25 mM sodium phosphate, 1.5 M (NH4)2SO4, pH 7.0 (buffer D). The running gradient was 0% to 100% buffer D from 3 min to 53 min. Peak retention was detected with UV light at wavelengths of 280 nm and 230 nm. Retention times were analyzed using the HIC-HPLC analytical method to integrate all peak areas from 20 min to 50 min. The operating and analysis software was OpenLab CDS Workstation (v2.3.0.443).

[0374] The retention time was 24.74 min, indicating that W308032-U5T6.E17-57.uIgG1 had normal hydrophobicity ( Figure 6 ).

[0375] Table 6: HIC-HPLC characterization results

[0376]

[0377] 3.5 Surface Plasmon Resonance (SPR)

[0378] Biacore 8K was used to detect binding affinity to human HER2. Test antibodies were captured on a CM5 sensor chip pre-fixed with anti-human IgG Fc antibodies. Different concentrations of human HER2 protein were injected onto the sensor chip at a flow rate of 30 μL / min for an association period of 18 s, followed by a dissociation period of 3600 s. After each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5). The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgram. Experimental data were fitted by a 1:1 binding model using Langmiur analysis. The molar concentration of human HER2 protein as an analyte was calculated using a molecular weight of 71.0 kDa.

[0379] Binding affinity to human CD47 was detected using Biacore 8K. Human CD47 protein was captured on a CM5 sensor chip pre-fixed with streptavidin. Different concentrations of test antibodies were injected onto the sensor chip at a flow rate of 30 μL / min for an association period of 240 s, followed by a dissociation period of 600-3600 s. After each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5). The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgram. The experimental data were fitted using a 1:1 binding model using Langmiur analysis. The molecular weight of 147 kDa was used to calculate the molar concentration of the test antibody as an analyte.

[0380] The results showed that W308032-U5T6.E17-57.uIgG1 maintained high affinity for human Her2 and relatively low affinity for human CD47. The affinity of W308032-U5T6.E17-57.uIgG1 for HER2 was about 50 times higher than that for CD47 ( Figures 7A-7F ). Figures 7A-7C Three independent SPR results for HER2 binding are shown and Figure 7D-7F Three independent SPR results for binding to CD47 are shown.

[0381] Table 7: SPR characterization summary

[0382]

[0383]

[0384] 3.6 Antigen binding to tumor cells after sinking

[0385] Serially diluted antibodies were mixed with human whole blood (1:10 dilution) or Jurkat cells (1.2-1.8×10 6 The cells were incubated at 4°C for 1 hr, then centrifuged and the pre-sunk supernatant was transferred to a new plate. CFSE-labeled human tumor cells (0.8x10 5Cells / well) were inoculated into a 96-well U-bottom plate and centrifuged at 1500rpm for 4min at 4°C, and the supernatant was removed. The pre-sinking supernatant was then added to resuspend the cells, incubated at 4°C for 1hr, and then washed twice with 180μL 1% BSA-PBS. Secondary antibody Alexa647-conjugated goat anti-human IgG Fc was added to resuspend the cells and incubated at 4°C in the dark for 0.5hr, then washed twice with 180μL 1% BSA-PBS. The mean fluorescence intensity (MFI) was measured by FACS (BD Canto II) and analyzed by FlowJo Version software. Tumor cells were gated by CFSE, and the binding EC on tumor cells was calculated using GraphPad Prism software. 50 : Non-linear regression (curve fitting) - log(agonist) vs. response - variable slope.

[0386] After being stained with human blood, W308032-U5T6.E17-57.uIgG1 showed binding to CD47 / Her2 double-positive SK-BR-3 cells, EC 50 is ~27.69nM, and the binding (EC 50 =24.36 nM), which was not affected. However, the binding of mololimab on SK-BR-3 (EC 50 =0.36nM) after being submerged in human blood (EC 50 =6.41 nM) were significantly affected, with EC 50 The offset is about 18 times ( Fig. 8A -B).

[0387] Table 8: Summary of antibody binding to SK-BR-3 cells in the absence or presence of human blood cells as sink

[0388]

[0389] After being saturated by Jurkat cells, W308032-U5T6.E17-57.uIgG1 showed binding on SK-BR-3 cells, EC 50 was ∼13.29 nM, which was comparable to the binding of Jurkat cells not pre-incubated (EC 50 =12.55 nM), which was not affected. However, the binding of mololimab to SK-BR-3 (EC 50 =0.19 nM) after being sunken by Jurkat cells (EC 50 =1.53 nM) was significantly affected, with EC 50 The offset is about 8 times ( Fig. 9A -B).

[0390] Table 9: Summary of antibody binding to SK-BR-3 cells in the absence or presence of Jurkat cells as sinks

[0391]

[0392] The effect of antigen sinking on the binding of antibodies to CD47 / Her2 double positive HCC1954 cells was also tested. After being sunk by Jurkat cells, W308032-U5T6.E17-57.uIgG1 showed binding on HCC1954 cells, EC 50 was ∼30.29 nM, which was comparable to the binding of Jurkat cells not pre-incubated (EC 50 =26.07 nM), which was not affected. However, the binding of mololimab to HCC1954 (EC 50 =0.44 nM) were affected after being sunken by Jurkat cells (EC 50 =1.68 nM), where EC 50 The offset is about 4 times ( Fig. 10A -B).

[0393] Table 10: Summary of antibody binding to HCC1954 cells in the absence or presence of Jurkat cells as sinks

[0394]

[0395] 3.7 Binding on CD47 single positive cells

[0396] Compared to mololimab, W308032-U5T6.E17-57.uIgG1 showed negligible binding on human blood cells ( Fig.11 ) and about 200-fold reduction in binding on Jurkat cells ( Fig.12 ).

[0397] Table 11: Summary of antibody binding to human blood cells

[0398]

[0399] Table 12: Summary of antibody binding to Jurkat cells

[0400]

[0401] 3.8 Blockade of CD47 ligand on tumor cells after antigen sinking

[0402] Serially diluted antibodies were mixed with human whole blood (1:10 dilution) or Jurkat cells (1.2-1.8×10 6The cells were incubated at 4°C for 1 hr, then centrifuged and the pre-sunk supernatant was transferred to a new plate. CFSE-labeled human tumor cells (0.8x10 5 Cells / well) were inoculated into a 96-well U-bottom plate and centrifuged at 1500rpm for 4min at 4°C, and the supernatant was removed. The pre-sunk supernatant and mFc-labeled SIRPα (1μg / mL) were then added to resuspend the cells and incubated at 4°C for 2hr, then washed twice with 180μL 1% BSA-PBS. Secondary antibody Alexa647-conjugated goat anti-mouse IgG Fc was added to resuspend the cells and incubated at 4°C in the dark for 0.5hr, then washed twice with 180μL 1% BSA-PBS. MFI was measured by FACS (BDCanto II) and analyzed by FlowJo Version software. Tumor cells were gated by CFSE, and the inhibition IC of tumor cells was calculated using GraphPad Prism software. 50 : Nonlinear regression (curve fitting) - log(antagonist) vs. response - variable slope.

[0403] After being stained with human blood, W308032-U5T6.E17-57.uIgG1 blocked the binding of CD47 ligand on CD47 / Her2 double-positive SK-BR-3 cells, where IC 50 The blocking rate was ~0.30nM, which was different from that of the cells not pre-incubated with human blood (IC 50 =0.25 nM), which was not affected. However, mololimab was not affected in SK-BR-3 (IC 50 =0.06 nM) on CD47 ligand blocked by human blood (IC 50 =0.93nM) was significantly affected after sinking, among which IC 50 The offset is about 15 times ( Fig.13A -B).

[0404] Table 13: Summary of CD47 ligand blockade in the absence or presence of human blood cells as sinks

[0405]

[0406] After being sunken by Jurkat cells, W308032-U5T6.E17-57.uIgG1 blocked the binding of CD47 ligand to CD47 / Her2 double-positive SK-BR-3 cells, where IC 50 The blockade (IC 50 =1.24 nM), which was not affected. However, mololimab was not affected in SK-BR-3 (IC 50=0.17 nM) on Jurkat cells (IC 50 =0.44nM) was affected after sinking, among which IC 50 The offset is about 3 times ( Fig.14A -B).

[0407] Table 14: Summary of CD47 ligand blockade in the absence or presence of Jurkat cells as sinks

[0408]

[0409] Furthermore, W308032-U5T6.E17-57.uIgG1 showed little CD47 ligand blocking effect on Jurkat cells compared with morolimumab ( Fig.15 ).

[0410] Table 15: Summary of CD47 ligand blocking effects on Jurkat cells

[0411]

[0412] 3.9 Receptor occupancy on tumor cells

[0413] Tumor cells were seeded into a 96-well U-bottom plate and centrifuged at 1500rpm for 4min at 4°C, and the supernatant was removed. Then a serial dilution of the bispecific antibody was added to resuspend the cells, incubated at 4°C for 1hr, and then washed twice with 180μL 1% BSA-PBS. The detection antibody (saturating concentration of biotin-labeled parent antibody) was added to the resuspended cells and incubated at 4°C for 1hr, then washed twice with 180μL 1% BSA-PBS. The secondary antibody streptavidin PE was added to resuspend the cells, incubated at 4°C in the dark for 0.5hr, and then washed twice with 180μL 1% BSA-PBS. MFI was measured by FACS (BD Canto II) and analyzed by FlowJo Version software. Receptor occupancy (%) was calculated as follows: 100-(MFI 样品 -MFI 阴性 ) / (MFI 饱和 -MFI 阴性 )x100. EC was calculated using GraphPad Prism software 50 : Non-linear regression (curve fitting) - log(agonist) vs. response - variable slope.

[0414] With the help of the Her2 binding arm, W308032-U5T6.E17-57.uIgG1 showed a maximal CD47 occupancy of more than 30% higher than that of mololimab on CD47 / Her2 double-positive SK-BR-3 cells ( Fig.16A -B).

[0415] Table 16: Summary of Her2 and CD47 occupancy on SK-BR-3 cells

[0416]

[0417] 3.10 ADCP against tumor cells

[0418] Monocytes were isolated from PBMC using human CD14 microbeads and then treated with rhM-CSF (50-100 ng / mL) for 6-8 days to differentiate into macrophages. In short, 30 μL of CFSE-labeled tumor cells, 50 μL of macrophages, and 20 μL of serially diluted antibodies were inoculated into a 96-well U-bottom ultra-low plate. The phagocytosis of tumor cells was performed at 37°C for 2-3 hours. The cells were then stained with APC-conjugated CD14 antibodies at 4°C for 45 min and washed with 1% BSA-PBS, then detected with FACS (BD CantoII) and analyzed with FlowJo Version software. The phagocytic activity was calculated as follows: Index % = percentage CFSE+ / CD14-APC+ / (percentage CFSE+ / CD14-APC+ +Percent CFSE- / CD14-APC+ ) x 100%. Phagocytic EC was calculated using GraphPad Prism software 50 : Non-linear regression (curve fitting) - log(agonist) vs. response - variable slope.

[0419] like Fig.17A -B, in two independent experiments, W308032-U5T6.E17-57.uIgG1 showed increased ADCP efficacy on CD47 / Her2 double-positive SK-BR-3 cells compared to the combination of trastuzumab and mololimab.

[0420] Table 17A: Summary of Antibodies on SK-BR-3 Cells (Study 1)

[0421]

[0422] Table 17B: Summary of Antibodies on SK-BR-3 Cells (Study 2)

[0423]

[0424] like Fig.18 As shown, W308032-U5T6.E17-57.uIgG1 showed efficacy in combination with trastuzumab and mololimab on CD47 / Her2 double-positive HCC1954 cells (EC 50=1.28nM, maximum phagocytic index =38.1%) with comparable ADCP efficacy (EC 50 =1.25nM, maximum phagocytic index =39.3%). Fig.19 As shown, W308032-U5T6.E17-57.uIgG1 showed ADCP efficacy (EC50 = 1.77 nM, maximum phagocytic index = 77.5%) comparable to that of the combination of trastuzumab and mololimab on CD47 / Her2 double-positive NCI-N87 cells. 50 =0.13 nM, maximum phagocytic index =77.8%).

[0425] Table 18: Summary of antibodies against HCC1954

[0426]

[0427]

[0428] Table 19: Summary of antibodies on NCI-N87

[0429]

[0430] ADCP against CD47 single positive cells

[0431] W308032-U5T6.E17-57.uIgG1 showed little ADCP efficacy on human blood cells ( Fig. 20 ) and much lower ADCP activity on Jurkat cells compared with mololimab ( Fig.21 ).

[0432] Table 20: Summary of Antibodies on Human Blood Cells

[0433]

[0434] Table 21: Summary of antibodies on Jurkat cells

[0435]

[0436]

[0437] 3.11 ADCC against tumor cells

[0438] NK cells were isolated from PBMCs using a CD56 positive selection kit. Briefly, 40 μL of tumor cells, 40 μL of NK cells, and 20 μL of serially diluted antibodies were seeded into a 96-well U-bottom plate. After incubation at 37°C for 4 hr, the cell mixture was centrifuged at 1500 rpm for 5 min, and 75 μL of the supernatant was transferred for detection. Cell death was measured and calculated using the LDH cytotoxicity detection kit (Roche) according to the manufacturer's instructions. Cytotoxic EC was calculated using GraphPad Prism software. 50 : Non-linear regression (curve fitting) - log(agonist) vs. response - variable slope.

[0439] like Fig. 22 As shown, W308032-U5T6.E17-57.uIgG1 showed effective ADCC efficacy on CD47 / Her2 double-positive SK-BR-3 cells (EC 50 =21.42 pM, maximum cytotoxicity =61.9%).

[0440] Table 22: Summary of ADCC on SK-BR-3

[0441]

[0442] W308032-U5T6.E17-57.uIgG1 also expressed in CD47 / Her2 double positive HCC1954 cells ( Fig.23 ) and NCI-N87 cells ( Fig.24 ) showed effective ADCC efficacy.

[0443] Table 23: Summary of ADCC on HCC1954

[0444]

[0445] Table 24: Summary of ADCC on NCI-N87

[0446]

[0447] ADCC against CD47 single positive cells

[0448] W308032-U5T6.E17-57.uIgG1 showed very weak ADCC efficacy on Jurkat cells only at high concentrations ( Fig.25 ).

[0449] Table 25: Summary of ADCC on Jurkat cells

[0450]

[0451] 3.12 Inhibitory effect on tumor cell proliferation

[0452] Briefly, 80 μL of tumor cells were seeded into 96-well black-walled plates. The next day, 20 μL of serially diluted antibodies were added to the cells and incubated at 37°C for 5-6 days. Then 50 μL / well of CTG solution was added to the cells and incubated at room temperature (RT) for 10 min before detection using EnVision. Inhibition IC50 was calculated using GraphPadPrism software: nonlinear regression (curve fit) - log (antagonist) vs. response - variable slope.

[0453] W308032-U5T6.E17-57.uIgG1 against CD47 / Her2 double positive SK-BR-3 cells ( Fig.26 ) and NCI-N87 cells ( Fig. 27 ) showed effective inhibitory effect on proliferation.

[0454] Table 26: Summary of the inhibitory effect on SK-BR-3

[0455]

[0456]

[0457] Table 27: Summary of Inhibitory Effects on NCI-N87

[0458]

[0459] 3.13 Blood coagulation

[0460] Anticoagulated fresh human whole blood was centrifuged at 2000rpm for 10min, and the plasma was discarded. Human blood cells were rinsed twice with DPBS. In brief, 25μL of 1:25 diluted human blood cells (i.e., cells were resuspended using 25x original blood volume) and 25μL of serially diluted antibodies were added to a U-shaped bottom 96-well plate and incubated at 37°C for 2hr before taking photos for detection.

[0461] W308032-U5T6.E17-57.uIgG1 is HA-negative on human blood cells, while mololimab is HA-positive on human blood cells ( Fig.28 ).

[0462] 3.14 Serum stability

[0463] Fresh human whole blood was incubated at room temperature (RT) for at least 30 min in a tube without anticoagulant. Serum was collected after centrifugation of blood at 4000 rpm for 10 min. The antibody was gently mixed with serum and incubated at 37°C. An aliquot of serum-treated samples was collected on days 0, 1, 4, 7, and 14 and snap-frozen with liquid nitrogen; and stored at -80°C until ready for analysis. The binding ability of the samples to tumor cells was evaluated to reflect their stability. Briefly, tumor cells were seeded into 96-well U-bottom plates and centrifuged at 1500 rpm for 4 min at 4°C, and the supernatant was removed. Then different concentrations of test samples were added to resuspend the cells, incubated at 4°C for 1 hr, and then washed twice with 180 μL 1% BSA-PBS. Secondary antibody Alexa647-conjugated goat anti-human IgG Fc was added to resuspend the cells and incubated at 4°C for 0.5 hr in the dark, and then washed twice with 180 μL 1% BSA-PBS. MFI was measured by FACS (BD Canto II) and analyzed by FlowJo Version software. Binding EC was calculated using GraphPad Prism software. 50 : Non-linear regression (curve fitting) - log(agonist) vs. response - variable slope.

[0464] W308032-U5T6.E17-57.uIgG1 was stable in human serum for at least 7 days and showed no reduction in binding on CD47 / Her2 double positive SK-BR-3 cells. When incubated with human serum for 14 days, W308032-U5T6.E17-57.uIgG1 showed a slight (less than 2-fold) reduction in binding potency on CD47 / Her2 double positive SK-BR-3 cells ( Fig.29 ).

[0465] Table 28: Summary of serum stability results

[0466]

[0467] Example 4

[0468] In vivo characterization of the W308032 antibody

[0469] 4.1 Rodent Efficacy

[0470] 4.1.1 HCC1954 xenograft model

[0471] This study used 6-8 week old female CB-17SCID mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.). HCC1954 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum as monolayer cultures in vitro at 37°C and 5% CO2 in air. For the xenograft model, HCC1954 tumor cells in the exponential growth phase were harvested and inoculated subcutaneously in the right forearm axilla of each mouse (3.0×10 6 cells / 0.2 mL, 1:1 DPBS and Matrigel (Corning). When the average tumor volume reached ∼147 mm 3 Animals were randomized into 12 groups and received the first dose of antibody treatment (day 0) followed by intraperitoneal treatment twice a week for a total of 10 injections. Mice were weighed and tumor growth was measured twice a week using a caliper. The formula (1 / 2(length × width) 2 ) Tumor volume was calculated. Results are expressed as mean ± SEM. Data were analyzed using Prism's two-way ANOVA with Bonferroni post-test, and p<0.05 was considered statistically significant. All procedures related to animal handling, care, and treatment in the study were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi Biologics and followed the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Grouping and treatment details of the HCC1954 xenograft model are shown in Table 29.

[0472] Table 29: Grouping and treatment of HCC1954 xenograft model

[0473]

[0474] In addition to two mice (one from the W308032-U5T6.E17-57.uIgG1 3 mg / kg group and one from the W308032-U5T6.E17-57.uIgG1 1 mg / kg group) that were found dead on days 28 and 17, one mouse in the Pertuzumab group died due to a tumor volume of 2500 mm 3 All other mice appeared normal and slowly gained weight during the experiment, indicating that the antibody was not toxic (data not shown).

[0475] like Fig.30As shown and summarized in Table 30, compared with the PBS group, trastuzumab + mololimab (day 36, TGI 76.8%) was able to significantly inhibit tumor growth, with significant differences from day 7; while trastuzumab (day 36, TGI 33.6%) and mololimab (day 36, TGI 18.5%) monotherapy groups showed slight inhibition of tumor growth, indicating the synergistic effect of CD47 and Her2. W308032-U5T6.E17-57.uIgG1 (10 mg / kg) showed slightly better efficacy than the trastuzumab + mololimab combination, and significantly better efficacy than trastuzumab or mololimab monotherapy ( Fig.30 ).

[0476] Table 30: Summary of TGI and P values ​​on day 36 of HCC1954 xenograft model (Note: two-way ANOVA Bonferroni post-test, *p<0.05; **p<0.01)

[0477]

[0478]

[0479] W308032-U5T6.E17-57.uIgG1 (30 mg / kg, 10 mg / kg, 3 mg / kg, 1 mg / kg) could inhibit tumor growth (TGI on day 36 was 100.1%, 90.7%, 75.6%, 36.7%, respectively), and there were significant differences from day 3 (30 mg / kg), day 7 (10 mg / kg), and day 10 (3 mg / kg), respectively, indicating that W308032-U5T6.E17-57.uIgG1 has a dose-dependent antitumor effect ( Fig.31 ).

[0480] The combination therapy of trastuzumab and pertuzumab has been approved for clinical use and has shown improved anti-tumor efficacy compared with trastuzumab monotherapy. To evaluate whether W308032-U5T6.E17-57.uIgG1 also has the potential to be used together with pertuzumab to further enhance the anti-tumor activity, the anti-tumor activity of the combination therapy was also examined in the HCC1954 xenograft model. Fig.32As shown and summarized in Table 30, compared with the PBS group, the combination groups of trastuzumab + pertuzumab (day 36, TGI 50.7%) and trastuzumab + mololimab + pertuzumab (day 36, TGI 105.2%) were able to significantly inhibit tumor growth, with significant differences from day 7; while the monotherapy groups of trastuzumab (day 36, TGI 33.6%) and pertuzumab (day 36, TGI 8.76%) showed only slight inhibition of tumor growth. The combination group of W308032-U5T6.E17-57.uIgG1 + pertuzumab (day 36, TGI 106.0%) showed comparable anti-tumor efficacy to the combination group of trastuzumab + mololimab + pertuzumab (day 36, TGI 105.2%).

[0481] All the above conclusions were also confirmed by the tumor weight data obtained on day 36 after the first treatment ( Fig.33 ). Data are shown as mean + SEM.

[0482] 4.1.2 NCI-N87 xenograft model

[0483] This study used female CB-17SCID mice aged 4-8 weeks (Beijing Weitong Lihua Laboratory Research Model and Service). NCI-N87 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum as monolayer cultures in vitro at 37°C and 5% CO2 air environment. For the xenograft model, NCI-N87 tumor cells in the exponential growth phase were harvested and inoculated subcutaneously in the right anterior abdominal region of each mouse with NCI-N87 tumor cells (1×10 7 cells / 0.1 mL, 1:1 PBS and Matrigel). When the average tumor volume reached ∼157 mm 3 Animals were randomized into 10 groups and received the first dose of treatment (day 0) followed by twice weekly intraperitoneal injections of antibody treatment for a total of 11 injections, except for Euheride treatment which was intraperitoneally injected once a week for a total of 6 injections. Mice were weighed and tumor growth was measured twice a week using a caliper. The formula (1 / 2(length × width 2 ) to calculate tumor volume.

[0484] All results are presented as mean ± SEM. Data were analyzed using two-way ANOVA Bonferroni post-test or one-way ANOVA Dunnett multiple comparison test in the RA language and statistical computing and graphics environment (version 3.3.1), and p < 0.05 was considered statistically significant. All procedures related to animal care and use were approved by the CrownBio Institutional Animal Care and Use Committee (IACUC) in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The grouping and treatment details of the NCI-N87 xenograft model are shown in Table 31.

[0485] Table 31. Grouping and treatment of NCI-N87 xenograft model

[0486]

[0487] like Fig.34 As shown and summarized in Table 32, Herceptin (trastuzumab, 10 mg / kg) + mololimab (10 mg / kg) can significantly inhibit tumor growth compared with the vehicle control group (day 38, TGI 111.92%), and W308032-U5T6.E17-57.uIgG1 (10 mg / kg and 20 mg / kg) showed comparable efficacy to Herceptin + mololimab combination therapy (day 38, TGI 116.04% and 118.17%, respectively). Fig.35 As shown, W308032-U5T6.E17-57.uIgG1 (5 mg / kg, 10 mg / kg, 20 mg / kg, 40 mg / kg) showed a dose-dependent anti-tumor efficacy (on day 38, TGI were 112.22%, 116.04%, 118.17%, 119.99%, respectively).

[0488] The combination of trastuzumab and chemotherapy has been approved for clinical use and has shown improved anti-tumor efficacy compared with trastuzumab monotherapy. To evaluate whether W308032-U5T6.E17-57.uIgG1 could also be used with chemotherapy to further enhance anti-tumor activity, the anti-tumor activity of the combination therapy was also examined in the NCI-N87 xenograft model. Fig.36 As shown and summarized in Table 32, W308032-U5T6.E17-57.uIgG1 (10 mg / kg) combined with paclitaxel (10 mg / kg) produced significant anti-tumor efficacy with a TGI of 117.57%, which is comparable to the efficacy of Euheride (10 mg / kg) with a TGI of 124.19%.

[0489] Table 32. Summary of TGI and P values ​​on day 38 in the NCI-N87 xenograft model

[0490]

[0491]

[0492] 4.1.3 JIMT-1 xenograft model

[0493] The efficacy of another trastuzumab-resistant breast cancer cell JIMT-1 xenograft model was also evaluated. Female CB-17SCID mice (Shanghai Lingchang Biotechnology Co., Ltd.) aged 9-10 weeks were used in the study. JIMT-1 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum as a monolayer culture in vitro at 37°C and 5% CO2 in an air environment. For the xenograft model, JIMT-1 tumor cells in the exponential growth phase were harvested and inoculated subcutaneously in the upper right flank region of each mouse with JIMT-1 tumor cells (5×10 6 cells / 0.1 mL PBS). When the average tumor volume reached ∼134 mm 3 Animals were randomized into 6 groups and received the first dose of treatment (day 0), followed by intraperitoneal injections of antibody twice a week for a total of 10 injections and then observed for 4 weeks after the last injection. Mice were weighed and tumor growth was measured twice a week using a caliper. The formula (1 / 2(length × width 2 ) to calculate tumor volume.

[0494] All results are presented as mean ± SEM. Data were analyzed using Prism's two-way ANOVA with Bonferroni post-test or one-way ANOVA with Dunnett's multiple comparison test, and p < 0.05 was considered statistically significant. All procedures related to animal care and use were approved by the CrownBio Institutional Animal Care and Use Committee (IACUC) in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Grouping and treatment details of the JIMT-1 xenograft model are shown in Table 33.

[0495] Table 33. Grouping and treatment of JIMT-1 xenograft model

[0496]

[0497]

[0498] Animals were treated twice weekly for a total of 10 doses over 5 weeks and then observed for 4 weeks without treatment. On day 35 (three days after the last dose), W308032-U5T6.E17-57.uIgG1 showed dose-dependent inhibition of tumor growth and was comparable to the efficacy of the combination of trastuzumab and mololimab (data not shown). Fig.37 As shown and summarized in Table 34, W308032-U5T6.E17-57.uIgG1 in combination with paclitaxel showed better efficacy in tumor growth inhibition than the bispecific antibody or trastuzumab monotherapy, which was comparable to the efficacy of Uretaxel.

[0499] like Fig.38 As shown, on day 63 (thirty-one days after the last dose), the combination of W308032-U5T6.E17-57.uIgG1 and paclitaxel showed almost no rebound in tumor volume, with 4 of 8 mice in each group being tumor-free. However, Euheride showed a significant rebound relative to the initial tumor volume, with only 1 of 8 mice in each group being tumor-free.

[0500] Table 34. Summary of TGI and P values ​​on day 35 of JIMT-1 xenograft model

[0501]

[0502]

[0503] 4.2 Pharmacokinetics in rodents

[0504] Female SD rats aged 9-11 weeks were used in this study (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). Animals were housed in a specific pathogen-free (SPF) barrier at Shanghai Model Biology Center Co., Ltd., with 2 animals per separate ventilated cage (IVC), and adapted for one week after arrival. For pharmacokinetic (PK) studies, each rat was given a single dose of antibody (15 mg / kg, IV push) on day 0, and blood samples were collected from the eyes and transferred to EDTA tubes. The test tubes were centrifuged at 8000 rpm (6010 g) for 5 minutes at 4 ° C, and then plasma was collected and stored at -20 ° C. All samples were uniquely identified to indicate the source and collection time. The sample collection schedule for each rat per dose is shown in Table 35.

[0505] Table 35: Sample Collection Schedule for Rat PK Study

[0506]

[0507] The concentration of analytes in serum was determined using a bioanalytical ELISA method. Briefly, 96-well ELISA plates were coated overnight at 2–8°C with recombinant human HER2 / ErB2 protein (His tag) in carbonate-bicarbonate buffer as the capture antibody. After washing and blocking, serially diluted plasma samples were added, and then biotin-labeled recombinant human CD47 protein (G1 for HER2+CD47 method) or goat anti-human IgG-biotin (G2 for HER2+Fc method) was used as the detection antibody. HRP-streptavidin and TMB substrate were used for color development. The reaction was terminated after approximately 5–10 minutes by adding 2 M HCl. The plate was analyzed using a microplate spectrophotometer ( M5e) read the absorbance at 450nm and 540nm. The OD value of the sample was substituted into the standard curve to obtain the plasma antibody concentration. Non-compartmental pharmacokinetic analysis of serum concentration of the antibody in rats was performed using Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA). The linear / log trapezoidal rule was applied to obtain PK parameters. All PK parameters were calculated when the antibody concentration was less than 1% of Cmax.

[0508] like Fig.39 As shown, W308032-U5T6.E17-57.uIgG1 showed rodent PK (t1 / 2 = 153 h, Table 36) comparable to trastuzumab (t1 / 2 = 184 h, Table 37).

[0509] Table 36: Summary of pharmacokinetic parameters of W308032-U5T6.E17-57.uIgG1

[0510]

[0511] Table 37: Summary of Pharmacokinetic Parameters of Trastuzumab

[0512]

[0513]

[0514] It will also be appreciated by those skilled in the art that the present disclosure may be embodied in other specific forms without departing from the spirit or core attributes of the present disclosure. Since the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it will be appreciated that other variations should be considered to fall within the scope of the present disclosure. Therefore, the present disclosure is not limited to the specific embodiments described in detail herein. On the contrary, reference should be made to the appended claims to indicate the scope and content of the present disclosure.

[0515] References

[0516] [1]Meike E W Logtenberg,Ferenc A Scheeren,Ton N Schumacher.The CD47-SIRPa Immune Checkpoint.Immunity.2020May 19;52(5):742-752.

[0517] [2]E J Brown,W A Frazier.Integrin-associated protein(CD47)and itsligands.Trends Cell Biol.2001Mar;11(3):130-5.

[0518] [3]Mark P Chao,Irving L Weissman,Ravindra Majeti.The CD47-SIRPαpathway in cancer immune evasion and potential therapeutic implications.CurrOpin lmmunol.2012Apr;24(2):225-32.

[0519] [4]I Rubin,Y Yarden.The basic biology of HER2.Ann Oncol.2001;12Suppl1:S3-8

[0520] [5]Li-Chung Tsao et.al.CD47 blockade augmentation of trastuzumabantitumor efficacy dependent on antibody-dependent cellular phagocytosis.JCIInsight.2019Dec 19;4(24):e131882.

[0521] [6]Tiia J Honkanen et.al.Prognostic and predictive role of spatiallypositioned tumour infiltrating lymphocytes in metastatic HER2 positive breastcancer treated with trastuzumab.Sci Rep.2017Dec 21;7(1):18027.

Claims

1. A polypeptide complex or an antigen binding portion thereof, comprising a first antigen binding portion that specifically binds to HER2 and a second antigen binding portion that specifically binds to CD47, wherein: The first antigen binding portion comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), the VH1 comprising a heavy chain complementary determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and the VL1 comprising a light chain complementary determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and The second antigen binding portion comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2), the VH2 comprising a HCDR1 comprising the amino acid sequence of SEQ ID NO:7, a HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:9, and the VL2 comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO:10, a LCDR2 comprising the amino acid sequence of SEQ ID NO:11, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

12.

2. The polypeptide complex or antigen binding portion thereof according to claim 1, wherein the first antigen binding portion and the second antigen binding portion are independently selected from the following forms: immunoglobulins and antigen binding portions thereof, such as Fab, F(ab)'2 or scFv.

3. The polypeptide complex or antigen binding portion thereof according to claim 1 or 2, wherein: The first antigen binding moiety comprises VH1 operably linked to a first T cell receptor (TCR) constant region (C1) and VL1 operably linked to a second TCR constant region (C2), and the second antigen binding moiety comprises VH2 operably linked to an antibody heavy chain CH1 domain and VL2 operably linked to an antibody light chain constant (CL) domain; or The first antigen binding portion comprises VH1 operably linked to the antibody heavy chain CH1 domain and VL1 operably linked to the antibody light chain constant (CL) domain, and the second antigen binding portion comprises VH2 operably linked to the C1 region and VL2 operably linked to the C2 region; Wherein C1 and C2 are capable of forming one or more non-native interchain disulfide bonds, said non-native interchain disulfide bonds improving the stability of C1, C2 and / or the interface between C1 and C2.

4. The polypeptide complex or antigen binding portion thereof according to any one of claims 1 to 3, wherein VH1 comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 13, and VL1 comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 14; and / or VH2 comprises the amino acid sequence of SEQ ID NO:15, or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO:15, and VL2 comprises the amino acid sequence of SEQ ID NO:16, or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO:

16.

5. The polypeptide complex or antigen binding portion thereof according to claim 3 or 4, wherein C1 comprises the amino acid sequence of SEQ ID NO:17 or an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO:17, and C2 comprises the amino acid sequence of SEQ ID NO:18 or an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO:

18.

6. The polypeptide complex or antigen binding portion thereof according to any one of the preceding claims, wherein the polypeptide complex comprises an Fc region, such as a human IgG Fc region.

7. The polypeptide complex or antigen binding portion thereof according to claim 6, wherein the Fc region is a human IgG1, IgG2, IgG3 or IgG4 Fc region.

8. The polypeptide complex or antigen binding portion thereof according to claim 6 or 7, wherein the Fc region is a native Fc region or an engineered Fc region, such as an Fc region comprising one or more substitutions selected from the group consisting of "knob-into-hole" substitution, S228P, M252Y / S254T / T256E and combinations thereof.

9. The polypeptide complex or antigen binding portion thereof according to any one of the preceding claims, comprising one, two or more CD47 binding moieties and one, two or more HER2 binding moieties.

10. The polypeptide complex or antigen binding portion thereof according to claim 9, comprising two heavy chains and two light chains, wherein from N-terminus to C-terminus: The first heavy chain comprises domains operably linked as in VH1-C1-hinge-Fc, The second heavy chain comprises domains operably linked as in VH2-CH1-hinge-Fc, The first light chain comprises the operably linked domains as in VL1-C2, and The second light chain comprises an operably linked domain as in VL2-CL; or The first heavy chain comprises domains operably linked as in VH1-CH1-hinge-Fc, The second heavy chain comprises domains operably linked as in VH2-C1-hinge-Fc, The first light chain comprises the operably linked domains as in VL1-CL, and The second light chain comprises the operably linked domains as in VL2-C2.

11. The polypeptide complex or antigen binding portion thereof according to claim 9, comprising two heavy chains and four light chains, wherein from N-terminus to C-terminus: The first heavy chain and the second heavy chain each comprise an operably linked domain as in VH1-C1-VH2-CH1-hinge-Fc, VH2-CH1-VH1-C1-hinge-Fc, VH1-C1-hinge-Fc-VH2-CH1 or VH2-CH1-hinge-Fc-VH1-C1, The first and second light chains each comprise an operably linked domain as in VL1-C2, and the third and fourth light chains each comprise an operably linked domain as in VL2-CL; or The first heavy chain and the second heavy chain each comprise an operably linked domain as in VH1-CH1-VH2-C1-hinge-Fc, VH2-C1-VH1-CH1-hinge-Fc, VH1-CH1-hinge-Fc-VH2-C1 or VH2-C1-hinge-Fc-VH1-CH1, The first and second light chains each comprise an operably linked domain as in VL1-CL, and the third and fourth light chains each comprise an operably linked domain as in VL2-C2.

12. The polypeptide complex or antigen binding portion thereof according to claim 9, comprising two heavy chains and three light chains, wherein from N-terminus to C-terminus: The first heavy chain comprises operably linked domains as in VH1-C1-VH2-CH1-hinge-Fc or VH2-CH1-hinge-Fc-VH1-C1, The second heavy chain comprises domains operably linked as in VH2-CH1-hinge-Fc, The first light chain comprises the operably linked domains as in VL1-C2, and The second light chain and the third light chain each comprise an operably linked domain as in VL2-CL; or The first heavy chain comprises operably linked domains as in VH2-CH1-VH1-C1-hinge-Fc or VH1-C1-hinge-Fc-VH2-CH1, The second heavy chain comprises domains operably linked as in VH1-C1-hinge-Fc, The first light chain and the second light chain each comprise an operably linked domain as in VL1-C2, and The third light chain comprises an operably linked domain as in VL2-CL; or The first heavy chain comprises operably linked domains as in VH1-CH1-VH2-C1-hinge-Fc or VH2-C1-hinge-Fc-VH1-CH1, The second heavy chain comprises domains operably linked as in VH2-C1-hinge-Fc, The first light chain comprises the operably linked domains as in VL1-CL, and The second and third light chains each comprise an operably linked domain as in VL2-C2; or The first heavy chain comprises operably linked domains as in VH2-C1-VH1-CH1-hinge-Fc or VH1-CH1-hinge-Fc-VH2-C1, The second heavy chain comprises domains operably linked as in VH1-CH1-hinge-Fc, The first and second light chains each comprise an operably linked domain as in VL1-CL, and the third light chain comprises an operably linked domain as in VL2-C2.

13. The polypeptide complex or antigen binding portion thereof according to any one of claims 9 to 12, wherein the C1 and C2 domains are interchangeable.

14. The polypeptide complex or antigen binding portion thereof according to any one of claims 3 to 13, wherein the operably linked is via a direct link or via a peptide linker comprising 1 to 30 amino acids in length.

15. The polypeptide complex or antigen binding portion thereof according to claim 14, wherein the peptide linker is a GS linker, such as (GS)n, (GGS)n, (GGGS)n, (GGGS)n, (GGGGS)n, (GGGS)n, (GGGS)n, (GGGSS)n, wherein n is an integer from 1 to 9.

16. The polypeptide complex or antigen binding portion thereof according to claim 10, comprising: a first heavy chain and a second heavy chain comprising SEQ ID NOs: 19 and 20, respectively, and a first light chain and a second light chain comprising SEQ ID NOs: 21 and 22, respectively.

17. The polypeptide complex or antigen binding portion thereof according to any one of the preceding claims, wherein the polypeptide complex is a humanized antibody or a fully human antibody.

18. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide complex or antigen binding portion thereof according to any one of claims 1 to 17.

19. A vector comprising the nucleic acid molecule according to claim 18.

20. A host cell comprising the nucleic acid molecule according to claim 18 or the vector according to claim 19.

21. A pharmaceutical composition comprising the polypeptide complex or antigen-binding portion thereof according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

22. A method for producing a polypeptide complex or an antigen binding portion thereof according to any one of claims 1 to 17, comprising the following steps: - culturing a host cell comprising a nucleic acid sequence encoding the polypeptide complex or an antigen-binding portion thereof under suitable conditions; and - isolating said polypeptide complex from said host cell.

23. A method for modulating a HER2 and / or CD47-related immune response in a subject, comprising administering to the subject a polypeptide complex or antigen binding portion thereof according to any one of claims 1 to 17 or a pharmaceutical composition according to claim 21.

24. A method for inhibiting tumor cell growth in a subject, comprising administering to the subject an effective amount of the polypeptide complex or antigen binding portion thereof according to any one of claims 1 to 17 or the pharmaceutical composition according to claim 21.

25. A method for diagnosing, preventing or treating cancer in a subject, comprising administering to the subject an effective amount of the polypeptide complex or antigen-binding portion thereof according to any one of claims 1 to 17 or the pharmaceutical composition according to claim 21.

26. The method of claim 25, wherein the cancer is a HER2 and / or CD47 positive cancer.

27. The method of claim 25 or 26, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, breast cancer, lung cancer, cervical cancer, kidney cancer, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, gastric cancer, melanoma, liver cancer, head and neck cancer, skin cancer, bladder cancer, brain cancer, bronchial cancer, bile duct cancer, endometrial cancer, neuroblastoma, retinoblastoma, testicular cancer, thymoma, thyroid cancer, urachal cancer, uterine cancer, vaginal cancer, astrocytoma, basal cell carcinoma, and combinations thereof.

28. The method of claim 27, wherein the cancer is breast cancer or gastric cancer.

29. The method of any one of claims 24-28, wherein the polypeptide complex or antigen binding portion thereof according to any one of claims 1-17 is administered in combination with a chemotherapeutic agent, radiation and / or other agents used for cancer immunotherapy.

30. The polypeptide complex or antigen binding portion thereof according to any one of claims 1 to 17, for use i) Modulate HER2 and / or CD47-related immune responses; ii) inducing macrophage-mediated phagocytosis of tumor cells; iii) inducing natural killer cell-mediated tumor cell cytotoxicity; and / or iv) Inhibit the growth of tumor cells.

31. The polypeptide complex or antigen binding portion thereof according to any one of claims 1 to 17, for use in diagnosing, treating or preventing cancer.

32. Use of the polypeptide complex or antigen binding portion thereof according to any one of claims 1 to 17 in the preparation of a medicament for: i) Modulate HER2 and / or CD47-related immune responses; ii) inducing macrophage-mediated phagocytosis of tumor cells; iii) inducing natural killer cell-mediated tumor cell cytotoxicity; and / or iv) Inhibit the growth of tumor cells.

33. Use of the polypeptide complex or antigen binding portion thereof according to any one of claims 1 to 17 in the preparation of a medicament for treating or preventing cancer associated with CD47 and / or HER2.

34. A kit, wherein the kit comprises a container comprising the polypeptide complex or antigen binding portion thereof according to any one of claims 1-17.

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