Anti-HER2 / anti-CD47 molecules and uses thereof

Abstract

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

Description

[Technical field]

[0001] <Cross reference> This application claims priority to International Patent Application No. PCT / CN2022 / 082951, filed March 25, 2022. The entire contents of this application are incorporated herein by reference.

[0002] <Technical field> This application relates generally to antibodies. More specifically, this application relates to bispecific antibodies that specifically bind to HER2 and CD47, methods for their preparation and uses. [Background technology]

[0003] Cluster of differentiation 47 (CD47) is an approximately 50 kDa immunoglobulin superfamily membrane protein consisting of a single extracellular V-set IgSF 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, to exert an antiphagocytic ("don't eat me") signal 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). Thus, CD47 may function as an innate immune checkpoint target for cancer therapy by blocking the CD47-SIRPα interaction to turn off the "don't eat me" signal [3].

[0004] 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. Functioning as homodimers or heterodimers, these receptors activate multiple cellular pathways, including the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways, to stimulate cell growth, survival and differentiation [4].

[0005] High CD47 expression is associated with poor prognosis in patients with various cancers, and co-expression of CD47 and Her2 is associated with poor prognosis in patients with Her2-targeted therapy. + It may contribute to disease progression in cancer (e.g., BC, GC) [5-6]. Specially designed CD47xHer2 bispecific antibodies (BsAbs) may preferentially target Her2+ / CD47+ double-positive tumor cells and minimize the effect on CD47 single-positive normal cells, thereby reducing systemic CD47 antigen-mediated sink effect and hematologic toxicity. By blocking CD47 / SIRPα signaling, it may increase antibody-dependent cellular phagocytosis (ADCP) of tumor cells to enhance the antitumor effect of Her2-targeted therapy and further stimulate adaptive immunity. Furthermore, the IgG1 Fc of BsAbs may enhance the antitumor effect of Her2-targeted therapy by increasing antibody-dependent cellular phagocytosis (ADCP) of tumor cells. + Antibody-dependent cellular cytotoxicity (ADCC) on tumor cells may be maintained.

[0006] Patients with metastatic breast and gastric cancers that overexpress Her2 initially respond to Her2-targeted therapy, but the majority of patients with advanced Her2-positive solid tumors (e.g., breast cancer) that initially respond to trastuzumab eventually acquire resistance to therapy and relapse despite persistent Her2 gene amplification or overexpression, leaving a large unmet medical need for patients with Her2-positive relapsed / refractory cancers. Therefore, CD47xHer2 BsAb may provide a novel therapeutic option in Her2-positive BC, GC and other solid tumors.

[0007] The present disclosure provides bispecific antibodies that dually target CD47 and HER2 and block the dual functions of CD47 and HER2. Summary of the Invention [Means for solving the problem]

[0008] These and other objectives are provided by the present disclosure, which in its broadest sense relates to compounds, methods, compositions and articles of manufacture that provide antibodies with improved efficacy. The advantages provided by the present disclosure are broadly applicable in the field of antibody therapy and diagnostics, and can be used in combination with other therapeutic and diagnostic agents, such as antibodies reactive with a variety of targets.

[0009] The present disclosure provides bispecific polypeptide complexes or bispecific antibodies against CD47 and HER2. The present invention also provides nucleic acid molecules encoding anti-CD47 / anti-HER2 antibodies, expression vectors and host cells used for the expression of the bispecific antibodies. The present disclosure further provides methods for preparing anti-CD47 / anti-HER2 antibodies (CD47xHer2 BsAb) and validating their function in vivo and in vitro. The bispecific antibodies of the present disclosure provide highly potent agents for preventing or treating diseases including proliferative diseases and immune disorders.

[0010] In one aspect, the 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).

[0011] In some embodiments, the present disclosure provides a bispecific polypeptide complex, or 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 is: a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; The second antigen-binding portion It comprises HCDR1 having the amino acid sequence of SEQ ID NO: 7, HCDR2 having the amino acid sequence of SEQ ID NO: 8, HCDR3 having the amino acid sequence of SEQ ID NO: 9, LCDR1 having the amino acid sequence of SEQ ID NO: 10, LCDR2 having the amino acid sequence of SEQ ID NO: 11, and LCDR3 having the amino acid sequence of SEQ ID NO: 12.

[0012] In some embodiments, the first antigen-binding portion and the second antigen-binding portion are in a Fab format.

[0013] In some embodiments, the first antigen-binding moiety comprises a first heavy chain variable domain (VH1) operably linked to a first T cell receptor (TCR) constant region (designated C1 or C beta) and a first light chain variable domain (VL1) operably linked to a second TCR constant region (designated C2 or C alpha), and the second antigen-binding moiety 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 are interchangeable.

[0014] In some other embodiments, the first antigen-binding portion comprises a first heavy chain variable domain (VH1) operably linked to an antibody heavy chain CH1 domain and a first light chain variable domain (VL1) operably linked to an antibody light chain constant (CL) domain, and the second antigen-binding portion comprises a second VH (VH2) operably linked to a first T cell receptor (TCR) constant region (C1) and a second VL (VL2) operably linked to a second TCR constant region (C2), wherein C1 and C2 are capable of forming one or more non-native interchain disulfide bonds. The positions of the C1 and C2 domains in the bispecific polypeptide complex or antigen-binding portion thereof are interchangeable.

[0015] In some embodiments, the first VH comprises an amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO: 13, and the first VL comprises an amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO: 14, and / or The second VH comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO: 15, and the second VL comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO: 16.

[0016] The bispecific polypeptide complex may further comprise an Fc region, e.g., a human IgG (IgG1, IgG2, IgG3 or IgG4) Fc region, e.g., a human IgG1 Fc region, an IgG2 Fc region or an IgG4 Fc region. The Fc region may be a native Fc region or a modified Fc region. For example, a human IgG1 Fc region may be modified to include a "knobs-into-holes" 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 modified to include a "knobs-into-holes" structure, (b) a human IgG4 Fc region modified to include a "knobs-into-holes" structure and an S228P mutation, and (c) a human IgG4 Fc region modified to include an S228P mutation and an M252Y / S254T / T256E mutation.

[0017] In some embodiments, the bispecific polypeptide complex comprises one CD47 binding moiety and one HER2 binding moiety. For example, the bispecific polypeptide complex comprises two heavy chains and two light chains, where the first heavy chain comprises, from N-terminus to C-terminus, domains operably linked as VH1-C1-hinge-Fc, the second heavy chain comprises, from N-terminus to C-terminus, domains operably linked as VH2-CH1-hinge-Fc, the first light chain comprises, from N-terminus to C-terminus, domains operably linked as VL1-C2, and the second light chain comprises, from N-terminus to C-terminus, domains operably linked as VL2-CL. As an alternative example of a bispecific polypeptide complex, from N-terminus to C-terminus, a first heavy chain comprises operably linked domains as follows: VH1-CH1-hinge-Fc, a second heavy chain comprises operably linked domains as follows: VH2-C1-hinge-Fc, a first light chain comprises operably linked domains as follows: VL1-CL, and a second light chain comprises operably linked domains as follows: VL2-C2. The positions of the C1 and C2 domains in the bispecific polypeptide complex or antigen-binding portion thereof are interchangeable.

[0018] In some embodiments the bispecific polypeptide complex disclosed herein comprises: a first heavy chain comprising SEQ ID NO:19 and a second heavy chain comprising SEQ ID NO:20, a first light chain comprising SEQ ID NO:21 and a second light chain comprising SEQ ID NO:22.

[0019] 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, where from N-terminus to C-terminus, the first heavy chain and the second heavy chain each comprise domains operably linked as follows: 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 light chain and the second light chain each comprise domains operably linked as follows: VL1-C2, and the third light chain and the fourth light chain each comprise domains operably linked as follows: VL2-CL. Alternatively, the bispecific polypeptide complex comprises two heavy chains and four light chains, where from N-terminus to C-terminus the first heavy chain and the second heavy chain each comprise operably linked domains as follows: 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 light chain and the second light chain each comprise operably linked domains as follows: VL1-CL, and the third light chain and the fourth light chain each comprise operably linked domains as follows: VL2-C2. The positions of the C1 and C2 domains in the bispecific polypeptide complex or antigen-binding portion thereof are interchangeable.

[0020] 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, where from N-terminus to C-terminus, a first heavy chain comprises domains operably linked as VH2-CH1-VH1-C1-hinge-Fc, or VH1-C1-hinge-Fc-VH2-CH1, a second heavy chain comprises domains operably linked as VH1-C1-hinge-Fc, the first light chain and the second light chain each comprise domains operably linked as VL1-C2, and a third light chain comprises domains operably linked as VL2-CL. Alternatively, the first heavy chain comprises operably linked domains as follows: VH2-C1-VH1-CH1-hinge-Fc, or VH1-CH1-hinge-Fc-VH2-C1, the second heavy chain comprises operably linked domains as follows: VH1-CH1-hinge-Fc, the first light chain and the second light chain each comprise operably linked domains as follows: VL1-CL, and the third light chain comprises operably linked domains as follows: VL2-C2. The positions of the C1 and C2 domains in the bispecific polypeptide complex or antigen-binding portion thereof are interchangeable.

[0021] 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, where from N-terminus to C-terminus, a first heavy chain comprises domains operably linked as VH1-C1-VH2-CH1-hinge-Fc, or VH2-CH1-hinge-Fc-VH1-C1, a second heavy chain comprises domains operably linked as VH2-CH1-hinge-Fc, a first light chain comprises domains operably linked as VL1-C2, and a second light chain and a third light chain each comprise domains operably linked as VL2-CL. Alternatively, the first heavy chain comprises operably linked domains as follows: VH1-CH1-VH2-C1-hinge-Fc, or VH2-C1-hinge-Fc-VH1-CH1, the second heavy chain comprises operably linked domains as follows: VH2-C1-hinge-Fc, the first light chain comprises operably linked domains as follows: VL1-CL, and the second light chain and the third light chain each comprise operably linked domains as follows: VL2-C2. The positions of the C1 and C2 domains in the bispecific polypeptide complex or antigen-binding portion thereof are interchangeable.

[0022] The operably linked or "-" may be via a direct linkage or via a peptide linker, e.g., a GS linker, e.g., (GS)n, (GGS)n, (GGGS)n, (GGGGS)n, (GGSG)n, (GGGSS)n, where n is an integer from 1 to 9. The bispecific polypeptide complex may be a humanized antibody or a fully human antibody. In some embodiments, the bispecific polypeptide complex is a fully human antibody.

[0023] 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.

[0024] In one aspect, disclosed herein is a vector comprising the above-described nucleic acid molecule. In one aspect, disclosed herein is a host cell comprising the above-described nucleic acid molecule or vector.

[0025] In one aspect, disclosed herein is a pharmaceutical composition comprising a bispecific polypeptide complex, or an antigen-binding portion thereof, and a pharma- ceutically acceptable carrier.

[0026] In one aspect, there is provided a method of producing a bispecific polypeptide complex, comprising the steps of: culturing under suitable conditions a host cell containing a nucleic acid sequence encoding a polypeptide complex; isolating the polypeptide complex from the host cell; Disclosed herein is a method comprising:

[0027] In one aspect, disclosed herein is a method of modulating an immune response associated with HER2 and / or CD47 in a subject, the method comprising administering to the subject a bispecific polypeptide complex or an antigen-binding portion thereof or a pharmaceutical composition disclosed herein.

[0028] In one aspect, disclosed herein is a method of inhibiting the growth of tumor cells (e.g., tumor cells that are CD47 and / or HER2 positive) in a subject, the method comprising administering to the subject an effective amount of a bispecific polypeptide complex or antigen-binding portion thereof disclosed herein, or a pharmaceutical composition.

[0029] In one aspect, disclosed herein is a method of inducing macrophage-mediated phagocytosis of tumor cells in a subject, the method comprising administering to the subject an effective amount of a bispecific polypeptide complex or antigen-binding portion thereof disclosed herein or a pharmaceutical composition.

[0030] In one aspect, disclosed herein is a method of inducing natural killer cell mediated cytotoxicity towards (or against) tumor cells in a subject, the method comprising administering to the subject an effective amount of a bispecific polypeptide complex or antigen-binding portion thereof as disclosed herein or a pharmaceutical composition.

[0031] In one aspect, disclosed herein is a method of 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 such as 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, medulloblastoma, nasopharyngeal carcinoma, neuroblastoma, squamous cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thymoma, thyroid cancer, urachal cancer, uterine cancer, vaginal cancer, astrocytoma, basal cell carcinoma, and combinations thereof.

[0032] In some further embodiments, the cancer is breast cancer, gastric cancer, lung cancer, skin cancer, or colorectal cancer.

[0033] In one aspect, disclosed herein is a composition comprising an effective amount of a bispecific polypeptide complex disclosed herein, or an antigen-binding portion thereof, for use in diagnosing, preventing, or treating cancer in a subject.

[0034] In one aspect, i) modulation of immune responses associated with HER2 and / or CD47, and / or ii) induction of macrophage-mediated phagocytosis of tumor cells, and / or iii) induction of natural killer cell-mediated cytotoxicity of tumor cells, and / or iv) Inhibition of tumor cell proliferation, such as tumor cells that are CD47 and / or HER2 positive. Disclosed herein are bispecific polypeptide complexes, or antigen-binding portions thereof, for use in

[0035] In one aspect, disclosed herein is a bispecific polypeptide complex, or an antigen-binding portion thereof, for use in the diagnosis, treatment or prevention of cancer.

[0036] In one aspect, i) modulation of immune responses associated with HER2 and / or CD47; ii) induction of macrophage-mediated phagocytosis of tumor cells; iii) induction of natural killer cell-mediated cytotoxicity of tumor cells; and / or iv) Inhibition of tumor cell proliferation, such as tumor cells that are CD47 and / or HER2 positive. Disclosed herein is the use of a bispecific polypeptide complex, or an antigen-binding portion thereof, in the manufacture of a medicament for

[0037] In one aspect, disclosed herein is the use of a bispecific polypeptide complex, or an antigen-binding portion thereof, in the manufacture of a medicament for diagnosing, treating or preventing cancer.

[0038] The bispecific polypeptide complexes or antigen-binding portions thereof disclosed herein can be administered in combination with chemotherapeutic agents, radiation and / or other agents for use in cancer immunotherapy and may be for use in combination therapy with chemotherapeutic agents, radiation and / or other agents for use in cancer immunotherapy.

[0039] In one aspect, disclosed herein is a kit comprising a container comprising a bispecific polypeptide complex as described above, or an antigen-binding portion thereof.

[0040] The foregoing is a summary and therefore contains, by definition, simplifications, generalizations, and omissions of detail; thus, 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]

[0041] [Figure 1]Figure 1 shows a schematic diagram of the W308032 bispecific antibody in the WuXiBody E17 format. From N-terminus to C-terminus, the first heavy chain is VH1-C beta-hinge-Fc, the second heavy chain is VH2-CH1-hinge-Fc, the first light chain is VL1-C alpha, and the second light chain is VL2-CL. [Diagram 2] FIG. 2 shows the SDS-PAGE characterization of W308032-U5T6.E17-57.uIgG1. [Diagram 3] FIG. 3 shows SEC-HPLC characterization of W308032-U5T6.E17-57.uIgG1. [Figure 4] Figure 4 shows the DSF characterization of W308032-U5T6.E17-57.uIgG1. [Diagram 5] Figure 5 shows DLS characterization of W308032-U5T6.E17-57.uIgG1. [Figure 6] FIG. 6 shows HIC-HPLC characterization of W308032-U5T6.E17-57.uIgG1. [Figure 7A] FIG. 7A shows the binding affinity of W308032-U5T6.E17-57.uIgG1 to human Her2 protein as determined by SPR (Round No. 1). [Figure 7B] FIG. 7B shows the binding affinity of W308032-U5T6.E17-57.uIgG1 to human Her2 protein as determined by SPR (Round No. 2). [Figure 7C] FIG. 7C shows the binding affinity of W308032-U5T6.E17-57.uIgG1 to human Her2 protein as determined by SPR (Round No. 3). [Figure 7D] FIG. 7D shows the binding affinity of W308032-U5T6.E17-57.uIgG1 to human CD47 protein as determined by SPR (Round No. 1). [Figure 7E]FIG. 7E shows the binding affinity of W308032-U5T6.E17-57.uIgG1 to human CD47 protein as determined by SPR (Round No. 2). [Figure 7F] FIG. 7F shows the binding affinity of W308032-U5T6.E17-57.uIgG1 to human CD47 protein as determined by SPR (Round No. 3). [Figure 8A] FIG. 8A shows binding of W308032-U5T6.E17-57.uIgG1 to SK-BR-3 without an antigen sink. [Figure 8B] FIG. 8B shows binding of W308032-U5T6.E17-57.uIgG1 to SK-BR-3 after sunk by human blood cells. [Figure 9A] FIG. 9A shows binding of W308032-U5T6.E17-57.uIgG1 to SK-BR-3 without an antigen sink. [Figure 9B] FIG. 9B shows binding of W308032-U5T6.E17-57.uIgG1 to SK-BR-3 after sunk by Jurkat cells. [Figure 10A] FIG. 10A shows binding of W308032-U5T6.E17-57.uIgG1 to HCC1954 without an antigen sink. [Figure 10B] FIG. 10B shows binding of W308032-U5T6.E17-57.uIgG1 to HCC1954 after sunk by Jurkat cells. [Figure 11] FIG. 11 shows binding of W308032-U5T6.E17-57.uIgG1 to human blood cells. [Figure 12] FIG. 12 shows binding of W308032-U5T6.E17-57.uIgG1 to Jurkat cells. [Figure 13A] FIG. 13A shows CD47 ligand blocking of W308032-U5T6.E17-57.uIgG1 against SK-BR-3 without an antigen sink. [Figure 13B] FIG. 13B shows CD47 ligand blocking of W308032-U5T6.E17-57.uIgG1 on SK-BR-3 after sunk by human blood cells. [Figure 14A] FIG. 14A shows CD47 ligand blocking of W308032-U5T6.E17-57.uIgG1 against SK-BR-3 without an antigen sink. [Figure 14B] FIG. 14B shows CD47 ligand blocking of W308032-U5T6.E17-57.uIgG1 on SK-BR-3 after sunk by Jurkat cells. [Figure 15] FIG. 15 shows CD47 ligand blocking by W308032-U5T6.E17-57.uIgG1 on Jurkat cells. [Figure 16A] Figure 16A shows HER2 occupancy of W308032-U5T6.E17-57.uIgG1 versus SK-BR-3. [Figure 16B] Figure 16B shows CD47 occupancy of W308032-U5T6.E17-57.uIgG1 against SK-BR-3. [Figure 17A] FIG. 17A shows ADCP of W308032-U5T6.E17-57.uIgG1 against SK-BR-3 in two independent experiments. [Figure 17B] FIG. 17B shows ADCP of W308032-U5T6.E17-57.uIgG1 against SK-BR-3 in two independent experiments. [Figure 18] FIG. 18 shows ADCP of W308032-U5T6.E17-57.uIgG1 against HCC1954. [Figure 19] FIG. 19 shows ADCP of W308032-U5T6.E17-57.uIgG1 against NCI-N87. [Figure 20] FIG. 20 shows ADCP of W308032-U5T6.E17-57.uIgG1 against human blood cells. [Figure 21]FIG. 21 shows ADCP of W308032-U5T6.E17-57.uIgG1 against Jurkat cells. [Figure 22] Figure 22 shows ADCC of W308032-U5T6.E17-57.uIgG1 against SK-BR-3. [Diagram 23] Figure 23 shows ADCC of W308032-U5T6.E17-57.uIgG1 against HCC1954. [Figure 24] Figure 24 shows ADCC of W308032-U5T6.E17-57.uIgG1 against NCI-N87. [Diagram 25] Figure 25 shows ADCC of W308032-U5T6.E17-57.uIgG1 against Jurkat cells. [Figure 26] FIG. 26 shows the inhibitory effect of W308032-U5T6.E17-57.uIgG1 on the proliferation of SK-BR-3. [Figure 27] Figure 27 shows the inhibitory effect of W308032-U5T6.E17-57.uIgG1 on the proliferation of NCI-N87. [Figure 28] FIG. 28 shows hemagglutination of W308032-U5T6.E17-57.uIgG1 against human blood cells. [Figure 29] Figure 29 shows the thermal stability results for W308032-U5T6.E17-57.uIgG1. [Diagram 30] FIG. 30 shows the combined anti-tumor effect of anti-HER2 and anti-CD47 therapy in an HCC1954 xenograft model. [Diagram 31] FIG. 31 shows the anti-tumor effect of W308032-U5T6.E17-57.uIgG1 in HCC1954 xenograft model at different dosage levels. [Diagram 32] FIG. 32 shows the antitumor effect of combination therapy with pertuzumab in an HCC1954 xenograft model. [Diagram 33]Figure 33 shows a summary of tumor weight in HCC1954 xenograft model on day 36. Note: Two-way ANOVA Bonferroni post-hoc test, *p<0.05, **p<0.01. [Diagram 34] FIG. 34 shows the combined anti-tumor effect of anti-Her2 and anti-CD47 therapy in the NCI-N87 xenograft model. [Diagram 35] FIG. 35 summarizes the antitumor effects of W308032-U5T6.E17-57.uIgG1 in the NCI-N87 xenograft model. [Diagram 36] FIG. 36 summarizes the antitumor effects of combination therapy with paclitaxel in the NCI-N87 xenograft model. [Figure 37] FIG. 37 summarizes the antitumor effects of combination therapy with paclitaxel in the JIMT-1 xenograft model. [Figure 38] Figure 38 shows an overview of tumor rebound after the last dose. The dashed line represents the mean initial tumor volume for each group. [Figure 39] FIG. 39 shows a rodent pharmacokinetic summary for W308032-U5T6.E17-57.uIgG1 and trastuzumab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Although the present disclosure may be embodied in many different forms, certain exemplary embodiments thereof are disclosed herein that illustrate the principles of the present disclosure. It should be emphasized that the present disclosure is not limited to the specific embodiments illustrated. Moreover, the section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0043] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context dictates otherwise, singular terms shall include the plural and plural terms shall include the singular. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins, reference to "a cell" includes a mixture of cells, and so forth. In this application, the use of "or" means "and / or" unless otherwise indicated. Furthermore, the use of the term "comprising," as well as other forms such as "comprises" and "comprised," is not limiting. Additionally, the ranges set forth herein and in the appended claims include the endpoints and all points between the endpoints.

[0044] In general, 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 well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art, unless otherwise indicated, as described in the various general and more specific references cited and discussed throughout the 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 Coligan et 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 well known and commonly used in the art. Other aspects, features and advantages of the methods, compositions and / or devices and / or other subject matter described herein will become apparent in the teachings set forth herein. Additionally, the contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety.

[0045] definition To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0046] As used herein, the term "antibody" or "Ab" is used in the broadest sense and encompasses any form of antibody that exhibits the desired biological activity or binding activity, including, but not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single domain antibodies. The bispecific polypeptide complexes disclosed herein also belong to the antibody category. A typical antibody typically comprises a heavy chain and a light chain. Heavy chains can be classified as μ, δ, γ, α, and ε, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and light chain constant region (C L As demonstrated herein, various modifications may be made to the constant regions or they may be replaced with constant regions from other immunoglobulins. H Area and V L The region can be further divided into hypervariable regions (called complementarity determining regions (CDRs)) spaced by relatively conserved regions (called framework regions (FRs)). H and V L The variable region (V) of each heavy / light chain pair consists of three CDRs and four FRs in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. H and V L) each form an antigen binding site. The extent of the framework regions and CDRs can be precisely defined using methods known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, the EU definition, and / or the contact definition, all of which are well known in the art. 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. Correspondence or alignment between the numberings according to the 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 Ig superfamily V-like domains. Dev Comp Immunol. (2003) 27:55-77). The antibodies can be of different antibody isotypes, for example IgG (e.g. IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0047] The term "antigen-binding portion" as used herein refers to an antibody fragment formed from a portion of an antibody that includes one or more CDRs, or any other antibody fragment that binds to an antigen but does not include an intact 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. An antigen-binding portion can bind to the same antigen as the parent antibody binds. In certain embodiments, an antigen-binding portion can include one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies. Further detailed formats of antigen-binding moieties 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.

[0048] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody, which may be used interchangeably in the context of this application, 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 competes with the full-length antibody for binding to the same antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed. 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference for all purposes.

[0049] "Fab" with respect to an antibody refers to the portion of an antibody that consists of a single light chain (both variable and constant regions) bound by disulfide bonds to the variable region and first constant region of a single heavy chain. In certain embodiments, the constant regions of both the light and heavy chains are replaced with a TCR constant region.

[0050] "Fc" (short for crystallizable fragment) in reference to an antibody refers to the portion of an antibody that includes the second constant region (CH2) and the third constant region (CH3) of a first heavy chain linked to the second constant region and the third constant region of a second heavy chain via disulfide bonds. As used herein, the Fc region may also include a portion or the entire hinge region. The term "hinge" or "hinge region" refers to the central flexible stretch of amino acids of the heavy chains of the IgG and IgA immunoglobulin classes, which connects these two chains by disulfide bonds, and as used herein may include the entire native hinge region, a portion thereof, a homolog or a functional equivalent thereof. The Fc region of an antibody is responsible for various effector functions such as ADCC and CDC, but does not usually function in antigen binding. The ability of an antibody to initiate and control effector functions via the 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 has become 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.

[0051] As used herein, the term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences, within which further framework region modifications can be made.

[0052] The term "human antibody" or "fully human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from a human germline immunoglobulin sequence. A human antibody 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 mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0053] The terms "operably linked" and "operably linked" refer to the juxtaposition of two or more biological sequences of interest, with or without a spacer or linker, in a relationship that allows them to function as intended. When used in reference to a polypeptide, 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 may be operably linked to a constant region to result in a stable product with antigen-binding activity. The term may also be used in reference to polynucleotides. In one example, when a polynucleotide encoding a polypeptide is operably linked to a control sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequence is linked in a manner that allows for controlled expression of the polypeptide from the polynucleotide. The term operably linked, when used herein to describe domains linked to form a polypeptide, may be represented by "-" and may refer to a direct link between the domains or a linker having a length of 1 to 30 amino acids, such as a single amino acid or a series of (G4S)n linkers (n=1 to 5 (1, 2, 3, 4, or 5)).

[0054] As used herein, the term "Ka" is intended to refer to the on-rate of a particular antibody-antigen interaction, while the term "Kd" is intended to refer to the off-rate of a particular antibody-antigen interaction. D The term "dissociation constant" is intended to refer to the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). D A preferred method of determining is by the use of surface plasmon resonance, preferably a biosensor system such as a Biacore® system.

[0055] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen. As used herein, the term "high affinity" for an IgG antibody refers to an antibody that binds to a target antigen with an affinity of 1×10 -7 M or less, preferably 5×10 -8 M or less, and even more preferably 1×10 -8 M or less, and even more preferably 5×10 -9 M or less, and even more preferably 1×10 -9 M or less, and even more preferably 5×10 -10 K below M D It refers to an antibody having the following structure:

[0056] As used herein, "EC 50 The term "50% effective concentration," also called "medium effective concentration," refers to the concentration of a drug, antibody, or toxin that induces a response halfway between the baseline and maximum after a specific exposure time. In the context of this application, EC 50 is expressed in units of "nM".

[0057] As used herein, "IC 50 The term IC, also referred to as "50% inhibitory concentration," refers to the 50% 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, IC50 is expressed in units of "nM".

[0058] 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 the CD47 ligand SIRPα) to any detectable extent. In some embodiments, the antibodies disclosed herein have an IC of 1 nM or less, 0.8 nM or less, 0.6 nM or less, 0.4 nM or less, or 0.3 nM or less. 50 blocks the binding of CD47 to SIRPα.

[0059] The term "epitope" as used herein refers to a portion on an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also known as "antigenic determinant". Epitopes or antigenic determinants generally consist of chemically active surface groupings of molecules such as amino acids, carbohydrate or sugar side chains, and generally have a specific three-dimensional structure and specific charge characteristics. For example, an epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique conformation that can be "linear" or "conformational". See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996).

[0060] The term "isolated" as used herein refers to a state obtained by artificial means from a natural state. When a particular "isolated" substance or component exists in nature, it is possible that its natural environment has been altered or that the substance has been isolated from its natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide naturally exists in a particular animal organism, and the same polynucleotide or polypeptide in high purity isolated from such a natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude contaminating artificial or synthetic substances or other impurities that do not affect the activity of the isolated substance.

[0061] The term "isolated antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated bispecific antibody that specifically binds CD47 and HER2 proteins is substantially free of antibodies having different target antigens or epitopes). However, an isolated antibody that specifically binds human CD47 protein and HER2 protein may have cross-reactivity to other antigens, such as CD47 or HER2 proteins from other species. An isolated antibody may also be substantially free of other cellular material and / or chemicals.

[0062] The term "vector" as used herein refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can carry genetic material elements that are expressed in a host cell by transformation, transduction, or transfection into the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda phage or M13 phage, 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 (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (such as SV40). A vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0063] The term "host cell" as used herein refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as E. 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.

[0064] The term "identity" as used herein refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percent of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in the alignment (if any) are preferably addressed 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, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., 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.

[0065] The term "immunogenicity" as used herein 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 an antigen to stimulate certain immune cells to activate, proliferate and differentiate to ultimately produce immunological effector substances such as antibodies and sensitized lymphocytes, but also refers to the specific immune response that antibodies or sensitized T lymphocytes are formed in the immune system of an organism after stimulating the organism with the antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors: the property of the antigen, the reactivity of the host, and the immunization means.

[0066] The term "transfection" or "transfecting" as used herein refers to the process by which nucleic acid is introduced into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods, such as electroporation. Several transfection techniques are well known in the art and are 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.

[0067] As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes an optical phenomenon that allows analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, NJ). For further explanation, see Example 5 and Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0068] The term "fluorescence activated cell sorting" or "FACS" as used herein refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence properties of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". Retrieved 2017-11-09). Instruments for performing FACS are known to those of skill in the art and are generally commercially available. Examples of such instruments include FACS Star Plus, FACScan and FACSort instruments manufactured by Becton Dickinson (Foster City, CA), Epics C manufactured by Coulter Epics Division (Hialeah, FL), and MoFlo manufactured by Cytomation (Colorado Springs, CO).

[0069] The term "subject" includes any human or non-human animal, preferably humans.

[0070] The terms "HER2-associated" or "HER2-associated," as used herein with respect to a disease or condition, refer to any disease or condition that is caused by, exacerbated by, or otherwise associated with increased or decreased expression or activity of HER2 (e.g., human HER2).

[0071] The term "cancer" as used herein refers to any or tumor or malignant cell growth, proliferation or metastasis mediated solid and non-solid tumors, e.g., leukemia, that initiates a medical condition.

[0072] The terms "treatment," "treating," or "treated" as used herein in the context of treating a condition generally relate to treatments and therapies in which some desired therapeutic effect is achieved, whether in a human or animal, such as inhibition of progression of the condition, including slowing the rate of progression, stopping the rate of progression, regressing the condition, improving the condition, and curing the condition. In the case of cancer, "treating" can refer to attenuating or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or any combination thereof. In the case of tumors, "treatment" includes removal of all or part of a tumor, inhibition or slowing of tumor growth and metastasis, reduction in tumor number, prevention or slowing of tumor onset, or any combination thereof.

[0073] The terms "prevent", "preventing" or "prevention" as used herein in the context of preventing a condition generally refer to preventing or delaying the onset of a disease or preventing the manifestation of its clinical or subclinical symptoms in a subject (whether human or animal), e.g., preventing a disease from occurring in a subject who has a predisposition to the condition or disease but has not yet been diagnosed as having it.

[0074] The term "therapeutically effective amount" as used herein relates to an amount of an active compound or a material, composition or dosage form containing an active compound that is effective to produce some desired therapeutic effect commensurate with a reasonable benefit / risk ratio when administered in accordance with a desired treatment regimen. Specifically, "therapeutically effective amount" refers to an amount or concentration of an antibody effective to treat a human CD47 / HER2-associated disease or condition.

[0075] As used herein, the present disclosure in "host cell" refers to a cell used for introduction of an exogenous polynucleotide.

[0076] As used herein, the term "pharmaceutical acceptable" means that the vehicle, diluent, excipient and / or salts thereof are chemically and / or physically compatible with the other ingredients in the formulation and physiologically compatible with the recipient.

[0077] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active agent, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including, but not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80, and ionic strength enhancers include, but are not limited to, sodium chloride.

[0078] As used herein, the term "adjuvant" refers to a non-specific immune enhancing agent that can enhance the immune response to an antigen or change the type of immune response in an organism when delivered to an organism together with an antigen or delivered to an organism in advance. There are various 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 currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0079] Bispecific Polypeptide Complexes The bispecific polypeptide complexes provided herein include bispecific antibodies and antigen-binding portions thereof. As used herein, the term "polypeptide complex" may be used interchangeably with "antibody." In some embodiments, the bispecific antibodies and antigen-binding portions thereof 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, for example, as "anti-HER2 / anti-CD47" or "anti-CD47 / HER2" or "anti-CD47xHER2" or "CD47xHER2" bispecific antibodies, or other similar terms.

[0080] In some embodiments, the bispecific antibody herein comprises a first antigen-binding moiety that specifically binds to CD47 (CD47-binding moiety) and a second antigen-binding moiety that specifically binds to HER2 (HER2-binding moiety). The first and second antigen-binding moieties may be in Fab, scFv or VHH format, etc., taking into consideration the stability, expression level, binding ability and other functions of the assembled antibody. For example, the HER2-binding moiety is in Fab, scFv or VHH format, and the CD47-binding moiety is in Fab format. Alternatively, the CD47-binding moiety is in Fab, scFv or VHH format, and the HER2-binding moiety is in Fab format. In some embodiments, both the HER2-binding moiety and the CD47-binding moiety are in Fab format, forming the two arms of the bispecific antibody.

[0081] In some embodiments, the bispecific antibodies disclosed herein comprise multiple antigen-binding moieties that specifically bind to CD47 and / or multiple antigen-binding moieties that specifically bind to HER2. Typically, in the case of bispecific antibodies, the multiple antigen-binding moieties have the same variable region (and thus target the same antigen / epitope) or the variable and constant regions (if present) are completely the same. For example, an antibody may comprise two identical CD47-binding moieties and one HER2-binding moiety, or one CD47-binding moiety and two identical HER2-binding moieties, or two identical CD47-binding moieties and two identical HER2-binding moieties. Furthermore, if there are two CD47-binding moieties or two HER2-binding moieties, preferably the two CD47-binding moieties and / or the two HER2-binding moieties are not on the same chain.

[0082] In some specific embodiments, the CD47 binding moiety is in Fab format, 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 moiety is also in Fab format, 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 moiety, the commonly employed CH1 and CL domains are replaced by a pair of TCR constant regions. The positions of C1 and C2 are interchangeable.

[0083] Alternatively, the CD47 binding moiety 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), where the positions of C1 and C2 are swapped, while the HER2 binding moiety 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.

[0084] The introduction of TCR constant regions to replace the commonly used CH1 and CL domains has been shown to increase the stability and expression levels of the generated antibody formats. A detailed description of the utility of TCR constant regions in assembling two parent antibodies into bispecific molecules with desired valency and functionality is disclosed in WO 2019 / 057122, the entire contents of which are incorporated herein by reference. When replaced by TCR constant regions (Cbeta / Calpha), a chimeric Fab is generated with a unique light-heavy chain interface that is orthogonal to the conventional antibody Fab. The assembly of different formats of chimeric Fab and conventional Fab can create a variety of bispecific molecules with different structures and valencies.

[0085] The first and second TCR constant regions are linked via a non-natural interchain disulfide bond. The pair of TCR constant regions in the antigen-binding moiety comprises a TCR alpha constant region (wild-type or preferably modified) in the light chain and a TCR beta constant region (wild-type or preferably modified) in the heavy chain. The TCR constant regions in the bispecific antibody can link together via a non-natural disulfide bond to form a dimer.

[0086] TCR, or T cell receptor, is a heterodimeric T cell surface protein belonging to the immunoglobulin superfamily, similar to a half antibody with a single heavy chain and a single light chain. A native TCR has an extracellular portion, a transmembrane portion and an intracellular portion. The extracellular domain of the TCR has a membrane proximal constant region and a membrane distal variable region.

[0087] The sequences of the wild type human TCR beta and alpha chain constant regions can be found in NCBI Accession No. A0A5B9 (www.uniprot.org / uniprot / A0A5B9) and NCBI Accession No. P01848 (www.uniprot.org / uniprot / P01848). The pairs of TCR constant regions for constructing 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.

[0088] As exemplified in the present application, the bispecific antibody comprises a modified TCR beta chain constant region (sequence shown in SEQ ID NO: 17) and a modified TCR alpha chain constant region (sequence shown in SEQ ID NO: 18).

[0089] It is understood that the mutants 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. Several C1 and C2 mutants for constructing WuXiBody antibody formats are disclosed in PCT / CN2021 / 072601, the entire contents of which are incorporated herein by reference.

[0090] In some embodiments, the TCR beta chain constant region replaces the CH1 domain and the TCR alpha chain constant region replaces the CL domain, or they may be swapped, with the TCR beta chain constant region in the light chain and the TCR alpha chain constant region in the heavy chain.

[0091] The advantages of replacing the CH1 and CL domains with the TCR constant region 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 a desired conformation, thereby stabilizing the TCR constant region dimer while obtaining good antigen-binding activity of the antibody variable region. It has also been found that C1 and C2 containing antigen-binding portions tolerate well conventional antibody engineering, such as modification of glycosylation sites and removal of some native sequences. Moreover, bispecific antibodies in such formats can be easily expressed and assembled with minimal or virtually no mismatching of antigen-binding sequences due to the presence of the TCR constant region in the antigen-binding portion.

[0092] In some specific embodiments, the bispecific antibodies disclosed herein comprise one HER2 binding moiety and one CD47 binding moiety in each arm, or in other words, two HER2 binding moieties and two CD47 binding moieties per antibody. The Fc region may be located at the C-terminus of the antibody and operably linked to the CD47 binding moiety or the HER2 binding moiety at the N-terminus of the Fc region. Alternatively, the Fc region may be located between the CD47 binding moiety and the HER2 binding moiety. Such bispecific antibodies may be constructed as homodimers with two identical heavy chains.

[0093] According to some exemplary embodiments, the bispecific polypeptide complexes herein comprise two heavy chains and four light chains, arranged from N-terminus to C-terminus as follows: (a) the first heavy chain and the second heavy chain each comprise operably linked domains such as VH1-C1-VH2-CH1-hinge-Fc (wherein VH1-C1 is derived from the HER2 binding moiety and VH2-CH1 is derived from the CD47 binding moiety), the two light chains comprise operably linked domains such as VL1-C2 (wherein VL1-C2 is derived from the HER2 binding moiety), and the other two light chains comprise operably linked domains such as VL2-CL (wherein VL2-CL is derived from the CD47 binding moiety); or (b) the first heavy chain and the second heavy chain each comprise operably linked domains such as VH2-CH1-VH1-C1-hinge-Fc (wherein VH1-C1 is derived from the HER2 binding moiety and VH2-CH1 is derived from the CD47 binding moiety), and the two light chains comprise operably linked domains such as VL1-C2 (wherein VL1-C2 is derived from the HER2 binding moiety), and the other two light chains comprise operably linked domains such as VL2-CL (wherein VL2-CL is derived from the CD47 binding moiety); or (c) the first heavy chain and the second heavy chain each comprise operably linked domains such as VH1-C1-hinge-Fc-VH2-CH1 (wherein VH1-C1 is derived from the HER2 binding portion and VH2-CH1 is derived from the CD47 binding portion), and the two light chains comprise operably linked domains such as VL1-C2 (wherein VL1-C2 is derived from the HER2 binding portion), and the other two light chains comprise operably linked domains such as VL2-CL (wherein VL2-CL is derived from the CD47 binding portion); or (d) the first heavy chain and the second heavy chain each comprise operably linked domains such as VH2-CH1-hinge-Fc-VH1-C1 (wherein VH1-C1 is derived from the HER2 binding portion and VH2-CH1 is derived from the CD47 binding portion), and the two light chains comprise operably linked domains such as VL1-C2 (wherein VL1-C2 is derived from the HER2 binding portion), and the other two light chains comprise operably linked domains such as VL2-CL (wherein VL2-CL is derived from the CD47 binding portion); or (e) the first heavy chain and the second heavy chain each comprise operably linked domains as VH1-CH1-VH2-C1-hinge-Fc (wherein VH1-CH1 is derived from the HER2 binding moiety and VH2-C1 is derived from the CD47 binding moiety), and the two light chains each comprise operably linked domains as VL1-CL (wherein VL1-CL is derived from the HER2 binding moiety), and the other two light chains each comprise operably linked domains as VL2-C2 (wherein VL2-C2 is derived from the CD47 binding moiety); or (f) the first heavy chain and the second heavy chain each comprise operably linked domains as follows: VH2-C1-VH1-CH1-hinge-Fc (wherein VH1-CH1 is derived from the HER2 binding moiety and VH2-C1 is derived from the CD47 binding moiety); the two light chains each comprise operably linked domains as follows: VL1-CL (wherein VL1-CL is derived from the HER2 binding moiety); and the other two light chains each comprise operably linked domains as follows: VL2-C2 (wherein VL2-C2 is derived from the CD47 binding moiety); or (g) the first heavy chain and the second heavy chain each comprise operably linked domains such as VH1-CH1-hinge-Fc-VH2-C1 (wherein VH1-CH1 is derived from the HER2 binding moiety and VH2-C1 is derived from the CD47 binding moiety), the two light chains each comprise operably linked domains such as VL1-CL (wherein VL1-CL is derived from the HER2 binding moiety), and the other two light chains each comprise operably linked domains such as VL2-C2 (wherein VL2-C2 is derived from the CD47 binding moiety); or (h) the first heavy chain and the second heavy chain each comprise domains operably linked as VH2-C1-hinge-Fc-VH1-CH1 (wherein VH1-CH1 is derived from the HER2-binding portion and VH2-C1 is derived from the CD47-binding portion); the two light chains each comprise domains operably linked as VL1-CL (wherein VL1-CL is derived from the HER2-binding portion); and the other two light chains each comprise domains operably linked as VL2-C2 (wherein VL2-C2 is derived from the CD47-binding portion).

[0094] 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.

[0095] According to some exemplary embodiments, the bispecific polypeptide complexes herein comprise two heavy chains and three light chains, arranged from N-terminus to C-terminus as follows: (a) the first heavy chain comprises operably linked domains as follows: VH1-C1-VH2-CH1-hinge-Fc; the second heavy chain comprises operably linked domains as follows: VH2-CH1-hinge-Fc; the first light chain comprises operably linked domains as follows: VL1-C2; and the second light chain and the third light chain comprise operably linked domains as follows: VL2-CL; or (b) the first heavy chain comprises operably linked domains as follows: VH2-CH1-hinge-Fc-VH1-C1; the second heavy chain comprises operably linked domains as follows: VH2-CH1-hinge-Fc; the first light chain comprises operably linked domains as follows: VL1-C2; and the second light chain and the third light chain comprise operably linked domains as follows: VL2-CL; or (c) the first heavy chain comprises operably linked domains such as VH2-CH1-VH1-C1-hinge-Fc, the second heavy chain comprises operably linked domains such as VH1-C1-hinge-Fc, the first light chain and the second light chain each comprise operably linked domains such as VL1-C2, and the third light chain comprises operably linked domains such as VL2-CL; or (d) the first heavy chain comprises operably linked domains such as VH1-C1-hinge-Fc-VH2-CH1, the second heavy chain comprises operably linked domains such as VH1-C1-hinge-Fc, the first light chain and the second light chain each comprise operably linked domains such as VL1-C2, and the third light chain comprises operably linked domains such as VL2-CL; or (e) the first heavy chain comprises operably linked domains as follows: VH1-CH1-VH2-C1-hinge-Fc; the second heavy chain comprises operably linked domains as follows: VH2-C1-hinge-Fc; the first light chain comprises operably linked domains as follows: VL1-CL; and the second light chain and the third light chain comprise operably linked domains as follows: VL2-C2; or (f) the first heavy chain comprises operably linked domains as follows: VH2-C1-hinge-Fc-VH1-CH1; the second heavy chain comprises operably linked domains as follows: VH2-C1-hinge-Fc; the first light chain comprises operably linked domains as follows: VL1-CL; and the second light chain and the third light chain comprise operably linked domains as follows: VL2-C2; or (g) the first heavy chain comprises operably linked domains such as VH2-C1-VH1-CH1-hinge-Fc, the second heavy chain comprises operably linked domains such as VH1-CH1-hinge-Fc, the first light chain and the second light chain each comprise operably linked domains such as VL1-CL, and the third light chain comprises operably linked domains such as VL2-C2; or (h) the first heavy chain comprises domains operably linked as VH1-CH1-hinge-Fc-VH2-C1, the second heavy chain comprises domains operably linked as VH1-CH1-hinge-Fc, the first light chain and the second light chain each comprise domains operably linked as VL1-CL, and the third light chain comprises domains operably linked as VL2-C2.

[0096] In some specific embodiments, the bispecific antibodies disclosed herein comprise one HER2 binding moiety in one arm and one CD47 binding moiety in the other arm. Figure 1 shows a bispecific antibody comprising the following structure (E17): from N-terminus to C-terminus, VH1-C1-hinge-Fc in the first heavy chain, VH2-CH1-hinge-Fc in the second heavy chain, VL1-C2 in the first light chain, and VL2-CL in the second light chain. VH1 and VL1 refer to the first VH and VL, respectively, that form the HER2 binding site, and VH2 and VL2 refer to the second VH and VL, respectively, that form the CD47 binding site. "-" represents an operable linkage, typically via a linker comprising a peptide bond or a peptide linker of about 1-30 amino acids. The peptide linker can be a series of (G4S)n (n=1-5).

[0097] Depending on the bispecific format and / or numbering convenience, each numbered sequence disclosed herein, e.g., 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.

[0098] CDRs and variable regions of bispecific polypeptide complexes 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), the first and second antigen-binding portions being derived from an anti-CD47 antibody and an anti-HER2 antibody, respectively. The parent antibody may be a known, already developed one, or may be newly developed. "Derived" generally means herein that the antigen-binding portion comprises the CDR sequence of the parent antibody or a highly homologous CDR sequence, preferably comprising 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, compared to the original CDR sequence of the parent antibody, one or two amino acids in one or more CDR regions may be modified to reduce the risk of glycosylation and deamidation.

[0099] Specifically, in the bispecific antibodies exemplified herein, the HER2 binding moiety is A) (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having no more than one, two or three amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 1; (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having no more than one, two or three amino acid additions, deletions and / or substitutions compared to SEQ ID NO:2; and (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having no more than one, two or three amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 3; one or more heavy chain CDRs (HCDRs) selected from the group consisting of: B) (i) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having no more than one, two or three amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 4; (ii) an LCDR2 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having no more than one, two or three amino acid additions, deletions and / or substitutions compared to SEQ ID NO:5; and (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having no more than one, two or three amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 6; or one or more light chain CDRs (LCDRs) selected from the group consisting of: C) one or more HCDRs of A) and one or more LCDRs of B) Including, The CD47 binding moiety is A') (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having no more than one, two or three amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 7; (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having no more than one, two or three amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 8; and (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having no more than one, two or three amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 9; one or more HCDRs selected from the group consisting of: B') (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having not more than 1, 2 or 3 amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 10; (ii) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having no more than one, two or three amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 11; and (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having not more than 1, 2 or 3 amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 12; or one or more LCDRs selected from the group consisting of C') one or more HCDRs of A') and one or more LCDRs of B') Includes.

[0100] Variable regions and CDRs in an antibody sequence can be identified according to general rules developed in the art or by aligning the sequence against a database of known variable regions. CDRs are described by 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), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared with each other. However, application of any definition to represent the CDRs of the antibodies disclosed herein is intended to be within the scope of this application. The CDRs shown in Table 2 below are defined according to the Kabat and IMGT numbering systems. However, Chothia, MacCallum, and other methods known in the art could 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 and can be accessed through the "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, described in Retter et al., Nucl. Acids Res., 33(Database issue):D671-D674(2005)).Preferably, the sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT and Protein Data Bank (PDB) with structure data from PDB. See the chapter on Protein Sequence and Structure Analysis of Antibody Variable Domains in the book Antibody Engineering Lab Manual by Dr. Andrew C. R. Martin (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13:978-3540413547, also available on the website at bioinforg.uk / abs). The Abysis database website further includes general rules developed for identifying CDRs, which can be used according to the teachings herein.

[0101] In some particular embodiments, the HER2 binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL): (a) VH comprises an HCDR1 as set forth in SEQ ID NO: 1, an HCDR2 as set forth in SEQ ID NO: 2, and an HCDR3 as set forth in SEQ ID NO: 3, and (b) VL comprises an LCDR1 shown in SEQ ID NO:4, an LCDR2 shown in SEQ ID NO:5, and an LCDR3 shown in SEQ ID NO:6.

[0102] In some specific embodiments, the CD47 binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), (a) VH comprises an HCDR1 as set forth in SEQ ID NO: 7, an HCDR2 as set forth in SEQ ID NO: 8, and an HCDR3 as set forth in SEQ ID NO: 9; and (b) VL comprises an LCDR1 shown in SEQ ID NO:10, an LCDR2 shown in SEQ ID NO:11, and an LCDR3 shown in SEQ ID NO:12.

[0103] In some embodiments, the bispecific antibody, or antigen-binding portion thereof, comprises a first antigen-binding portion that specifically binds to HER2, the first antigen-binding portion comprising: (A) (i) comprising the amino acid sequence of SEQ ID NO: 13; (ii) comprises an amino acid sequence that is at least 85%, 90% or 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 13; or (iii) An amino acid sequence in which one or more (e.g., 1, 2, 3 or more, preferably 1, 2 or 3, more preferably 1 or 2) amino acids have been added, deleted and / or substituted compared to SEQ ID NO: 13. heavy chain variable region, and / or (B) (i) comprising the amino acid sequence of SEQ ID NO: 14; (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 14; or (iii) An amino acid sequence in which one or more (e.g., 1, 2, 3 or more, preferably 1, 2 or 3, more preferably 1 or 2) amino acids have been added, deleted and / or substituted compared to SEQ ID NO: 14. Contains the light chain variable region.

[0104] In some further embodiments, the bispecific antibody, or antigen-binding portion thereof, comprises a second antigen-binding portion that specifically binds to CD47, the second antigen-binding portion comprising: (A) (i) comprising the amino acid sequence of SEQ ID NO: 15; (ii) comprises an amino acid sequence that is at least 85%, 90% or 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 15; or (iii) An amino acid sequence in which one or more (e.g., 1, 2, 3 or more, preferably 1, 2 or 3, more preferably 1 or 2) amino acids have been added, deleted and / or substituted compared to SEQ ID NO: 15. heavy chain variable region, and / or (B) (i) comprising the amino acid sequence of SEQ ID NO: 16; (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 16; or (iii) An amino acid sequence in which one or more (e.g., 1, 2, 3 or more, preferably 1, 2 or 3, more preferably 1 or 2) amino acids have been added, deleted and / or substituted compared to SEQ ID NO: 16. Contains the light chain variable region.

[0105] 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)) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residual table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percentage identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6 or 4, and length weights of 1, 2, 3, 4, 5 or 6.

[0106] Additionally or alternatively, the protein sequences of the present disclosure can be further used as a "query sequence" to perform searches against public databases, for example to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program using a score=50 and wordlength=3 to obtain amino acid sequences homologous to the antibody molecules of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized 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) can be used. See www.ncbi.nlm.nih.gov.

[0107] In some specific embodiments, the heavy chain variable region of the HER2 binding moiety consists of the amino acid sequence of SEQ ID NO: 13, the light chain variable region of the HER2 binding moiety consists of the amino acid sequence of SEQ ID NO: 14, and / or the heavy chain variable region of the CD47 binding moiety consists of the amino acid sequence of SEQ ID NO: 15 and the light chain variable region of the CD47 binding moiety consists of the amino acid sequence of SEQ ID NO: 16.

[0108] In other embodiments, the amino acid sequence 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 respective sequences set forth above.

[0109] In some further embodiments, the bispecific antibodies or antigen-binding portions thereof may include conservative amino acid substitutions or modifications in the variable regions of the heavy and / or light chains. It is understood in the art that certain conservative sequence modifications can be made that do 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.

[0110] As mentioned above, the term "conservative substitution" as used herein refers to an amino acid substitution that will not adversely affect or change the essential properties of the protein / polypeptide containing 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 an amino acid residue is replaced with another amino acid residue having a similar side chain, e.g., a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties (including the ability to form covalent 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 alkaline 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 β-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 replaced with 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).

[0111] Antigen Binding of Bispecific Polypeptide Complexes Preferably, the bispecific polypeptide complex of the present disclosure is capable of binding to human HER2 and CD47. Binding of the antibody to HER2 and CD47 can be assessed using one or more techniques well established in the art, such as ELISA or FACS, which measure 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 assays in which the antibody is reacted with a cell line expressing human HER2 or human CD47, such as CHO cells transfected to express HER2 or CD47 on the cell surface, or a HER2 or CD47 positive cell line, or a HER2 and CD47 double positive cell line.

[0112] Additionally or alternatively, the binding of the antibody, e.g., the binding kinetics (e.g., K D The binding affinity (value) can be tested in a BIAcore binding assay. For example, the antibodies of the disclosure have a binding affinity of 1×10 as measured by surface plasmon resonance. -9 K below M D binds to human HER2 protein at 8 × 10 -10 K below M D binds to human HER2 protein at 6 × 10 -10 K below M D binds to human HER2 protein at 4 × 10 -10 K below M D binds to human HER2 protein at 2 × 10 -10 K below M D or 1.5 × 10 -10 K below M D It binds to the human HER2 protein.

[0113] Functionality of BsAb The bispecific antibodies disclosed herein are characterized by particular functional features or properties. In some embodiments, the antibodies have one or more of the following properties: (a) specifically binds to HER2 with high affinity and specifically binds to CD47 with relatively low affinity compared to a control antibody; (b) binding to target cells (e.g., human blood cells or Jurkat cells) without being affected by the antigen sink effect; (c) negligible binding to red blood cells (RBCs), avoiding induction of hemagglutination and phagocytosis of human RBCs; (d) binding to Jurkat cells is significantly reduced compared to a control antibody (e.g., about 1 / 200); (e) efficiently blocking the binding of CD47 to SIRPα on CD47 / HER2 double positive cells without being affected by the antigen sink effect (e.g., on human blood cells or Jurkat cells); (f) There is almost no effect of CD47 ligand blocking on CD47 single positive cells. (g) The CD47 occupancy rate on CD47 / HER2 double positive cells is significantly higher than that of the control antibody. (h) demonstrating significantly increased ADCP efficacy against CD47 / HER2 double positive cells compared to the combination of anti-CD47 and anti-HER2 monotherapy, while showing little ADCP efficacy against human blood cells; (i) It has strong ADCC efficacy against CD47 / HER2 double positive cells, but weak ADCC efficacy against Jurkat cells; (j) having a strong inhibitory effect on the proliferation of CD47 / HER2 double-positive cells; (k) significantly inhibiting tumor cell proliferation in HER2-positive cancer models; and (l) having better or equivalent rodent pharmacokinetics compared to a control antibody;

[0114] 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 magrolimab. 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.

[0115] In some embodiments, the bispecific polypeptide complexes disclosed herein have a higher binding affinity to HER2 compared to monospecific anti-HER2 antibodies or other anti-HER2 / CD47 bispecific antibodies. In some embodiments, the bispecific polypeptide complexes disclosed herein have a binding affinity to HER2 of less than K D is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higher than a monospecific anti-HER2 antibody or another anti-HER2 / CD47 bispecific antibody, as measured by

[0116] In some embodiments, the bispecific polypeptide complexes disclosed herein have a lower binding affinity for CD47 compared to 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 greater than or equal to K D at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% lower than a monospecific anti-CD47 antibody or another anti-HER2 / CD47 bispecific antibody, as measured by

[0117] In some embodiments, the bispecific polypeptide complexes disclosed herein have a higher binding affinity to HER2 compared to a monospecific anti-HER2 antibody and / or a lower binding affinity to CD47 compared to a monospecific anti-CD47 antibody. In some embodiments, the bispecific polypeptide complexes disclosed herein have a higher or equal binding affinity to HER2 compared to trastuzumab and a lower binding affinity to CD47 compared to magrolimab.

[0118] In some embodiments, the bispecific polypeptide complexes disclosed herein have a higher binding affinity to HER2 compared to other anti-HER2 / CD47 bispecific antibodies and / or a lower binding affinity to CD47 compared to other anti-HER2 / CD47 bispecific antibodies.

[0119] Not affected by antigen sink effect Antibodies against cell membrane-associated antigens usually undergo target-mediated clearance, known as the antigen sink effect. The widespread expression of CD47 is known to reduce the bioavailability of anti-CD47 mAbs in tumors due to the antigen sink effect, and it also carries the risk of off-target toxicity, most substantially due to cross-linking of red blood cells that show high expression of CD47.

[0120] Expression of CD47 on normal tissues may create an "antigen sink" 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 use BsAbs with reduced affinity for CD47. These BsAbs retain the ability to block CD47-SIRPα interaction but require binding to a second tumor antigen for high affinity binding.

[0121] The BsAbs disclosed herein demonstrate that by targeting CD47 and HER2, CD47-SIRPα blockade is specifically induced in cells co-expressing HER2, and the BsAbs exhibit therapeutic synergy not observed with monospecific anti-CD47 or monospecific anti-HER2 antibodies. The BsAbs herein specific for CD47 (low affinity) and HER2 (high affinity) exhibit binding specificity and potent functional effects against CD47 and HER2 double positive cancer cells with negligible RBC antigen sink.

[0122] Blocking CD47 binding to SIRPα CD47 is normally expressed on the surface of healthy normal cells, mobilizing hematopoietic stem cells to prevent phagocytosis, and is upregulated in almost all hematological and solid tumors to evade immune surveillance and avoid phagocytosis. Preventing the interaction of CD47 with SIRPa allows phagocytes to "eat" and destroy cancer cells. CD47 blockade repolarizes tumor-associated macrophages to a proinflammatory antitumor state, and the elimination of malignant cells by phagocytes provides an additional route for neoantigen presentation to the adaptive immune system.

[0123] Signal regulatory protein alpha (SIRPα; also known as CD172a) is a receptor for CD47 and is expressed primarily on the surface of macrophages. CD47 is known to interact with SIRPα, allowing it to evade immune surveillance. The antibodies of the present disclosure may modulate, e.g., block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the binding of CD47 to SIRPα. Blockade of the CD47-SIRPα interaction using the antibodies described herein may ameliorate or overcome immune evasion, with potential clinical benefit.

[0124] As demonstrated in the Examples, the bispecific antibodies described herein can effectively block CD47 ligand binding to CD47 / HER2 double-positive cell lines, in other words, can block the interaction of CD47 with SIRPα, without showing CD47 ligand blocking to CD47 single-positive cell lines (e.g., Jurkat cells). Furthermore, the blocking effect on CD47 / HER2 double-positive cell lines was not affected by the antigen sink effect caused by human blood cells or Jurkat cells, etc.

[0125] Weak binding to human red blood cells (RBC) to avoid hemagglutination 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 red blood cells. In preclinical studies, transient hemolytic anemia was associated with anti-CD47 therapy due to increased RBC clearance.

[0126] The antibodies of the present disclosure have negligible binding to human red blood cells, avoiding the undesirable effect of red blood cell aggregation.Compared to the control anti-CD47 antibody, the bispecific antibodies of the present disclosure also have about 200-fold reduced binding to Jurkat cells.Consistently, the phagocytosis of human RBCs caused by the bispecific antibodies of the present disclosure will be much milder than that of the control antibody.

[0127] Mediating ADCP or ADCC activity The term "antibody-dependent cellular phagocytosis" or "ADCP" is a cellular process by which effector cells with phagocytic capabilities, such as monocytes and macrophages, can internalize target cells. Once phagocytosed, the target cell resides within a phagosome, which fuses with a lysosome to initiate the target cell's degradation via oxygen-dependent or -independent mechanisms. This function relies on opsonization, or the identification of the target cell by an antibody, which also acts as a bridge between the target cell and the phagocyte. Mechanistically, an antibody binds to its cognate antigen on the target cell through its antigen recognition domain and then recruits the phagocyte to the target by its Fc region. Upon binding to the phagocyte's Fc receptor, the target cell is ingested and degraded. This process also results in the production of soluble factors by the effector cells that help initiate and drive the immune response.

[0128] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" as used herein refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" the cytotoxic cells and are absolutely necessary for such killing. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas 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). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, such as those described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model, for example in the animal model disclosed in Clynes et al. PNAS (USA), 95:652-656 (1998).

[0129] As disclosed herein, ADCP may be the mechanism of action of anti-CD47 therapeutic antibodies. The bispecific antibodies herein exhibit strong ADCP efficacy against CD47 / HER2 double positive cells. These antibodies of the present disclosure could also induce strong ADCC activity against CD47 / HER2 double positive tumor cells. On the other hand, BsAbs have little or very weak ADCP or ADCC efficacy against CD47 single positive cells, indicating that binding to HER2 is necessary for the effector function of these antibodies herein.

[0130] Synergy The bispecific antibodies disclosed herein, which combine the dual binding activities of CD47 and HER2, exhibit synergistic effects compared to monotherapies such as trastuzumab, panitumumab, or magrolimab alone.

[0131] The present disclosure demonstrates that administration of the bispecific antibodies disclosed herein achieved significantly improved tumor growth inhibition compared to anti-HER2 mAb or anti-CD47 mAb alone in HCC1954 breast cancer model or NCI-N87 xenograft model in mice. Combination of the bispecific antibodies disclosed herein with another anti-HER2 mAb further improved the anti-tumor effect at the same dosage level.

[0132] Fc area 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 a specific embodiment, the Fc region is of the IgG1 isotype.

[0133] In the context of the bispecific antibodies of the present disclosure, the Fc region may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type Fc region. The present disclosure encompasses bispecific antigen-binding molecules that contain one or more modifications to obtain a desired functionality in the Fc region, such as a "knobs-into-holes" structure to promote heterodimerization, or a modified Fc region to alter the binding interaction of Fc with FcRn or FcγR.

[0134] The term "knobs-into-holes" as used herein refers to modifying the CH3 domain of an antibody Fc region to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization. Knobs can be obtained by substituting a small amino acid residue in the first CH2 / CH3 polypeptide with a large one, and holes can be obtained by substituting a large residue with a small one. For details of the mutation sites for knobs-into-holes, see Spiess et al., 2015, supra, and Brinkmann et al., 2017, supra, and US Patent Publication No. 2003078385. Generally, a "knob" is created by substituting the bulky residue W on one heavy chain with T366 according to EU numbering as in Kabat et al., and the corresponding "hole" is created by a triple mutation of T366S, L368A, and Y407V on the other heavy chain. In some embodiments, the "hole" mutations are Y349C, T366S, L368A and / or Y407V and the "knob" mutations are 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.

[0135] 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 an 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 an IgG4 isotype comprising S354C and T366W substitutions (knob) and the second heavy chain of the bispecific antibody comprises an Fc region of an IgG4 isotype comprising Y349C, T366S, L368A and Y407V substitutions (hole).

[0136] In addition, the Fc region may contain 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 comprises a LALA mutation, i.e., L234A and L235A mutations, according to EU numbering as in Kabat et al. The LALA mutation is perhaps the most commonly used mutation to impede antibody effector function, e.g., to eliminate Fc binding to specific FcγRs and reduce ADCC activity mediated by PBMCs and monocytes. Furthermore, it has been found that the introduction of YTE (M252Y / S254T / T256E) and LS (M428L / N434S) into the Fc region may increase therapeutic potential as a result of increased half-life and increased protection period. The S228P mutation was also found to prevent IgG4 Fab arm exchange in vivo and in vitro, as demonstrated using a novel quantitative immunoassay in combination with a physiological matrix preparation (J Biol Chem 2015 Feb 27;290(9):5462-9).

[0137] The bispecific antibodies disclosed herein may comprise an Fc region selected from one of the following: (a) a human IgG1 Fc region engineered to contain a "knob-into-hole" structure; (b) a human IgG4 Fc region that has been modified to include a "knob-into-hole" structure and, optionally, a S228P mutation; and (c) A human IgG4 Fc region that has been modified to include the S228P mutation and optionally the M252Y / S254T / T256E mutations.

[0138] "EU numbering system" or "EU index" is generally used when referring to residues in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "Kabat-like EU numbering" or "Kabat-like EU index" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system.

[0139] Nucleic acid molecules encoding BsAbs In some embodiments, the present 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.

[0140] Nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. In the case of antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described in more detail below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding such antibodies can be retrieved from the gene library.

[0141] The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding nucleic acid to another DNA molecule encoding a heavy chain constant region (CH1, CH2 and CH3, or a TCR beta constant region). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), supra), and DNA fragments encompassing 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 is more preferably an IgG1 or IgG4 constant region.

[0142] The isolated nucleic acid encoding the VL region can be converted to a full-length light chain gene (and a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region, CL, or the TCR alpha constant region. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., supra), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In some embodiments, the light chain constant region can be a kappa constant region or a lambda constant region.

[0143] Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In these manipulations, a VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used in this context is intended to mean that the two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0144] In certain embodiments, the isolated nucleic acid molecule comprises one or more nucleic acid sequences selected from the group consisting of: (A) a nucleic acid sequence encoding a heavy chain sequence of a CD47 binding moiety or a heavy chain sequence of a HER2 binding moiety; (B) a nucleic acid sequence encoding a heavy chain sequence of a CD47 binding moiety operably linked to a heavy chain sequence of a HER2 binding moiety; (C) a nucleic acid sequence encoding the light chain sequence of the CD47 binding portion; (D) a nucleic acid sequence encoding the light chain sequence of the HER2 binding moiety; (E) Any combination of (A) to (D), and (F) A nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of the nucleic acid sequence of (A) to (E).

[0145] In some particular embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence that encodes SEQ ID NO: 19. In some particular embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence that encodes SEQ ID NO: 20. In some particular embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence that encodes SEQ ID NO: 21. In some particular embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence that encodes SEQ ID NO: 22.

[0146] Exemplary high stringency conditions include hybridization at 45°C in 5xSSPE and 45% formamide, and a final wash at 65°C in 0.1xSSC. It is understood in the art that equivalent stringency conditions can be achieved by varying 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 in hybridization conditions can be empirically determined or precisely calculated based on the length and guanosine / cytosine (GC) base pairing percentage 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.

[0147] Vectors and host cells The nucleic acid molecule encoding the bispecific polypeptide complex can be inserted into a vector for further cloning (amplification of DNA) or expression using recombinant techniques known in the art. In some embodiments, the antibody can be produced by homologous recombination, as known in the art. The DNA encoding the monoclonal antibody is easily isolated and sequenced using conventional procedures (e.g., using an oligonucleotide probe capable of specifically binding to a gene encoding the heavy chain of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of 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. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0148] In some embodiments, vector systems include mammalian, bacterial, yeast systems, and the like, including but not limited to plasmids such as 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, and other experimental and commercially available vectors. Suitable vectors can include plasmid vectors or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). In one embodiment of the present disclosure, the vector can be pET, e.g., pETbac, which contains genes for hexa-histidine- and c-Myc-tags.

[0149] A vector comprising a nucleic acid sequence encoding a bispecific polypeptide complex can be introduced into a host cell for cloning or gene expression. Thus, 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.

[0150] Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryote, 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 described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, e.g., used with a DHFR selection marker as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159:601-621), COS cells, and SP2 cells. Another expression system, particularly for use with NSO myeloma cells, includes the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841.In addition, SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651), human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL2), dog kidney cells (MDCK, ATCC CCL34), buffalo rat hepatocytes (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human hepatocytes (Hep G2, HB8065), mouse mammary tumor cells (MMT060562, ATCC CCL51), TRI cells (Mather et al., 1982, Annals NYAcad. Sci. 383:44-68), MRC5 cells, FS4 cells, mouse myeloma cells, such as NSO (e.g., RCB0213, 1992, Bio / Technology 10:169), and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC CRL1581), rat myeloma cells such as YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL1662), PER.C6 cells, and human hepatoma cell lines (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, they can be modified to lack the ability to fucosylate expressed proteins.

[0151] Suitable prokaryotes for this purpose include eubacteria, for example gram-negative or gram-positive organisms, for example Enterobacteriaceae, for example Escherichia, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, for example Serratia marcescans, and Shigella, as well as Bacilli, for example Bacillus subtilis and B. licheniformis, Pseudomonas, for example Pseudomonas aeruginosa, and Streptomyces.

[0152] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for bispecific antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, many other genera, species and strains, 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 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger are commonly available and useful herein.

[0153] Other host cells suitable for the expression of the bispecific polypeptide complexes provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and mutants and corresponding permissive insect host cells have been identified from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. Various virus strains for transfection are publicly available, such as the L-1 mutant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as viruses herein in accordance with the present disclosure, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco may also be utilized as hosts.

[0154] Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0155] The host cells used to produce the bispecific polypeptide complexes provided herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing the host cells. Furthermore, any of the media described in 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. Reissue Patent No. 30,985 may be used as a culture medium for the host cells. Any of these media may be supplemented as necessary 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 the GENTAMYCIN™ drug), 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 may also be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions such as temperature, pH, and the like are those previously used with the host cell selected for expression and will be apparent to one of skill in the art.

[0156] When using recombinant techniques, antibodies can be produced in the periplasmic space intracellularly or directly secreted into the medium. If the antibody is produced intracellularly, the first step is to remove particulate debris, either host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0157] The antibody prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting precipitation, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0158] After any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5 to 4.5, preferably performed at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0159] Pharmaceutical Compositions In some aspects, the present disclosure relates to a pharmaceutical composition comprising a bispecific polypeptide complex as disclosed herein and a pharma- ceutically acceptable carrier.

[0160] Ingredients of the composition The pharmaceutical composition may optionally include one or more additional pharmacoactive ingredients, such as another antibody or drug. The pharmaceutical composition of the present disclosure may also be administered in combination therapy with another immunostimulant, anti-cancer agent, anti-viral agent, or vaccine, such that the bispecific polypeptide complex enhances the immune response to the vaccine. Pharmaceutically acceptable carriers can include, for example, pharmacologic acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, combinations or combinations of various other ingredients known in the art.

[0161] Suitable ingredients can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorings, emulsifiers or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propylgalactosyl. As disclosed herein, compositions comprising antibodies include one or more antioxidants, such as methionine, to inhibit oxidation of the antibody. Inhibition of oxidation can prevent or reduce loss of binding affinity, thereby enhancing antibody stability and shelf life. Thus, in some embodiments, the present disclosure provides compositions comprising one or more antibodies and one or more antioxidants, such as methionine. The disclosure further provides various methods in which antibodies can be mixed with one or more antioxidants, such as methionine, to prevent oxidation of the antibodies and thereby extend their shelf life and / or increase their activity.

[0162] By way of further example, pharma- ceutically acceptable carriers 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 in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate buffers or citrate buffers; antioxidants such as sodium bisulfate; Examples of suitable excipients include local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antibacterial agents used as carriers may be added to pharmaceutical compositions in multi-dose containers including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients include, for example, water, saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0163] Administration, Formulation and Dosage The pharmaceutical compositions of the present disclosure may be administered in vivo to a subject in need thereof by a variety of routes, including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or otherwise by implantation or inhalation. The subject compositions may be formulated into preparations in solid, semi-solid, liquid or gas form, including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and route of administration may be selected according to the intended application and treatment regimen.

[0164] Preparations suitable for enteral administration include hard or soft gelatin capsules, pills, tablets such as coated tablets, elixirs, suspensions, syrups or inhalations, and controlled release formulations thereof.

[0165] Formulations 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 further include other pharma- ceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Similarly, the particular dosing regimen, i.e., dosage, timing, and repetition, will depend on the particular individual and the individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, and the like).

[0166] The frequency of administration may be determined and adjusted over the course of treatment, but is based on reducing the number of proliferative or tumorigenic cells, maintaining a reduction in such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the onset of metastasis. In some embodiments, the dosage administered may be adjusted or attenuated to manage potential side effects and / or toxicity. Alternatively, sustained continuous release formulations of the subject therapeutic compositions may be appropriate.

[0167] Those skilled in the art will understand that appropriate dosages may vary from patient to patient. Determining optimal dosages will generally involve balancing the level of therapeutic benefit with any risk or adverse side effects. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds and / or materials used in combination, the severity of the condition, and the patient's species, sex, age, weight, condition, general health and previous medical history. Dosages are generally selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial adverse side effects, but the amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian or clinician.

[0168] In general, bispecific polypeptide complexes may be administered in a variety of ranges. In some embodiments, the bispecific polypeptide complexes provided herein may be administered in a therapeutically effective dosage 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 certain of these embodiments, the antibody is administered at a dosage of about 50 mg / kg or less, and in certain 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 may vary over the course of treatment. For example, in certain embodiments, an initial dosage may be higher than subsequent dosages. In certain embodiments, the dosage may vary over the course of treatment depending on the subject's response.

[0169] In either case, the antibodies of the present disclosure are preferably administered as needed to a subject in need thereof. The frequency of administration can be determined by one of skill in the art, such as the attending physician, taking into consideration the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, etc.

[0170] In certain preferred embodiments, a course of treatment involving the antibody of the present disclosure will include multiple administrations of the selected formulation over a period of several weeks or months. More specifically, the antibody of the present disclosure may be administered once a day, once every two days, once every four days, once a week, once every 10 days, once every two weeks, once every three weeks, once a month, once every six weeks, once every two months, once every ten weeks, or once every three months. In this regard, it will be understood that dosages may be modified or intervals may be adjusted based on patient response and clinical practice.

[0171] Dosages and regimens may also be empirically determined for the disclosed therapeutic compositions in individuals who have received one or more doses. For example, an individual may be given incremental doses of the therapeutic composition manufactured as described herein. In selected embodiments, the dosage may be gradually increased or decreased or attenuated based on empirically determined or observed side effects or toxicity, respectively. To assess the effectiveness of the selected composition, markers of the particular disease, disorder, or condition may be tracked as described above. In the case of cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques, improvement as assessed by direct tumor biopsy and microscopic examination of tumor samples, measurement of indirect tumor markers (e.g., PSA for prostate cancer) or tumorigenic tumor antigens identified according to the methods described herein, reduction in pain or paralysis, improvement in speech, vision, breathing, or other disability associated with the tumor, increased appetite, or improved quality of life or increased survival as measured by approved tests. It will be apparent to one of skill in the art that dosages will vary depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has become metastatic to other locations in the individual, and any previous and concomitant treatments used.

[0172] Suitable formulations for parenteral administration (e.g., intravenous injection or infusion) may comprise a bispecific polypeptide complex provided herein at a concentration of about 10 μg / ml to about 100 mg / ml. 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 2mg / ml, 3mg / ml, 4mg / ml, 5mg / ml, 6mg / ml, 8mg / ml, 10mg / ml, 12mg / ml, 14mg / ml, 16mg / ml, 18mg / ml, 20mg / ml, 25mg / ml, 30mg / ml, 35mg / ml, 40mg / ml, 45mg / ml, 50mg / ml, 60mg / ml, 70mg / ml, 80mg / ml, 90mg / ml or 100mg / ml.

[0173] Uses / Indications The bispecific polypeptide complexes of the present disclosure have numerous in vitro and in vivo utilities. For example, these molecules can be administered to cells in culture in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of situations. The immune response can be enhanced, stimulated, or upregulated.

[0174] Preferred subjects include human patients in need of an enhanced immune response. The method is particularly suitable for treating human patients with disorders that can be treated by enhancing immune responses (e.g., T cell-mediated immune responses, phagocytosis of tumor cells). The method is particularly suitable for treating cancer in vivo. To achieve antigen-specific enhancement of immunity, the bispecific antibody can be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a tumor-bearing subject or a virus-bearing subject). When the bispecific antibody is administered together with another agent, the two can be administered in either order or simultaneously.

[0175] Treatment of disorders such as cancer In some aspects, the disclosure provides a method of treating a disorder in a subject, comprising administering to a subject (e.g., a human) in need of treatment a therapeutically effective amount of an antibody or antigen-binding portion thereof disclosed herein. For example, the disorder is cancer.

[0176] A variety of cancers in which CD47 and / or HER2 are implicated, whether malignant or benign, primary or secondary, can be treated or prevented with the methods provided by the present disclosure. The cancer can be a solid cancer or a hematological malignancy. Examples of such cancers include lung cancer, such as bronchogenic carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondroitin hamartoma (non-cancerous) and sarcoma (cancerous); cardiac cancer, such as myxoma, fibroma, and rhabdomyoma; osteochondroma, chondroma, chondroblastoma, chondromyxofibroma, osteoid, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's tumor), and other cancers. bone cancers, such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymoma, meningioma, pituitary adenoma, pinealoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germinoma, teratoma, dermoid cyst, and hemangioma; colon cancers, leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colon polyps and colonic cysts Cancers of the digestive system, such as colorectal cancer; liver cancer, such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma and hemangiosarcoma; kidney cancer, such as renal adenocarcinoma, renal cell carcinoma, hypernephroma and transitional cell carcinoma of the renal pelvis; bladder cancer; skin cancer, 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; benign prostate Male reproductive system cancers, such as 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 (such as papillary, follicular, anaplastic, or medullary carcinoma); pheochromocytoma (adrenal gland); non-cancerous proliferation of the parathyroid gland; and pancreatic cancer. In some particular embodiments, the cancer is skin cancer (such as cutaneous squamous cell carcinoma), colon cancer, colorectal cancer (such as colorectal adenocarcinoma), lung cancer (such as lung adenocarcinoma), or breast cancer (such as triple-negative breast adenocarcinoma).

[0177] In some other embodiments, the disorder is an autoimmune disease. Examples of autoimmune diseases that can be treated with the antibody or antigen-binding portion thereof include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. The antibody or antigen-binding portion thereof can also be used to treat or prevent infectious diseases, inflammatory diseases (such as allergic asthma), and chronic graft-versus-host disease.

[0178] Combined with chemotherapy The antibodies may be used in combination with anti-cancer, cytotoxic or chemotherapeutic agents.

[0179] The term "anti-cancer agent" or "anti-proliferative agent" refers to any agent that can be used to treat a cell proliferation disorder, such as cancer, including, but not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapy, radiotherapy and anti-metastatic agents, and immunotherapeutic agents. It will be understood that in selected embodiments as described above, such anti-cancer agents may comprise a conjugate or may be bound to the disclosed site-specific antibody prior to administration. More specifically, in some embodiments, the selected anti-cancer agent is linked to an unpaired cysteine ​​of the modified antibody to provide the modified conjugate described herein. Thus, such modified conjugates are expressly contemplated as being within the scope of the present disclosure. In other embodiments, the anti-cancer agent of the present disclosure is given in combination with a site-specific conjugate comprising a different therapeutic agent as described above.

[0180] As used herein, the term "cytotoxic agent" means a substance that is toxic to cells, reduces or inhibits the function of cells, and / or causes destruction of cells. 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 from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxins and exotoxins, Staphylococcal enterotoxin A), fungi (e.g., alpha-sarcin, restrictocin), plants (e.g., abrin, ricin, modeccin, viscumin, pokeweed antiviral protein, saporin, gelonin, momolisin, trichosanthin, barley toxin, Abrasiongum curcas protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitegelin, restrictocin, phenomycin, neomycin, and the trichothecenes) or animals (e.g., cytotoxic RNases such as extracellular pancreatic RNase, DNase I (including fragments and / or variants thereof)).

[0181] For purposes of this disclosure, a "chemotherapeutic agent" includes chemical compounds (e.g., cytotoxic or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation and / or survival of cancer cells. Such chemical agents often target intracellular processes necessary for cell growth or division, and are therefore particularly effective against cancerous cells, which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules, thus inhibiting cells from entering mitosis. In general, chemotherapeutic agents may include any chemical agent that inhibits or is designed to inhibit cancerous cells, or cells that are likely to become cancerous or produce tumorigenic progeny (e.g., TICs). Such agents are often administered and are often most effective in combination, for example, in regimens such as CHOP or FOLFIRI.

[0182] Examples of anti-cancer drugs that may be used in combination with the site-specific constructs of the present disclosure (either as components of a site-specific conjugate or in unconjugated form) include alkylating agents, alkylsulfonates, aziridines, ethylenimines and methylameramines, acetogenins, camptothecins, bryostatins, kallistatins, CC-1065, cryptophycins, dolastatins, duocarmycins, eleutherobin, pancratistatins, sarcodictins, spongiostatins, nitrogen mustards, antibiotics, enediamines, and the like. Antibiotics, dynemicins, bisphosphonates, esperamicins, chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycins, ausramycins, azaserines, bleomycins, cactinomycins, carabicins, carminomycins, carzinophilins, chromomycins, dactinomycins, daunorubicins, detrevicins, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN(R) doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomas Isin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogues, purine analogues, androgens, antiadrenals, folic acid supplements such as folinic acid, aceglatone, aldophosphamide glycosides, aminolecithin, cefolate ... Vrinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diazicon, elfornitine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidynin, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraelin, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK(R) polysaccharide complex (JHS NaturalProducts, Eugene, Oregon), razoxane, rhizoxin, schizofiran, spirogermanium, tenuazonic acid, triazicon, 2,2',2"-trichlorotriethylamine, trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidin), urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, taxoids, chlorambucil, GEMZAR® gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogs, vinblastine, platinum, etoposide (V P-16), ifosfamide, mitoxantrone, vincristine, NAVELBINE® vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan (Camptosar, CPT-11), topoisomerase inhibitors RFS2000, difluoromethylornithine, retinoids, capecitabine, combretastatin, leucovorin, oxaliplatin, inhibitors of PKC-alpha, Raf, H-Ras, HER2 and VEGF-A that reduce cell proliferation, and pharma- ceutical acceptable salts, acids or derivatives of any of the above. This definition also includes anti-hormonal agents that act to control or inhibit hormone action on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors which inhibit the enzyme aromatase which controls estrogen production in the adrenal glands, and anti-androgens, as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog), antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors, vaccines, PROLEUKIN® rIL-2, LURTOTECAN® topoisomerase 1 inhibitors, ABARELIX® rmRH, vinorelbine and esperamicin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0183] Combined with radiation therapy The present disclosure also provides for the combination of antibodies with radiation therapy (i.e., any mechanism for inducing DNA damage locally in tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, electronic emissions, etc.). Combination therapy using directed delivery of radioisotopes to tumor cells is also contemplated, and the conjugates of the present disclosure may be used in conjunction with targeted anticancer drugs or other targeting means. Typically, radiation therapy is administered in multiple pulses over a period of about 1 to about 2 weeks. Radiation therapy may be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiation therapy may be administered as a single dose or as multiple consecutive doses.

[0184] Pharmaceutical Packs and Kits Pharmaceutical packs and kits are also provided that include one or more containers containing one or more doses of the antibody. In some embodiments, a unit dose is provided that includes a predetermined amount of a composition that includes, for example, the antibody, with or without one or more additional agents. In other embodiments, such a unit dose is provided as a single-use pre-filled syringe for injection. In still other embodiments, the composition included in the unit dose may include a buffer, such as saline, sucrose, or the like, a phosphate, and / or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the conjugate composition may be provided as a lyophilized powder that can be reconstituted upon addition of an appropriate liquid, such as sterile water or saline. In certain preferred embodiments, the composition includes one or more substances that inhibit protein aggregation, such as, but not limited to, sucrose and arginine. An optional label on or associated with the container indicates that the enclosed conjugate composition is used to treat a selected neoplastic disease condition.

[0185] The present disclosure also provides kits for obtaining single or multiple dose units of the site-specific conjugate and optionally one or more anti-cancer agents. The kits include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers can be formed from a variety of materials, such as glass or plastic, and can contain a pharma- ceutical effective amount of the conjugates of the present disclosure in conjugated or unconjugated form. In other preferred embodiments, the containers include a sterile access port (e.g., the containers can be intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle). Such kits will generally include a pharma- ceutical acceptable formulation of the modified conjugate in a suitable container, and optionally one or more anti-cancer agents in the same or different containers. The kits can also include other pharma- ceutical acceptable formulations for either diagnosis or combination therapy. For example, in addition to the antibody, such a kit may include any one or more of a variety of anti-cancer agents, such as chemotherapeutic or radiotherapeutic agents, anti-angiogenic agents, anti-metastatic agents, targeted anti-cancer agents, cytotoxic agents, and / or other anti-cancer agents.

[0186] More specifically, the kit may have a single container containing the antibody of the present disclosure with or without additional components, or may have separate containers for each desired agent. When combination therapeutics are provided for conjugation, a single solution may be premixed in a molar equivalent combination or with one component in excess of the other. Alternatively, the conjugate and any anti-cancer agent of the kit may be kept separate in separate containers prior to administration to a patient. The kit may also include second / third container means for containing a sterile pharma- ceutically acceptable buffer or other diluent, such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and dextrose solution.

[0187] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, with sterile aqueous solution or saline being particularly preferred.However, the components of the kit may also be provided as dry powder.When a reagent or component is provided as dry powder, the powder can be reconstituted by adding a suitable solvent.It is envisioned that the solvent may also be provided in a separate container.

[0188] As briefly mentioned above, the kits may also include a means for administering the antibodies and any components to a patient, such as one or more needles, IV bags or syringes, or eye droppers, pipettes, or other such devices by which the formulations may be injected or introduced into an animal or applied to a diseased area of ​​the body. Kits of the present disclosure will also typically include a means for containing vials and the like or other components in close confinement for commercial sale, such as injection or blow molded plastic containers into which the desired vials or other devices are placed and held.

[0189] Sequence Listing Overview A sequence listing containing several amino acid sequences is attached to this application. Table A below provides an overview of the sequences included.

[0190] [Table 0-1] [Table 0-2] [Table 0-3] EXAMPLES

[0191] The present disclosure, having been broadly described above, will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the disclosure, and are not intended to represent that the following experiments are all or the only experiments performed.

[0192] Example 1 Preparation of materials Table 1 provides information on commercially available materials used in the examples.

[0193] [Table 1]

[0194] Example 2 W308032 Generation of bispecific antibodies For the generation of BsAbs, a CD47-binding moiety (CD47:T6) from an anti-CD47 antibody and a HER2-binding moiety (HER2:U5) from an anti-HER2 antibody were combined and used to construct a bispecific antibody.

[0195] The bispecific antibody was constructed using the WuXiBody® E17 format (Figure 1). The C alpha and C beta genes were synthesized by Genewiz Inc. Light chain 1 was the VL sequence of HER2:U5 inserted into a linearized vector containing C alpha, and light chain 2 was the VL sequence of CD47:T6 inserted into a linearized vector containing the constant region of the lambda light chain. Heavy chain 1 was the VH(HER2:U5)-C beta DNA fragment inserted into a linearized vector containing the human IgG1 constant region CH2-CH3 with S354C-T366W mutations, and heavy chain 2 was the 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.

[0196] The heavy and light chain expression plasmids were co-transfected into Expi293 cells using the Expi293 Expression System Kit according to the manufacturer's instructions. Five days after transfection, the supernatant of Expi293 cells expressing the target protein was collected and purified by filtration using a Protein A column. Fractions from Protein A elution were collected and adjusted to pH 5.0 for IEX purification using a CEX column. The CEX column was equilibrated with 50 mM NaAc (pH 5.0) before and after loading. Peak fractions were collected in a linear step with UA detection using 50 mM NaAc, 500 mM NaCl (pH 5.0) and then dialyzed against PBS buffer. The concentration of the purified protein was measured by absorbance at 280 nm. The size and purity of the purified protein were tested by SDS-PAGE and SEC-HPLC, respectively.

[0197] The resulting antibody is designated W308032-U5T6.E17-57.uIgG1 (or abbreviated as "W308032-U5T6.E17").

[0198] [Table 2]

[0199] Example 3 In vitro characterization of W308032 antibody 3.1 Protein analysis The size and purity of the W308032 antibody were assessed by SDS-PAGE and size exclusion chromatography (SEC-HPLC). Figures 2 and 3 and Table 3 show the SDS-PAGE and SEC-HPLC characterization of the purified W308032-U5T6.E17-57.uIgG1. Figure 2 shows the apparent molecular weight of approximately 150 kDa under non-reducing conditions, approximately 50 kDa, and 25 kDa under reducing conditions. The results showed that the bispecific molecule assembled as expected.

[0200] [Table 3]

[0201] 3.2 Differential scanning fluorometry (DSF) The melting temperature (Tm) of the antibody was examined by DSF assay using 7500Fast Real-Time PCR system (Applied Biosystems). Briefly, 19 μL of antibody solution was mixed with 1 μL of 62.5×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 was collected. Data collection and Tm calculation were performed automatically by the operating software (QuantStudio® Real-Time PCR Software v1.3).

[0202] DSF results showed that Tm1 reached 63.1°C and Tm2 reached 70.1°C, indicating that W308032-U5T6.E17-57.uIgG1 has good thermal stability (Figure 4).

[0203] [Table 4]

[0204] 3.3 Diffusion interaction parameter (kD) measurement by dynamic light scattering (DLS) The kD measurements were performed using a DynaPro Plate Reader III (Wyatt Dynapro™). The kD parameter is the first-order diffusion interaction parameter obtained in the DLS assay and serves as an indicator of the colloidal and thermal stability of the molecules. The samples were first filtered through a 0.02 μm filter and concentrated to more than 20 mg / mL. The samples were diluted to final concentrations of 2.5, 5, 10, 15 and 20 mg / mL with PBS buffer. Then, 7.5 μL of the sample solution was added to a 1536-well microplate. The plate was sealed with ClearSeal Film and centrifuged at 3000 rpm for 5 minutes to allow the samples to fall to the bottom of the wells. Each sample was tested in duplicate wells. The plate was placed in the corresponding position and data collection was performed by the DYNAMICS operating software (v7.8.1.3). Five acquisitions were collected for each protein sample, with each acquisition time of 5 seconds. For each measurement, the diffusion coefficient was determined and plotted against the protein concentration. The kD values ​​were automatically calculated by the software.

[0205] The determined kD was -13.0 mL / g, indicating that W308032-U5T6.E17-57.uIgG1 has good colloidal stability (Figure 5).

[0206] [Table 5]

[0207] 3.4 Hydrophobic Interaction Chromatography HPLC (HIC-HPLC) The hydrophobic properties of the antibodies were detected by an HPLC1260 Infinity II system (Agilent Technologics™) equipped with a TSKgel Butyl-NPR column (Tosoh-0042168). 20 μL of sample was injected into the column and separated for 61 min at a flow rate of 0.5 ml / min. The running buffers were 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 by UV light at wavelengths of 280 nm and 230 nm. Retention times were analyzed by HIC-HPLC analysis method, integrating all peak areas from 20 min to 50 min. The operating and analysis software was OpenLab CDS Workstation (v2.3.0.443).

[0208] The determined retention time was 24.74 min, indicating that W308032-U5T6.E17-57.uIgG1 has normal hydrophobicity (Figure 6).

[0209] [Table 6]

[0210] 3.5 Surface Plasmon Resonance (SPR) Biacore 8K was used to detect binding affinity to human HER2. Test antibodies were captured on a CM5 sensor chip with pre-immobilized anti-human IgG Fc antibody. Different concentrations of human HER2 protein were injected into the sensor chip at a flow rate of 30 μL / min for a 180 s binding phase followed by 3600 s dissociation. 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 sensorgrams. The experimental data was fitted by a 1:1 binding model using Langmuir analysis. The molar concentration of the analyte human HER2 protein was calculated using a molecular weight of 71.0 kDa.

[0211] Binding affinity to human CD47 was detected using Biacore 8K. Human CD47 protein was captured on a CM5 sensor chip pre-immobilized with streptavidin. Different concentrations of test antibodies were injected onto the sensor chip at a flow rate of 30 μL / min for a 240 s binding phase followed by 600-3600 s dissociation. 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 sensorgrams. The experimental data were fitted by a 1:1 binding model using Langmuir analysis. The molar concentration of the analyte test antibody was calculated using a molecular weight of 147 kDa.

[0212] The results showed that W308032-U5T6.E17-57.uIgG1 maintained high affinity for human Her2 and relatively low affinity for human CD47, with the affinity of W308032-U5T6.E17-57.uIgG1 being approximately 50-fold higher for HER2 compared to CD47 (Figures 7A-7F). Figures 7A-7C show three independent SPR results for binding to HER2, and Figures 7D-7F show three independent SPR results for binding to CD47.

[0213] [Table 7]

[0214] 3.6 Binding to tumor cells after antigen sink Serially diluted antibodies were applied to human whole blood (1:10 dilution) or Jurkat cells (1.2–1.8 × 10 6 The plates were preincubated with CFSE-labeled human tumor cells (0.8 × 10 cells / well) for 1 h at 4 °C, followed by centrifugation and transferring the absorbed supernatant to a new plate. 5Cells / well) were seeded in 96-well U-bottom plates and centrifuged at 1500 rpm for 4 min at 4°C before removing the supernatant. The absorbed supernatant was then added to resuspend the cells, incubated for 1 h at 4°C, and washed twice with 180 μL 1% BSA-PBS. Secondary antibody Alexa647-conjugated goat anti-human IgG Fc was added to resuspend the cells, incubated for 0.5 h at 4°C in the dark, and washed twice with 180 μL 1% BSA-PBS. Mean fluorescence intensity (MFI) was measured by FACS (BD Canto II) and analyzed by FlowJo Version software. Tumor cells were gated by CFSE and binding ECs to tumor cells were analyzed. 50 was calculated using GraphPad Prism software: nonlinear regression (curve fit)-log(agonist) vs. response-variable slope.

[0215] After being absorbed by human blood, W308032-U5T6.E17-57.uIgG1 had an EC of approximately 27.69 nM. 50 The antibody showed binding to CD47 / Her2 double positive SK-BR-3 cells at 100 ng / mL, which was consistent with the non-absorbed binding by human blood (EC 50 = 24.36 nM). However, the binding of magrolimab to SK-BR-3 (EC 50 =0.36 nM) was dramatically affected after absorption by human blood (EC 50 = 6.41nM), EC 50 was shifted approximately 18-fold (Figures 8A-8B).

[0216] [Table 8]

[0217] After uptake by Jurkat cells, W308032-U5T6.E17-57.uIgG1 had an EC of approximately 13.29 nM. 50 , which shows binding to SK-BR-3 cells, compared with non-absorbed binding by Jurkat cells (EC 50= 12.55 nM). However, the binding of magrolimab to SK-BR-3 (EC 50 =0.19 nM) was dramatically affected after uptake by Jurkat cells (EC 50 = 1.53 nM), EC 50 was shifted by approximately 8-fold (Figures 9A-9B).

[0218] [Table 9]

[0219] The antigen sink effect on antibody binding to CD47 / Her2 double positive HCC1954 cells was also examined. After uptake by Jurkat cells, W308032-U5T6.E17-57.uIgG1 had an EC 50 showed binding to HCC1954 cells at 100 ng / mL, which was comparable to the non-absorbed binding by Jurkat cells (EC 50 = 26.07 nM). However, the binding of magrolimab to HCC1954 (EC 50 = 0.44 nM) was affected after uptake by Jurkat cells (EC 50 = 1.68 nM), EC 50 was shifted approximately four-fold (Figures 10A to 10B).

[0220] [Table 10]

[0221] 3.7 Binding to CD47 single positive cells W308032-U5T6.E17-57.uIgG1 showed negligible binding to human blood cells (Figure 11) and approximately 200-fold reduced binding to Jurkat cells compared to magrolimab (Figure 12).

[0222] [Table 11]

[0223] [Table 12]

[0224] 3.8 CD47 ligand blocking on tumor cells after antigen sink Serially diluted antibodies were applied to human whole blood (1:10 dilution) or Jurkat cells (1.2–1.8 × 10 6 The plates were preincubated with CFSE-labeled human tumor cells (0.8 × 10 cells / well) for 1 h at 4 °C, followed by centrifugation and transferring the absorbed supernatant to a new plate. 5 Cells (cells / well) were seeded in 96-well U-bottom plates and centrifuged at 1500 rpm for 4 min at 4°C before removing the supernatant. The absorbed supernatant and mFc-tagged SIRPα (1 μg / mL) were then added to resuspend the cells and incubated for 2 h at 4°C, followed by washing twice with 180 μL of 1% BSA-PBS. The secondary antibody, Alexa647-conjugated goat anti-mouse IgG Fc, was added to resuspend the cells and incubated for 0.5 h at 4°C in the dark, followed by washing twice with 180 μL of 1% BSA-PBS. MFI was measured by FACS (BD Canto II) and analyzed by FlowJo Version software. Tumor cells were gated by CFSE and the inhibitory IC against tumor cells was calculated. 50 was calculated using GraphPad Prism software: nonlinear regression (curve fit)-log(antagonist) vs. response-variable slope.

[0225] After being absorbed by human blood, W308032-U5T6.E17-57.uIgG1 had an IC of approximately 0.30 nM. 50 Blocking of CD47 ligand binding to CD47 / Her2 double positive SK-BR-3 cells was achieved using human blood-absorbed blocking (IC 50 = 0.25 nM). However, CD47 ligand blocking (IC 50= 0.06 nM) was dramatically affected after absorption by human blood (IC 50 =0.93nM), IC 50 was shifted by approximately 15-fold (FIGS. 13A-13B).

[0226] [Table 13]

[0227] After uptake by Jurkat cells, W308032-U5T6.E17-57.uIgG1 had an IC of approximately 0.98 nM. 50 Blocking CD47 ligand binding to CD47 / Her2 double positive SK-BR-3 cells was achieved using 100% IgG4-dependent ELISA, which was shown to be a potent inhibitor of CD47 ligand binding in the absence of uptake by Jurkat cells (IC 50 = 1.24 nM). However, the CD47 ligand blocking effect of magrolimab on SK-BR-3 (IC 50 = 0.17 nM) was not affected after uptake by Jurkat cells (IC 50 =0.44nM), IC 50 was shifted by approximately three times (FIGS. 14A-14B).

[0228] [Table 14]

[0229] Furthermore, W308032-U5T6.E17-57.uIgG1 showed little CD47 ligand blocking effect on Jurkat cells compared to magrolimab (Figure 15).

[0230] [Table 15]

[0231] 3.9 Receptor occupancy on tumor cells Tumor cells were seeded in 96-well U-bottom plates and centrifuged at 1500 rpm for 4 min at 4 °C, and the supernatant was removed. Then, serially diluted bispecific antibodies were added to resuspend the cells, incubated at 4 °C for 1 h, and washed twice with 180 μL of 1% BSA-PBS. Saturating concentrations of biotin-labeled parent antibodies, which were detection antibodies, were added to resuspend the cells, incubated at 4 °C for 1 h, and washed twice with 180 μL of 1% BSA-PBS. Secondary antibodies, streptavidin PE, were added to resuspend the cells, incubated at 4 °C in the dark for 0.5 h, and washed twice with 180 μL of 1% BSA-PBS. MFI was measured by FACS (BD Canto II) and analyzed by FlowJo Version software. Receptor occupancy (%) was calculated as 100-(MFI 試料 -MFI neg ) / (MFI 飽和 -MFI neg ) × 100. EC 50 was calculated using GraphPad Prism software: nonlinear regression (curve fit)-log(agonist) vs. response-variable slope.

[0232] Thanks to its Her2-binding arm, W308032-U5T6.E17-57.uIgG1 showed >30% higher maximum CD47 occupancy than magrolimab on CD47 / Her2 double positive SK-BR-3 cells (Figures 16A-B).

[0233] [Table 16]

[0234] 3.10 ADCP for tumor cells Monocytes were isolated from PBMCs using human CD14 microbeads and then differentiated into macrophages by treatment with rhM-CSF (50-100ng / mL) for 6-8 days. Briefly, 30μL of CFSE-labeled tumor cells, 50μL of macrophages and 20μL of serially diluted antibodies were seeded in 96-well U-bottom ultra-low plates. Phagocytosis of tumor cells was performed for 2-3 hours at 37°C. Cells were then stained with APC-conjugated CD14 antibody for 45 minutes at 4°C, washed with 1% BSA-PBS, and then detected by FACS (BD Canto II) and analyzed by FlowJo Version software. Phagocytic activity was calculated as follows: Index% = percentage CFSE+ / CD14-APC+ / (percentage CFSE+ / CD14-APC+ +Percentage CFSE- / CD14-APC+ ) × 100%. 50 was calculated using GraphPad Prism software: nonlinear regression (curve fit)-log(agonist) vs. response-variable slope.

[0235] As shown in Figures 17A-B, W308032-U5T6.E17-57.uIgG1 demonstrated increased ADCP efficacy compared to the combination of trastuzumab and magrolimab against CD47 / Her2 double positive SK-BR-3 cells in two independent experiments.

[0236] [Table 17A]

[0237] [Table 17B]

[0238] As shown in Figure 18, W308032-U5T6.E17-57.uIgG1 inhibited the combination of trastuzumab and magrolimab (EC 50 = 1.28 nM, maximum phagocytosis index = 38.1%) and equivalent ADCP efficacy (EC50 = 1.25 nM, maximum phagocytosis index = 39.3%). As shown in Figure 19, W308032-U5T6.E17-57.uIgG1 inhibited the phagocytosis of CD47 / Her2 double positive NCI-N87 cells in combination with trastuzumab and magrolimab (EC 50 = 1.77 nM, maximum phagocytosis index = 77.5%) and equivalent ADCP efficacy (EC 50 = 0.13 nM, maximum phagocytosis index = 77.8%).

[0239] [Table 18]

[0240] [Table 19]

[0241] ADCP for CD47 single positive cells W308032-U5T6.E17-57.uIgG1 showed little ADCP efficacy against human blood cells (FIG. 20) and showed much lower ADCP activity than magrolimab against Jurkat cells (FIG. 21).

[0242] [Table 20]

[0243] [Table 21]

[0244] 3.11 ADCC against tumor cells 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 4 h of incubation at 37°C, 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 an LDH cytotoxicity detection kit (Roche) according to the manufacturer's instructions. Cytotoxic EC 50 was calculated.

[0245] As shown in Figure 22, W308032-U5T6.E17-57.uIgG1 exhibited potent ADCC efficacy (EC 50 = 21.42 pM, maximum cytotoxicity = 61.9%).

[0246] [Table 22]

[0247] W308032-U5T6.E17-57.uIgG1 also demonstrated potent ADCC efficacy against CD47 / Her2 double positive HCC1954 cells (Figure 23) and NCI-N87 cells (Figure 24).

[0248] [Table 23]

[0249] [Table 24]

[0250] ADCC against CD47 single-positive cells W308032-U5T6.E17-57.uIgG1 showed very weak ADCC efficacy against Jurkat cells only at high concentrations (Figure 25).

[0251] [Table 25]

[0252] 3.12 Inhibitory effect on tumor cell proliferation Briefly, 80 μL of tumor cells were seeded in a 96-well black-walled plate. 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, followed by detection using EnVision. Inhibitory IC50 was calculated using GraphPad Prism software: nonlinear regression (curve fit)-log (antagonist) vs. response-variable slope.

[0253] W308032-U5T6.E17-57.uIgG1 showed a strong inhibitory effect on the proliferation of CD47 / Her2 double positive SK-BR-3 cells (Figure 26) and NCI-N87 cells (Figure 27).

[0254] [Table 26]

[0255] [Table 27]

[0256] 3.13 Red blood cell agglutination Anticoagulated fresh human whole blood was centrifuged at 2000 rpm for 10 min and the plasma was discarded. Human blood cells were rinsed twice with DPBS. Briefly, 25 μL of 1:25 diluted human blood cells (i.e., cells were resuspended in 25 times the original blood volume) and 25 μL of serially diluted antibodies were added to a U-bottom 96-well plate and incubated at 37°C for 2 hours, after which a photograph was taken for detection.

[0257] W308032-U5T6.E17-57.uIgG1 was HA negative on human blood cells, whereas magrolimab was HA positive on human blood cells (Figure 28).

[0258] 3.14 Serum stability Fresh human whole blood was statically incubated at room temperature (RT) for at least 30 min in tubes without anticoagulant. Blood was centrifuged at 4000 rpm for 10 min, and serum was collected. Antibodies were gently mixed with serum and incubated at 37°C. One aliquot of serum-treated sample was taken and flash frozen by liquid nitrogen on days 0, 1, 4, 7, and 14, respectively, and stored at -80°C until ready for analysis. The binding ability of the samples to tumor cells was evaluated to show their stability. Briefly, tumor cells were seeded in 96-well U-bottom plates and centrifuged at 1500 rpm for 4 min at 4°C, after which the supernatant was removed. Various concentrations of test samples were then added to resuspend the cells and incubated at 4°C for 1 h, followed by washing twice with 180 μL of 1% BSA-PBS. Secondary antibody Alexa647-conjugated goat anti-human IgG Fc was added, resuspended and incubated for 0.5 h at 4° C. in the dark, then washed twice with 180 μL 1% BSA-PBS. MFI was measured by FACS (BD Canto II) and analyzed by FlowJo Version software. Binding EC50 was calculated using GraphPad Prism software: nonlinear regression (curve fit)-log(agonist) vs. response-variable slope.

[0259] W308032-U5T6.E17-57.uIgG1 was stable in human serum for at least 7 days and showed no reduction in binding to CD47 / Her2 double positive SK-BR-3 cells. When incubated with human serum for 14 days, W308032-U5T6.E17-57.uIgG1 showed a slight (>2-fold) reduction in binding avidity to CD47 / Her2 double positive SK-BR-3 cells (Figure 29).

[0260] [Table 28]

[0261] Example 4 In vivo characterization of W308032 antibody 4.1 Rodent Efficacy 4.1.1 HCC1954 xenograft model Six to eight-week-old female CB-17 SCID mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the experiments. HCC1954 cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C under an atmosphere of 5% CO2 in air. For the xenograft model, exponentially growing HCC1954 tumor cells were harvested and implanted (3.0 × 10 6 Cells were inoculated subcutaneously with 0.2 mL of DPBS and Matrigel (Corning) at a ratio of 1:1. The mean tumor volume was approximately 147 mm. 3 Once the tumor volume reached 100 mg / kg, animals were randomized into 12 groups and administered the first dose of antibody treatment (day 0) followed by treatments intraperitoneally twice weekly for a total of 10 injections. Mice were weighed and tumor growth was measured twice weekly using calipers. Tumor volume was calculated using the formula (1 / 2(length x width) / (length x width). 2 )) was used to calculate the mean and standard error (mean ± SEM). Data were analyzed by two-way ANOVA with Bonferroni post-hoc test using Prism, and p<0.05 was considered statistically significant. All procedures related to animal handling care and treatment in the experiments were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi Biologics, which follows the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Details of grouping and treatment of HCC1954 xenograft model are shown in Table 29.

[0262] [Table 29]

[0263] Two mice were confirmed dead on days 28 and 17 (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), and one with a tumor volume of 2500 mm 3 With the exception of one mouse from the pertuzumab group that was euthanized because it reached 1 mg / kg, all other mice appeared normal during the experiment and slowly gained weight, indicating that the antibody was not toxic (data not shown).

[0264] As shown in Figure 30 and summarized in Table 30, compared with the PBS group, trastuzumab + magrolimab (day 36, TGI 76.8%) could significantly inhibit tumor growth, with a significant difference from day 7, while the monotherapy groups of trastuzumab (day 36, TGI 33.6%) and magrolimab (day 36, TGI 18.5%) showed only slight inhibition on tumor growth, indicating the synergistic effect of CD47 and Her2. W308032-U5T6.E17-57.uIgG1 (10 mg / kg) showed slightly better efficacy than the combination of trastuzumab + magrolimab, and significantly better efficacy than the monotherapy of trastuzumab or magrolimab (Figure 30).

[0265] [Table 30]

[0266] W308032-U5T6.E17-57.uIgG1 (30 mg / kg, 10 mg / kg, 3 mg / kg, 1 mg / kg) was able to inhibit tumor growth (on day 36, TGI was 100.1%, 90.7%, 75.6%, 36.7%, respectively), which was significantly different from days 3 (30 mg / kg), 7 (10 mg / kg), and 10 (3 mg / kg), respectively, indicating that W308032-U5T6.E17-57.uIgG1 has a dose-dependent anti-tumor effect (Figure 31).

[0267] Combination therapy with trastuzumab and pertuzumab has been approved for clinical use and has shown improved antitumor efficacy compared with trastuzumab monotherapy. To evaluate whether W308032-U5T6.E17-57.uIgG1 could also be used with pertuzumab to further increase antitumor activity, the antitumor activity of the combination therapy was also examined in the HCC1954 xenograft model. As shown in Figure 32 and summarized in Table 30, compared with the PBS group, the combination groups of Trastuzumab + Pertuzumab (day 36, TGI 50.7%) and Trastuzumab + Magrolimab + Pertuzumab (day 36, TGI 105.2%) could 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 on tumor growth. The combination group of W308032-U5T6.E17-57.uIgG1 + Pertuzumab (day 36, TGI 106.0%) showed comparable antitumor effects to the combination group of Trastuzumab + Magrolimab + Pertuzumab (day 36, TGI 105.2%).

[0268] All the above conclusions were confirmed by the tumor weight data obtained 36 days after the first treatment (Figure 33). Data are presented as mean + SEM.

[0269] 4.1.2 NCI-N87 xenograft model Four- to eight-week-old female CB-17 SCID mice (Beijing Vital River Laboratories Research Models and Services) were used for the experiments. NCI-N87 cells were maintained in vitro as monolayer cultures at 37 °C in an atmosphere of 5% CO2 in air in RPMI-1640 medium supplemented with 10% fetal bovine serum. For the xenograft model, exponentially growing NCI-N87 tumor cells were harvested and each mouse was inoculated with NCI-N87 tumor cells (1 × 10 7 The tumors were inoculated subcutaneously with 0.1 mL of cells (1:1 PBS and Matrigel). The mean tumor volume was approximately 157 mm. 3 Once the tumor reached 100 mg / kg, animals were randomly grouped into 10 groups and given the first dose of treatment (day 0) followed by intraperitoneal antibody treatment twice weekly for a total of 11 injections (excluding intraperitoneal Enherz treatment once weekly for a total of 6 injections). Mice were weighed and tumor growth was measured twice weekly using calipers. Tumor volume was calculated using the formula (1 / 2(length x width)). 2 ) was used for the calculation.

[0270] All results were expressed as mean and standard error (mean ± SEM). Data were analyzed using two-way ANOVA Bonferroni post-hoc test or one-way ANOVA Dunnett multiple comparison test in R (A Language and Environment for Statistical Computing and Graphics (Version 3.3.1)), and p<0.05 was considered statistically significant. All procedures regarding animal care and use were approved by CrownBio's Institutional Animal Care and Use Committee (IACUC) in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) regulations. Details of grouping and treatment of NCI-N87 xenograft model are shown in Table 31.

[0271] [Table 31]

[0272] As shown in Figure 34 and summarized in Table 32, compared to the vehicle control group, Herceptin (Trastuzumab, 10 mg / kg) + Magrolimab (10 mg / kg) was able to significantly inhibit tumor growth (day 38, TGI 111.92%), and W308032-U5T6.E17-57.uIgG1 (10 mg / kg and 20 mg / kg) showed comparable efficacy to the Herceptin + Magrolimab combination therapy (day 38, TGI 116.04% and 118.17%, respectively). As shown in Figure 35, W308032-U5T6.E17-57.uIgG1 (5 mg / kg, 10 mg / kg, 20 mg / kg, 40 mg / kg) showed dose-dependent anti-tumor effects (day 38, TGI was 112.22%, 116.04%, 118.17%, 119.99%, respectively).

[0273] The combination of trastuzumab and chemotherapy has been approved for clinical use and has shown improved antitumor effects compared to trastuzumab monotherapy. To evaluate whether W308032-U5T6.E17-57.uIgG1 can also be used with chemotherapy to further increase antitumor activity, the antitumor activity of the combination therapy was also examined in the NCI-N87 xenograft model. As shown in Figure 36 and summarized in Table 32, W308032-U5T6.E17-57.uIgG1 (10 mg / kg) combined with paclitaxel (10 mg / kg) produced a significant antitumor effect of 117.57% TGI, which was comparable to the effect of Enherz (10 mg / kg) of 124.19% TGI.

[0274] [Table 32]

[0275] 4.1.3 JIMT-1 xenograft model Efficacy in another trastuzumab-resistant breast cancer cell JIMT-1 xenograft model was also evaluated. Nine to ten week old female CB-17 SCID mice (Shanghai Lingchang Bio-Technology. Co., Ltd.) were used for the experiments. JIMT-1 cells were maintained in vitro as monolayer cultures at 37°C in an atmosphere of 5% CO2 in air in DMEM medium supplemented with 10% fetal bovine serum. For the xenograft model, exponentially growing JIMT-1 tumor cells were harvested and implanted (5 × 10) JIMT-1 tumor cells into the right upper flank region of each mouse. 6 The average tumor volume was approximately 134 mm. 3 Once the tumor volume reached 100 mg / kg, animals were randomly grouped into 6 groups and administered the first dose of treatment (day 0), followed by intraperitoneal antibody treatment twice weekly for a total of 10 injections, with an additional 4-week observation period following the final injection. Mice were weighed and tumor growth was measured twice weekly using calipers. Tumor volume was calculated using the formula (1 / 2(length x width) / (length x width). 2 ) was used for the calculation.

[0276] All results were expressed as mean and standard error (mean ± SEM). Data were analyzed by two-way ANOVA Bonferroni post hoc test or one-way ANOVA Dunnett multiple comparison test using Prism, and p<0.05 was considered statistically significant. All procedures related to animal care and use were approved by the Institutional Animal Care and Use Committee (IACUC) of CrownBio in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Details of grouping and treatment of the JIMT-1 xenograft model are shown in Table 33.

[0277] [Table 33]

[0278] Animals were treated twice weekly for a total of 10 doses over 5 weeks, followed by 4 weeks of observation without treatment. On day 35 (3 days after the last dose), W308032-U5T6.E17-57.uIgG1 showed dose-dependent inhibition of tumor growth and efficacy comparable to the combination of trastuzumab and magrolimab (data not shown). As shown in Figure 37 and summarized in Table 34, W308032-U5T6.E17-57.uIgG1 in combination with paclitaxel showed better efficacy in tumor growth inhibition than single agent treatment with bispecific antibody or trastuzumab, which was comparable to the efficacy of Enhertz.

[0279] As shown in Figure 38, on day 63 (31 days after the last dose), W308032-U5T6.E17-57.uIgG1 in combination with paclitaxel showed little rebound in tumor volume, with 4 of 8 mice in each group being tumor-free. However, Enhertz showed a clear rebound over the initial tumor volume, with only 1 of 8 mice in each group being tumor-free.

[0280] [Table 34]

[0281] 4.2 Pharmacokinetics in rodents Female SD rats (Beijing Vital River Laboratory Animal Technology Co.,Ltd) aged 9-11 weeks were used for the experiments. The animals were housed in specific pathogen-free (SPF) barriers at the Shanghai Model Organisms Center,Inc., two per individual ventilated cage (IVC), and allowed to acclimate for one week after arrival. For pharmacokinetic (PK) experiments, each rat received a single dose of antibody (15 mg / kg, IV bolus) on day 0, and blood samples were collected from the eye and transferred to EDTA tubes. The tubes were centrifuged at 8000 rpm (6010 g) for 5 min at 4°C, and then plasma was collected and stored at -20°C. All samples were uniquely identified to indicate origin and collection time. The schedule of sample collection per rat per dose is shown in Table 35.

[0282] [Table 35]

[0283] The concentrations of analytes in serum were determined using a bioanalytical ELISA method. Briefly, 96-well ELISA plates were coated with the capture antibody, recombinant human HER2 / ErB2 protein (His tag) in carbonate-bicarbonate buffer overnight at 2-8 °C. After washing and blocking, serially diluted plasma samples were added, and then biotin-labeled recombinant human CD47 protein (HER2+CD47 method for G1) or goat anti-human IgG-biotin (HER2+Fc method for G2) was used as the detection antibody. HRP-streptavidin and TMB substrate were used for color development. The reaction was stopped by adding 2 M HCl after about 5-10 min. The absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer (SpectraMax® M5e). The OD values ​​of the samples were substituted into the standard curve to obtain the plasma antibody concentrations. The serum concentrations of the antibodies in rats were subjected to noncompartmental pharmacokinetic analysis using Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA). The linear / logarithmic trapezoidal rule was applied in obtaining the PK parameters. All PK parameters were calculated without antibody concentrations below 1% of Cmax.

[0284] As shown in Figure 39, W308032-U5T6.E17-57.uIgG1 demonstrated comparable rodent PK (t = 153 hours, Table 36) to trastuzumab (t = 184 hours, Table 37).

[0285] [Table 36]

[0286] [Table 37]

[0287] Those skilled in the art will further appreciate that the present disclosure may be embodied in other specific forms without departing from its spirit or central characteristics. It is to be understood that the foregoing description of the present disclosure discloses only exemplary embodiments thereof, and other variations are also contemplated within the scope of the present disclosure. Thus, the present disclosure is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims as indicating the scope and content of the present disclosure.

[0288] References [1] Meike EW Logtenberg, Ferenc A Scheeren, Ton N Schumacher.The CD47-SIRPa Immune Checkpoint.Immunity.2020 May 19;52(5):742-752. [2] EJ Brown, WA Frazier. Integrin-associated protein (CD47) and its ligands. Trends Cell Biol. 2001 Mar;11(3):130-5. [3] Mark P Chao, Irving L Weissman, Ravindra Majeti. The CD47-SIRPα pathway in cancer immune evasion and potential therapeutic implications. Curr Opin lmmunol.2012 Apr;24(2):225-32. [4] I Rubin,Y Yarden.The basic biology of HER2.Ann Oncol.2001;12 Suppl 1:S3-8 [5] Li-Chung Tsao et.al.CD47 blockade augmentation of trastuzumab antitumor efficacy dependent on antibody-dependent cellular phagocytosis.JCI Insight.2019 Dec 19;4(24):e131882. [6] Tiia J Honkanen et.al.Prognostic and predictive role of spatially positioned tumour infiltrating lymphocytes in metastatic HER2 positive breast cancer treated with trastuzumab.Sci Rep.2017 Dec 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, The first antigen-binding portion comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 comprises a heavy chain complementarity 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, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and the VL1 comprises a light chain complementarity 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; A polypeptide complex or an antigen-binding portion thereof, wherein the second antigen-binding portion comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2), wherein the VH2 comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 9, and the VL2 comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

12.

2. The first antigen-binding moiety and the second antigen-binding moiety can be in the following formats: immunoglobulins and antigen-binding portions thereof, e.g., Fab, F(ab)' ２ or scFv.

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

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

16.

5. 5. A polypeptide complex or an 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 having at least 85%, 90%, or 95% identity to SEQ ID NO: 17, and C2 comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO:

18.

6. The polypeptide complex or antigen-binding portion thereof of any one of claims 1 to 5, wherein the polypeptide complex comprises an Fc region, such as a human IgG Fc region.

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

8. 8. The polypeptide complex or antigen-binding portion thereof of claim 6 or 7, wherein the Fc region is a native Fc region or a modified Fc region, e.g., an Fc region comprising one or more substitutions selected from "knob-into-hole" substitutions, S228P, M252Y / S254T / T256E, and combinations thereof.

9. A polypeptide complex or antigen-binding portion thereof according to any one of claims 1 to 8, comprising one, two or more CD47 binding moieties and one, two or more HER2 binding moieties.

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

11. It comprises two heavy chains and four light chains, from the N-terminus to the C-terminus: the first heavy chain and the second heavy chain each comprise domains operably linked as follows: 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 light chain and the second light chain each comprise operably linked domains such as VL1-C2; the third light chain and the fourth light chain each comprise operably linked domains as VL2-CL; or the first heavy chain and the second heavy chain each comprise domains operably linked as follows: 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 light chain and the second light chain each comprise operably linked domains such as VL1-CL; 10. The polypeptide complex or antigen-binding portion thereof of claim 9, wherein the third light chain and the fourth light chain each comprise an operably linked domain as VL2-C2.

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

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

14. A polypeptide complex or an antigen-binding portion thereof according to any one of claims 3 to 13, operably linked via a direct linkage or via a peptide linker having a length of 1 to 30 amino acids.

15. 15. The polypeptide complex or antigen-binding portion thereof of claim 14, wherein the peptide linker is a GS linker, e.g., (GS)n, (GGS)n, (GGGS)n, (GGGGS)n, (GGSG)n, (GGGSS)n, where n is an integer from 1 to 9.

16. 11. The polypeptide complex or antigen-binding portion thereof of claim 10, comprising a first heavy chain comprising SEQ ID NO: 19 and a second heavy chain comprising SEQ ID NO: 20, and a first light chain comprising SEQ ID NO: 21 and a second light chain comprising SEQ ID NO:

22.

17. A polypeptide complex or an antigen-binding portion thereof according to any one of claims 1 to 16, wherein the polypeptide complex is a humanized antibody or a fully human antibody.

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

19. A vector comprising the nucleic acid molecule of claim 18.

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

21. A pharmaceutical composition comprising a polypeptide complex or an antigen-binding portion thereof according to any one of claims 1 to 17 and a pharma- ceutically 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 steps of: Cultivating under suitable conditions a host cell comprising a nucleic acid sequence encoding the polypeptide complex or an antigen-binding portion thereof; isolating said polypeptide complex from said host cell; The method includes:

23. 22. A method of modulating an immune response associated with HER2 and / or CD47 in a subject, comprising administering to said subject a polypeptide complex or an antigen-binding portion thereof described in any one of claims 1 to 17, or a pharmaceutical composition described in claim 21.

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

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

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

27. 27. The method of claim 25 or 26, wherein the cancer is selected from 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, glioma, cancer of unknown primary, sarcoma, medulloblastoma, neuroblastoma, retinoblastoma, testicular cancer, thymoma, thyroid cancer, urachal cancer, uterine cancer, vaginal cancer, astrocytoma, basal cell carcinoma, and combinations thereof.

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

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

30. i) modulation of HER2 and / or CD47 associated immune responses; ii) induction of macrophage-mediated phagocytosis of tumor cells; iii) induction of natural killer cell-mediated cytotoxicity of tumor cells, and / or iv) Inhibition of tumor cell proliferation A polypeptide complex or an antigen-binding portion thereof according to any one of claims 1 to 17 for use in

31. A polypeptide complex or an antigen-binding portion thereof according to any one of claims 1 to 17 for use in the diagnosis, treatment or prevention of cancer.

32. i) modulation of HER2 and / or CD47 associated immune responses; ii) induction of macrophage-mediated phagocytosis of tumor cells; iii) induction of natural killer cell-mediated cytotoxicity of tumor cells, and / or iv) Inhibition of tumor cell proliferation 20. Use of a polypeptide complex, or an antigen-binding portion thereof, according to any one of claims 1 to 17 in the manufacture of a medicament for.

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

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

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