TW202330627A - Bispecific antibodies specifically binding to cd47 and her2, and uses thereof - Google Patents

Abstract

The invention relates to novel bispecific antibodies, comprising at least one binding domain capable of specifically binding to CD47 and at least one binding domain capable of specifically binding to HER2. The invention further relates to methods of producing these bispecific antibodies and to methods of using the same.

Description

Bispecific antibodies that specifically bind CD47 and HER2 and their uses

The present invention relates to novel bispecific antibodies, which comprise at least one binding domain capable of specifically binding to CD47 and at least one binding domain capable of specifically binding to HER2. The invention also relates to methods of producing these bispecific antibodies and methods of using them.

CD47 (differentiation group 47) was first identified as a tumor antigen in human ovarian cancer in the 1980s. CD47, also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-related antigen and MER6, is a multiplex membrane receptor belonging to the immunoglobulin superfamily with an immunoglobulin-like domain and five A transmembrane region, which serves as a ligand for signal regulatory protein α (SIRP α ) and binds to the V-like domain at the NH2 terminus of SIRP α . SIRP α is mainly expressed in bone marrow cells, including macrophages, granulocytes, dendritic cells (DCs), mast cells and their precursor cells, including hematopoietic stem cells. CD47 on normal cells binds to SIRP α on macrophages, releasing a “don’t eat me” signal and inhibiting the phagocytosis of macrophages. It is an important mechanism for macrophages in the innate immune system to identify self and non-self. CD47 is widely expressed in human tumor cells and tissues, including acute myeloid leukemia (AML), chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), multiple myeloma (MM) ), bladder cancer, and other solid tumors. Tumor cells bind to SIRP α on the surface of macrophages through highly expressed CD47, evading macrophage phagocytosis and promoting tumor growth. The CD47 immune checkpoint is considered a potentially effective and widely applicable target for tumor immunotherapy. Currently, a variety of specific blockers targeting the interaction between CD47 and SIRP α have been developed. A number of ongoing preclinical and clinical trials involve drugs for the treatment of diffuse large B lymphocyte lymphoma, acute myeloid leukemia, and advanced solid tumors, including anti-CD47 antibodies and SIRP alpha fusion proteins.

HER2, human epidermal growth factor receptor 2, also known as ErbB-2, is a member of the human epidermal growth factor receptor family and is encoded by the ERBB2 gene. HER2 overexpression has been shown to play a key role in the development and progression of some malignant breast cancers. In recent years, HER2 has been identified as a very important tumor marker and has become an important therapeutic target for approximately 15%-30% of breast cancer patients. Moreover, HER2 overexpression has also been found in ovarian, gastric, and lung adenocarcinomas, as well as malignant uterine cancer. For example, Her2 is overexpressed in approximately 7%-34% of gastric cancer patients. A variety of monoclonal antibodies targeting HER2 have been developed, such as trastuzumab (trade name: Herceptin). Trastuzumab was approved in the United States in 1998 and has It has shown efficacy in treating HER2-positive breast cancer or other cancers.

Although a large number of monoclonal antibodies targeting HER2 or CD47 have been developed for the treatment of cancer, some patients do not respond to monotherapy for a variety of reasons. Therefore, there is a need to develop bispecific antibodies against two separate and unique antigens, such as CD47 and HER2. The present invention provides novel anti-HER2/CD47 bispecific antibodies that specifically bind to CD47 and HER2, such as tumor cells co-expressing CD47 and HER2. Bispecific antibodies take advantage of their bifunctional characteristics to target HER2-expressing cancer cells while blocking the CD47 "don't eat me signal" on the surface of the same cancer cells, thereby activating innate Macrophages in the immune system specifically kill cancer cells that express both HER2 and CD47. At the same time, phagocytosis of cancer cells is expected to increase the presentation of tumor antigens, and indirectly stimulates the adaptive immune system to kill cancer cells. The bispecific antibody has high antitumor activity and does not cause significant agglutination of red blood cells expressing only CD47. Therefore, the present invention can meet more clinical needs.

The present invention provides a bispecific antibody comprising at least one antigen-binding domain capable of specifically binding to CD47 and at least one antigen-binding domain capable of specifically binding to HER2.

In one aspect, bispecific antibodies provided herein include:

(a) at least one antigen-binding domain capable of specifically binding CD47, and

(b) at least one antigen-binding domain capable of specifically binding HER2,

The antigen-binding domain capable of specifically binding to CD47 includes VH and VL, wherein VH includes: HCDR1 including or consisting of the amino acid sequence shown in SEQ ID NO: 11, and HCDR1 including the amino acid sequence shown in SEQ ID NO: 12 An HCDR2 containing or consisting of an amino acid sequence as shown in SEQ ID NO: 13 or 17 or 21, and an HCDR3 containing or consisting of an amino acid sequence as set forth in SEQ ID NO: 13 or 17 or 21; and VL includes: containing as set forth in SEQ ID NO: 14 LCDR1 containing the amino acid sequence shown or consisting of the amino acid sequence shown in SEQ ID NO: 15 or 18 or 22 or LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 15 or 18 or 22, and including the amine shown in SEQ ID NO: 16 amino acid sequence or LCDR3 consisting of it.

In some embodiments, the bispecific antibodies provided herein, wherein

The VH capable of specifically binding the antigen-binding domain of CD47 includes: HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11, and HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 12 or HCDR2 consisting of the same, and HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 17; and the VL capable of specifically binding the antigen-binding domain of CD47 includes: LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 18, and LCDR2 comprising or consisting of SEQ ID NO: 16 The amino acid sequence shown or LCDR3 consisting thereof.

In some embodiments, the bispecific antibodies provided herein, wherein

The VH capable of specifically binding the antigen-binding domain of CD47 comprises at least 90%, 91%, 92%, 93%, 94 of the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 6 or 7. A VL containing or consisting of an amino acid sequence with %, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and capable of specifically binding to the antigen-binding domain of CD47, as in SEQ ID NO: The amino acid sequence shown in 2, 4, 8, 9 or 10 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or An amino acid sequence with 100% sequence identity or consisting of an amino acid sequence.

In some embodiments, the bispecific antibodies provided herein, wherein the VH and VL capable of specifically binding the antigen-binding domain of CD47 are selected from

(1) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

(2) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4;

(3) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10;

(4) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 6, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9 or 10; or

(5) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10.

In some embodiments, the bispecific antibodies provided herein, wherein

The VH capable of specifically binding the antigen-binding domain of CD47 comprises or consists of the amino acid sequence shown in SEQ ID NO: 7, and the VL capable of specifically binding the antigen-binding domain of CD47 comprises SEQ ID NO: The amino acid sequence shown in 8 or consisting of it.

In some embodiments, the bispecific antibodies provided herein, wherein the antigen-binding domain capable of specifically binding HER2 comprises VH and VL, wherein the VH of the antigen-binding domain capable of specifically binding HER2 comprises: comprising SEQ ID NO. : an amino acid sequence shown in SEQ ID NO: 23 or an HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 24 or an HCDR2 consisting of an amino acid sequence shown in SEQ ID NO: 24, and an amino group including an amino group shown in SEQ ID NO: 25 an HCDR3 acid sequence or consisting of it; and a VL comprising: an LCDR1 comprising or consisting of an amino acid sequence shown in SEQ ID NO: 26, comprising an amino acid sequence shown in SEQ ID NO: 27 LCDR2 having a sequence or consisting of it, and LCDR3 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 28.

In some embodiments, the bispecific antibodies provided herein, wherein

The VH capable of specifically binding the antigen-binding domain of HER2 includes at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or consisting of an amino acid sequence with 97%, 98%, 99% or 100% sequence identity, and capable of specifically binding the VL of the antigen-binding domain of HER2 comprising an amino group as shown in SEQ ID NO: 30 An acid sequence has or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the bispecific antibodies provided herein, wherein the bispecific antibody comprises:

(a) at least one antigen-binding domain comprising VH and VL capable of specifically binding to CD47, wherein

The VH capable of specifically binding the antigen-binding domain of CD47 comprises at least 90%, 91%, 92%, 93%, 94 of the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 6 or 7. A VL containing or consisting of an amino acid sequence with %, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and capable of specifically binding to the antigen-binding domain of CD47, as in SEQ ID NO: The amino acid sequence shown in 2, 4, 8, 9 or 10 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or an amino acid sequence with 100% sequence identity or consisting of it, and

(b) at least one antigen-binding domain comprising VH and VL capable of specifically binding HER2, wherein

The VH capable of specifically binding the antigen-binding domain of HER2 includes at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or consisting of an amino acid sequence with 97%, 98%, 99% or 100% sequence identity, and capable of specifically binding the VL of the antigen-binding domain of HER2 comprising an amino group as shown in SEQ ID NO: 30 An acid sequence has or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the bispecific antibodies provided herein, wherein the antigen binding domain is chimeric, fully human, or humanized.

In some embodiments, the bispecific antibodies provided herein, wherein the antigen-binding domain comprises a Fab fragment or a scFv fragment.

In some embodiments, the bispecific antibodies provided herein, wherein the antigen-binding domain capable of specifically binding HER2 comprises a Fab fragment, and the antigen-binding domain capable of specifically binding CD47 comprises a scFv fragment or a Fab fragment.

In some embodiments, the bispecific antibodies provided herein comprise an Fc domain.

In some embodiments, the Fc domain is an IgG, particularly a human IgG1 Fc domain or a human IgG4 Fc domain.

In some embodiments, the two subunits of the Fc domain: the first subunit and the second subunit are the same or different.

In some embodiments, the Fc domain contains one or more amino acid modifications that alter the binding affinity and/or effector function of the antibody for the Fc receptor.

In some embodiments, the Fc domain contains one or more amino acid modifications that increase the binding affinity of the antibody for FcRn.

In some embodiments, the Fc domain contains amino acid substitutions at positions 428 and 434 (numbered according to the Kabat EU index).

In some embodiments, the Fc domain contains amino acid substitutions at positions 428 and 434, wherein the amino acid substitutions are M428L and N434S (according to Kabat EU index numbering).

In some embodiments, one or both of the subunits of the Fc domain (preferably both subunits are identical) comprise an amino acid as set forth in SEQ ID NO: 53, 54, 55 or 64 The sequence has or consists of an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, preferably This subunit contains M428L and N434S (numbered according to Kabat EU index).

In some embodiments, each of the two subunits of the Fc domain comprises at least 90%, 91%, 92%, 93%, 94%, or consisting of an amino acid sequence with 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, an Fc domain contains one or more amino acid modifications that promote association of the first and second subunits of the Fc domain.

In some embodiments, the first subunit of the Fc domain contains a knob and the second subunit of the Fc domain contains a hole according to the knob-into-hole approach.

In some embodiments, the first subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the Kabat EU index) and the second subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (According to Kabat EU index number).

In some embodiments, the first subunit of the Fc domain comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% similarity to the amino acid sequence set forth in SEQ ID NO: 57 %, 97%, 98%, 99% or 100% sequence identity to or consisting of an amino acid sequence, and/or the second subunit of the Fc domain contains an amine group as set forth in SEQ ID NO: 56 An acid sequence has or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the bispecific antibodies provided herein are bivalent, trivalent, or tetravalent.

In some embodiments, bispecific antibodies provided herein comprise:

(a) The first Fab fragment capable of specifically binding to CD47,

(b) a second Fab fragment capable of specifically binding to HER2, and

(c) An Fc domain consisting of a first subunit and a second subunit capable of stable association.

In some embodiments, one or both of the Fab fragments are CrossFab, such as CrossFab (CL-CH1).

In some embodiments, the first Fab fragment is CrossFab (CL-CH1).

In some embodiments, bispecific antibodies provided herein comprise:

(i) The first polypeptide chain has the structure: VH (anti-CD47)-CL-Fc domain subunit,

(ii) The second polypeptide chain has the structure: VL (anti-CD47)-CH1,

(iii) The third polypeptide chain has the structure: VH (anti-HER2)-CH1-Fc domain subunit, and

(iv) The fourth polypeptide chain has the structure: VL (anti-HER2)-CL.

In some embodiments, bispecific antibodies provided herein comprise:

(i) The first polypeptide chain has the structure: VH (anti-CD47)-CH1-Fc domain subunit,

(ii) The second polypeptide chain has the structure: VL (anti-CD47)-CL,

(iii) The third polypeptide chain has the structure: VH (anti-HER2)-CL-Fc domain subunit, and

(iv) The fourth polypeptide chain has the structure: VL (anti-HER2)-CH1.

In some embodiments, bispecific antibodies provided herein comprise:

(i) A first polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity,

(ii) a second polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity,

(iii) A third polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity, and

(iv) a fourth polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consists of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, bispecific antibodies provided herein comprise:

(a) Two scFv fragments capable of specifically binding to CD47, and

(b) Two heavy chains and two light chains of a whole antibody comprising two Fab fragments and an Fc domain capable of specifically binding HER2, to which each of the two scFv fragments is linked .

In some embodiments, each of the two scFv fragments is linked to the whole antibody by a peptide linker (eg, the C-terminus of the whole antibody).

In some embodiments, each of the two scFv fragments is linked to the C-terminus of both heavy chains of the whole antibody by a peptide linker.

In some embodiments, the peptide linker comprises a GS linker.

In some embodiments, the peptide linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, 32, 33, 34, 35, 36, or 37.

In some embodiments, the scFv fragment comprises a peptide linker connecting the C-terminus of VH to the N-terminus of VL, or a peptide linker connecting the N-terminus of VH to the C-terminus of VL.

In some embodiments, the scFv fragment comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38 or 39.

In some embodiments, bispecific antibodies provided herein comprise:

(a) Two scFv fragments capable of specifically binding to CD47, each of the two scFv fragments comprising: a VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, and a VH comprising the amino acid sequence shown in SEQ ID NO: 7 NO: The amino acid sequence shown in 8 or the VL composed thereof; and

(b) Two heavy chains and two light chains of a whole antibody comprising two Fab fragments and an Fc domain capable of specifically binding to HER2, each of the two Fab fragments comprising: comprising as in SEQ ID NO: 29 The amino acid sequence shown or VH consisting of it, and VL comprising the amino acid sequence shown in SEQ ID NO: 30 or consisting of it;

Each of the two scFv fragments is linked to the C-terminus of the two heavy chains of the whole antibody via a peptide linker.

In some embodiments, bispecific antibodies provided herein comprise two heavy chains and two light chains, wherein each of the heavy chains comprises an amine group as set forth in SEQ ID NO: 40, 41, 42, or 63 The acid sequence has or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and Each of the light chains includes at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or consists of an amino acid sequence with 99% or 100% sequence identity.

In some embodiments, bispecific antibodies provided herein comprise:

(a) Two scFv fragments capable of specifically binding to CD47, each of the two scFv fragments comprising at least 90%, 91%, 92%, 93 identical amino acid sequences as shown in SEQ ID NO: 38 or 39 or consisting of an amino acid sequence with %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and

(b) Two heavy chains and two light chains of a full antibody comprising two Fab fragments and an Fc domain capable of specifically binding to HER2, wherein

(1) Each heavy chain contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or consisting of an amino acid sequence with 98%, 99% or 100% sequence identity, and each of the light chains comprises at least 90%, 91% with the amino acid sequence set forth in SEQ ID NO: 43 , or consisting of an amino acid sequence with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity,

(2) Each of the heavy chains contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or consisting of an amino acid sequence with 98%, 99% or 100% sequence identity, and each of the light chains comprises at least 90%, 91% with the amino acid sequence set forth in SEQ ID NO: 43 , or consisting of an amino acid sequence with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity,

(3) Each of the heavy chains contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or consisting of an amino acid sequence with 98%, 99% or 100% sequence identity, and each of the light chains comprises at least 90%, 91% with an amino acid sequence as set forth in SEQ ID NO: 43 , or consisting of an amino acid sequence with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or

(4) Each of the heavy chains contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or consisting of an amino acid sequence with 98%, 99% or 100% sequence identity, and each of the light chains comprises at least 90%, 91% with an amino acid sequence as set forth in SEQ ID NO: 43 , or consisting of an amino acid sequence with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity,

Each of the two scFv fragments is linked to the C-terminus of the two heavy chains of the whole antibody via a peptide linker.

In some embodiments, bispecific antibodies provided herein comprise:

(a) two scFv fragments capable of specifically binding to CD47, each of the two scFv fragments comprising or consisting of the amino acid sequence shown in SEQ ID NO: 39, and

(b) Two heavy chains and two light chains of a whole antibody comprising two Fab fragments and an Fc domain capable of specifically binding HER2, wherein each of the heavy chains comprises an amine as set forth in SEQ ID NO: 42 an amino acid sequence or consisting of an amino acid sequence, and each of the light chains includes or consists of an amino acid sequence as shown in SEQ ID NO: 43,

Each of the two scFv fragments is linked to the C-terminus of the two heavy chains of the whole antibody via a peptide linker.

In some embodiments, bispecific antibodies provided herein comprise:

(a) two scFv fragments capable of specifically binding to CD47, each of the two scFv fragments comprising or consisting of the amino acid sequence shown in SEQ ID NO: 39, and

(b) Two heavy chains and two light chains of a whole antibody comprising two Fab fragments and an Fc domain capable of specifically binding HER2, wherein each of the heavy chains comprises an amine as set forth in SEQ ID NO: 63 an amino acid sequence or consisting of an amino acid sequence, and each of the light chains includes or consists of an amino acid sequence as shown in SEQ ID NO: 43,

Each of the two scFv fragments is linked to the C-terminus of the two heavy chains of the whole antibody via a peptide linker.

In one aspect, the invention also provides an isolated nucleic acid encoding a bispecific antibody of the invention.

In one aspect, the invention also provides a recombinant vector or expression vector comprising one or more nucleic acids of the invention, wherein the vector is suitable for recombinant production of a bispecific antibody of the invention.

In one aspect, the invention also provides a host cell comprising one or more recombinant or expression vectors of the invention.

In one aspect, the invention also provides immunoconjugates comprising a bispecific antibody of the invention.

In one aspect, the invention also provides a pharmaceutical composition comprising the bispecific antibody of the invention, the nucleic acid of the invention, the vector of the invention, the host cell of the invention or the immunoconjugate of the invention, and optionally Contains pharmaceutically acceptable excipients.

In one aspect, the invention provides a method for treating or preventing a disease or condition in a subject, comprising administering to the individual an effective amount of a bispecific antibody of the invention, a nucleic acid of the invention, a vector of the invention, a vector of the invention, The host cell of the invention, the immunoconjugate of the invention, or the pharmaceutical composition of the invention.

In some embodiments, the disease or condition is CD47-related and/or HER2-related.

In some embodiments, the disease or condition is cancer.

In some embodiments, the cancer is a blood cancer and may be selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), non-Hodgkin lymphoma (eg, Burkitt lymphoma), B lymphoma Blastic leukemia/lymphoma, B-cell chronic lymphocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), follicular lymphoma, small lymphocytic leukemia (SLL), central nervous system (CNS) Lymphoma, Richter's Syndrome, multiple myeloma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, and anaplastic large cell lymphoma.

In some embodiments, the cancer is a solid tumor, optionally selected from the group consisting of breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, head and neck squamous cell carcinoma, ovarian cancer, prostate cancer, pancreatic cancer, multiple Glioblastoma, gastric junction adenocarcinoma, esophagogastric junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing sarcoma, rhabdomyosarcoma, or neuroblastoma.

In one aspect, the invention also provides the bispecific antibody of the invention, the nucleic acid of the invention, the vector of the invention, the host cell of the invention, or the immunoconjugate of the invention, or the pharmaceutical composition of the invention. Use in the preparation of a medicament for the treatment of cancer in a subject.

Figure 1 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of Raji cells.

Figure 2 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of Toledo cells.

Figure 3 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of REC-1 cells.

Figure 4 shows the activity of antibody HMA02h14-48 in blocking the interaction of human CD47 with SIRP alpha .

Figure 5 shows the effect of antibody HMA02h14-48 on human MΦ phagocytic Raji cells.

Figure 6 shows the effect of antibody HMA02h14-48 on human MΦ phagocytosis of Toledo cells.

Figure 7 shows the effect of antibody HMA02h14-48 on human MΦ phagocytosis of REC-1 cells.

Figure 8 shows the effect of antibody HMA02h14-48 on human MΦ phagocytosis of HL-60 cells.

Figure 9 shows the effect of antibody HMA02h14-48 on the agglutination activity of red blood cells in vitro.

Figure 10 shows the ability of antibody HMA02h14-48 to bind to CD47 on the surface of human red blood cells.

Figure 11 shows the inhibition of Toledo tumor growth by Hu5F9 and HMA02h14-48.

Figure 12 shows the inhibition of REC-1 tumor growth by Hu5F9 and HMA02h14-48.

Figure 13 shows the binding activity of anti-HER2/CD47 bispecific antibodies to human CD47 protein based on ELISA.

Figure 14 shows the binding activity of anti-HER2/CD47 bispecific antibodies to human HER2 protein based on ELISA.

Figure 15 shows the binding activity of anti-HER2/CD47 bispecific antibodies to both human CD47 and human HER2 proteins on CHO-K1/hCD47 OE.

Figure 16 shows the binding activity of anti-HER2/CD47 bispecific antibodies to OE19 cells.

Figure 17 shows the blocking activity of anti-HER2/CD47 bispecific antibodies in CD47(OE19)/SIRPα interaction based on Jurkat SIRPα signaling assay.

Figure 18 shows the blocking activity of anti-HER2/CD47 bispecific antibodies in CD47 (CHO-K1/hCD47)/SIRPα interaction based on Jurkat SIRPα signaling assay.

Figure 19 shows the effect of anti-HER2/CD47 bispecific antibodies on phagocytosis of OE19 cells by human MΦ.

Figure 20 shows the effect of ani-HER2/CD47 bispecific antibodies on the agglutination reaction of red blood cells in vitro.

Figure 21 shows the ability of anti-HER2/CD47 bispecific antibodies to bind to CD47 on the surface of human red blood cells.

Figure 22 shows the effect of BsAb4 on OE19 tumor growth in NOD-SCID mice.

Figure 23 shows the effect of BsAb6 on the growth of OE19 tumors implanted in nude mice.

definition

Unless otherwise stated, the practice of the present invention will employ conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the scope of the art.

In order to make it easier to understand the present invention, some scientific and technical terms are defined as follows. Unless otherwise expressly defined elsewhere herein, all technical and scientific terms used herein have the meaning commonly understood by a person of ordinary skill in the technical field to which this invention belongs. For definitions and terminology in this field, professionals may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviation of an amino acid residue is the standard 3-letter and/or 1-letter code used in the art to refer to one of the 20 commonly used L-amino acids. Unless expressly stated otherwise, any singular form used in this application (including the claims) includes its corresponding plural form.

The term "and/or" when used to connect two or more options shall be understood to mean any one of the options or any two or more of the options.

As used herein, the term "comprises" or "includes" means the inclusion of a mentioned element, integer or step but not the exclusion of any other element, integer or step. As used herein, when the term "comprises" or "includes" is used, it also encompasses instances consisting of the mentioned elements, integers, or steps, unless otherwise stated. For example, when reference is made to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass an antibody variable region that consists of that particular sequence.

Unless otherwise stated, the term "CD47" or "CD47 protein" refers to a protein from any vertebrate source, including mammals (e.g., primates (e.g., humans) and rodents (e.g., mice and rats)). Any natural CD47. The term encompasses "full-length" unprocessed CD47 as well as any form of CD47 or any fragment thereof resulting from intracellular processing. The term also includes naturally occurring CD47 variants, such as splice variants or allelic variants. In some embodiments, CD47 is derived from human Full-length CD47 or fragments thereof (e.g., mature fragments thereof lacking a signal peptide). In some embodiments, human CD47 (hCD47) refers to CD47 or a fragment thereof that is identical to the amino acid sequence shown in NCBI accession number NP_001768.1. In some embodiments, the term also encompasses fusion proteins comprising CD47 or fragments thereof.

As used herein, unless otherwise stated, the term "HER2" or "HER2 protein" refers to proteins derived from any vertebrate source, including mammals (e.g., primates (e.g., humans)) and rodents (e.g., mice and rats). )) of any natural HER2. The term encompasses "full-length" unprocessed HER2 as well as any form of HER2 resulting from intracellular processing or any fragment thereof. The term also includes naturally occurring HER2 variants, such as splice variants or allelic variants. In some embodiments, HER2 refers to full-length HER2 from human or a fragment thereof (eg, a mature fragment thereof lacking a signal peptide). In some embodiments, human HER2 (hHER2) refers to HER2 or a fragment thereof that is identical to the amino acid sequence shown in NCBI accession number NP_004439.2. In some embodiments, the term also encompasses fusion proteins comprising HER2 or fragments thereof.

As used herein, the term "anti-HER2/CD47 bispecific antibody" refers to an antibody that contains multiple antigen-binding domains capable of specifically binding both CD47 and HER2.

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and Antibody fragments, provided they exhibit the desired antigen-binding activity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies composing the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. Modifier "single plant" Indicates the characteristics of antibodies obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibodies by any particular method.

As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each binding site to the same epitope of the same antigen. The term "bispecific" refers to the ability of an antibody to specifically bind to at least two different antigenic determinants. Bispecific antibodies contain at least two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antibody is capable of binding two epitopes simultaneously, particularly two epitopes from different antigens, e.g., expressed on two different cells. For example, the antibody of the invention is bispecific, comprising two antigen-binding domains capable of specifically binding to HER2 and two antigen-binding domains capable of specifically binding to CD47.

The terms "whole antibody" and "intact antibody" are used interchangeably herein and refer to an antibody that has a structure substantially similar to that of a native antibody. "Native antibodies" refer to naturally occurring immunoglobulin molecules of varying structures. For example, natural IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, consisting of two light and two heavy chains interconnected by disulfide bonds. Each of the heavy chains consists of a heavy chain variable region (herein abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each of the light chains consists of a light chain variable region (herein abbreviated as VL) and a light chain constant region. The light chain constant region consists of the domain CL. The VH and VL regions can be further divided into hypervariable regions (complementarity determining regions or CDRs), with more conservative regions (framework regions or FRs) inserted in between. Each VH or VL consists of three CDRs and four FRs, arranged in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in the binding of antibodies to antigens, but displays a variety of effector functions. The heavy chains of antibodies can be classified into one of five types (called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM)), some of which can be further divided into subtypes , such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). Based on the amino acid sequence of their constant regions, the light chains of antibodies can be classified into one of two types, called kappa (kappa) and lambda (lambda).

A "complementarity determining region" or "CDR region" or "CDR" is a region within an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop (a "hypervariable loop") and/or contains Antigen contact residues ("antigen contact points"). CDRs are mainly responsible for binding to epitopes. The CDRs of the heavy chain and light chain are usually called CDR1, CDR2 and CDR3, which are numbered sequentially starting from the N-terminus. The CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2 and HCDR3 respectively, while the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2 and LCDR3 respectively. Each VH or VL consists of three CDRs and 4 FRs, which are arranged in the following order from the amino end to the carboxyl end: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In a given VH or VL amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined by using any one or combination of various well-known schemes, including, for example: the Chothia scheme (Chothia et al. , Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987)); Kabat protocol (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) and Contact (University College London); North protocol (North et al., A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011 )). The boundaries of the CDRs of the antibodies in the present invention can be determined according to any scheme in the art or a combination thereof and human evaluation.

"Antibody fragment" refers to a molecule other than a whole antibody that contains a portion of the whole antibody that binds the antigen to which the whole antibody is bound. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, tribodies, tetrabodies, crossFab fragments; linear antibodies; Single chain antibody molecules (eg scFv); and single domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see for example Plückthun in The harmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 /16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having increased half-life in vivo, see US Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific, see, e.g., EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Tribodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production from recombinant host cells (eg, E. coli or phage), as described herein.

Papain digestion of a full-length antibody produces two identical antigen-binding fragments, termed "Fab" fragments, each containing the heavy and light chain variable domains, as well as the constant domain of the light chain and the first portion of the heavy chain. Constant domain (CH1). Thus, as used herein, the term "Fab fragment" refers to a light chain fragment comprising VL and the constant domain (CL) of the light chain, and an antibody fragment containing VH and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which one or more cysteine residues of the constant domain carry free thiol groups. Pepsin treatment produces an F(ab') ₂ fragment with two antigen binding sites (two Fab fragments) and part of the Fc domain. According to the present invention, the term "Fab fragment" also includes "crossFab fragment" or "crossover Fab fragment".

A "single chain variable fragment" or "scFv fragment" is a fusion protein of the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody linked by a short peptide linker of 10 to about 25 amino acids. . This peptide linker is typically rich in glycine for flexibility, and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of linkers, the protein retains the specificity of the original antibody. scFv antibodies are described, for example, in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96. Additionally, antibody fragments comprise single-chain polypeptides having characteristics of VH (i.e., capable of assembling with VL) or characteristics of VL (i.e., capable of assembling with VH), which together assemble into a functional antigen-binding domain and thereby provide a full-length Antigen-binding properties of antibodies.

The term "crossFab fragment" or "xFab fragment" or "cross-Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged with each other. Two different chain compositions of the crossed Fab molecule are possible and included in the bispecific antibodies of the invention: on the one hand, the variable regions of the Fab heavy and light chains are exchanged, i.e. the crossed Fab molecule is comprised by the light chain A peptide chain composed of a variable region (VL) and a heavy chain constant region (CH1), and a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). This cross-Fab molecule is also called CrossFab (VL-VH). On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the crossover Fab molecule contains a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL), and a light chain variable region. The peptide chain consists of the heavy chain constant region (VL) and the heavy chain constant region (CH1). This cross-Fab molecule is also called CrossFab (CL-CH1).

The term "valency" as used herein means that the specified number of binding domains is present in the antibody. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding domains in the antibody, respectively. The titer of an antibody can also be determined relative to a given antigen Expressed by the number of binding domains in a given cluster. For example, in some embodiments, an antibody of the invention is bivalent relative to CD47 and bivalent relative to HER2 (i.e., 2+2).

"Fusion," "connection," or "conjugation" means that the components (eg, the heavy chain of an antibody and the scFv fragment) are connected by a peptide bond, either directly or through one or more peptide linkers.

The term "peptide linker" refers to a peptide containing one or more amino acids (usually about 2 to 60 amino acids). Peptide linkers are known in the art or described herein. Suitably, non-immunogenic linkers include, for example, glycine polymer (G)n, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer. Peptide linkers comprising glycine-serine polymers, including, for example, (GS) _n , (GSGGS) _n , (GGGS) _n (SEQ ID NO: 37), (GGGGS) _n (SEQ ID NO: 31) or (GGGGSGGGS) _n , where "n" is an integer from 1 to 10, preferably 1 to 4, more preferably 3 or 4, and the peptide linker is collectively referred to as a GS linker. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 31, 32, 33, 34, 36, or 37. The peptide linker may also comprise naturally or non-naturally occurring sequences, such as native sequences derived from the CH1 domain (eg, an amino acid sequence consisting of ATG) or the hinge region of an IgG. In some embodiments, the peptide linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35.

The term "antigen-binding domain" or "antigen-binding site" refers to that portion of an antibody that contains a region that specifically binds and is complementary to some or all of the antigen. In the case of larger antigens, the antigen-binding molecule may bind only to a specific part of the antigen, called an epitope. The antigen binding domain may be provided, for example, by one or more variable domains (also called variable regions). Preferably, the antigen-binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of antigen-binding domains include, but are not limited to, diabodies, Fab, Fab', F(ab') ₂ , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) ₂ , bispecific dsFv ( dsFv-dsFv'), disulfide bond stabilized diabody (ds diabody), single chain antibody molecule (scFv) and scFv dimer (bivalent diabody).

"Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those of ordinary skill in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assay (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree of binding of the antibody to the unrelated protein is less than about 10% of the degree of binding of the antibody to the antigen, for example, as measured by SPR. In certain embodiments, the antibody that binds to the antigen has a dissociation constant (KD) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 ⁻⁷ M or more low, such as 10 ^-7 M to 10 ^-13 M, such as 10 ^-8 M to 10 ^-13 M).

"Affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the internal binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (Kd and Ka, respectively). Therefore, equivalent affinities can involve different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. One specific method used to measure affinity is surface plasmon resonance (SPR).

The term "Fc domain" or "Fc region" is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc domains and variant Fc domains. The IgG Fc domain contains an IgG CH2 domain and an IgG CH3 domain. The "CH2 junction" of the human IgG Fc domain "Domain" generally extends from an amino acid residue at position about 231 to an amino acid residue at about 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. CH2 Domains herein It may be a native sequence CH2 domain or a variant CH2 domain. "CH3 domain" includes the stretch of residues from the C-terminus to the CH2 domain in the Fc domain (i.e., from amino acid residues at about position 341 of the IgG to amino acid residue at about 447). In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the C-terminus of the heavy chain. In one embodiment, the C-terminal lysine of the Fc domain The acid (Lys447) may or may not be present. In one embodiment, the C-terminal lysine (Lys447) and glycine (Gln446) of the Fc domain may or may not be present. In one embodiment, with alanine Replace the C-terminal lysine (Lys447) of the Fc domain. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc domain or constant region is according to the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

As used herein, the term "chimeric" refers to an antibody or antigen-binding domain in which part of the heavy and/or light chain is derived from one species and the remainder of the heavy and/or light chain is derived from a different species. In illustrative embodiments, a chimeric antibody may comprise a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the antigen-binding domain capable of specifically binding to HER2 includes a variable region derived from a mouse and a constant region derived from a human.

As used herein, the term "humanized" means that the antibody or antigen-binding domain contains CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, constant regions derived from a human.

As used herein, the term "fully human" with respect to an antibody or antigen-binding domain means that the antibody or antigen-binding domain has or consists of one or more amino acid sequences that Columns correspond to amino acid sequences of antibodies produced by humans or human immune cells or derived from non-human sources (eg, transgenic non-human animals utilizing human antibody libraries or other human antibody coding sequences). In certain embodiments, fully human antibodies do not contain amino acid residues (especially antigen-binding residues) derived from non-human antibodies.

The term "carcinoma" or "cancer" refers to or describes a physiological disorder in mammals, often characterized by unregulated cell growth. This definition includes benign and malignant cancers as well as dormant tumors or micrometastases. "Cancer" includes, but is not limited to, solid tumors and cancers of the blood or hematologic system. Examples of various cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

As used herein, the terms "subject," "patient," or "individual" include any human or non-human animal. The term "non-human animals" includes all vertebrate animals, such as mammals, and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.

The terms therapeutically "effective amount", therapeutically "effective dose" and "effective amount" as used herein refer to when the anti-HER2/CD47 bispecific antibodies of the present invention are administered to cells, tissues or recipients alone or in combination with other therapeutic agents. An amount effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the development of a disease or condition. A therapeutically effective dose also refers to an amount of antibody or antigen-binding fragment thereof sufficient to result in an improvement in symptoms, such as an amount that treats, cures, prevents, or ameliorates a relevant medical condition or increases the rate of treatment, cure, prevention, or amelioration of such condition. When an active ingredient is administered to an individual alone, the therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of the therapeutic agent will result in an increase in the diagnostic criteria or parameters by at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

As used herein, "treatment" includes: 1) therapeutic measures (therapeutic treatment) that cure, alleviate and alleviate the symptoms of a diagnosed pathological condition or disease and/or stop the progression of a diagnosed pathological condition or disease, and 2 ) preventive or prophylactic measures (prophylaxis or prophylactic treatment), which prevent and/or mitigate the development of pathological conditions or diseases. Accordingly, subjects receiving treatment include individuals already suffering from the disease, individuals susceptible to the disease, and individuals in whom the disease is to be prevented. In some embodiments, the invention relates to the treatment of a disease or condition. In some other embodiments, the invention relates to the prevention of diseases or conditions.

In some embodiments according to the present invention, "treatment" of a disease or condition refers to amelioration of the disease or condition (ie, alleviation or prevention or reduction of at least one of the progression of the disease or its clinical symptoms). In some other embodiments, "treating" or "treating" refers to alleviating or improving at least one physical parameter, including those that may not be discernible to the patient. In some other embodiments, "treating" or "treating" refers to modulating a disease or condition physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of body parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.

In other embodiments according to the present invention, "prevention" of a disease or condition includes inhibition of the onset or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventive regimen. Generally speaking, in the context of cancer, the term "prevention" refers to the administration of a drug to a subject before the onset of signs or symptoms of cancer, particularly in subjects at risk for cancer.

In some embodiments, after "treating" cancer by the methods of the present invention, an individual patient is deemed to have been successfully treated if the individual exhibits one or more of the following: a reduction in the number of cancer cells or the complete disappearance of cancer cells; a reduction in the number of cancer cells or the complete disappearance of cancer cells; Tumor size; Inhibition or lack of cancer cell infiltration into surrounding organs, including, for example, spread of cancer cells to soft tissue and bone; Inhibition or lack of tumor metastasis; Inhibition or lack of tumor growth long-term; alleviates one or more symptoms associated with a specific cancer; reduces morbidity and mortality; improves quality of life; reduces tumor incidence, frequency, or tumorigenicity; reduces the number or frequency of cancer stem cells in tumors; causes tumor cells to differentiate into Non-tumorigenic state; or combination of effects.

"Inhibition of tumor growth" refers to any mechanism by which the growth of tumor cells can be inhibited. In some embodiments, tumor cell growth is inhibited by delaying tumor cell proliferation. In some embodiments, tumor cell growth is inhibited by halting tumor cell proliferation. In some embodiments, tumor cell growth is inhibited by killing tumor cells. In some embodiments, tumor cell growth is inhibited by inducing tumor cell apoptosis. In some embodiments, tumor cell growth is inhibited by inducing tumor cell differentiation. In some embodiments, tumor cell growth is inhibited by depriving the tumor cells of nutrients. In some embodiments, tumor cell growth is inhibited by preventing tumor cell migration. In some embodiments, tumor cell growth is inhibited by preventing tumor cell invasion.

As used herein, "sequence identity" refers to the degree of sequence identity based on comparing nucleotides or amino acids one by one within a comparison window. "(Percent) Sequence Identity" can be calculated as follows: compare two best aligned sequences in a comparison window and determine whether there are identical nucleic acid bases (e.g., A, T, C, G, I) in both sequences or of the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) Number of positions to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiply the result by 100 to produce percent sequence identity. For the purpose of determining percent sequence identity, optimal alignment can be achieved in various ways known in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. One of ordinary skill in the art will be able to determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximal alignment over the entire length of the sequences being compared or over regions of the target sequence. Law. In the present invention, for antibody sequences, in a preferred embodiment, two amino acid sequences are determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and then performing optimal alignment according to the numbering rules. percent identity.

As used herein, the term "agglutination" refers to the clumping of cells, and the term "hemagglutination" refers to the clumping of a specific class of cells, namely red blood cells. Therefore, hemagglutination is a type of agglutination.

As used herein, the term "conservative substitution" or "conservative amino acid substitution" refers to the replacement of one or more amino acids with one or more chemically or functionally similar amino acids. Conservative substitution tables providing similar amino acids are well known in the art. Polypeptide sequences having such substitutions are referred to as "conservatively modified variants." Such conservatively modified variants are in addition to and not exclusive of polymorphic variants, interspecies homologs, and alleles. Conservative substitutions for each other are illustrated in Table A.

“Hu5F9” herein is an anti-CD47 antibody in the form of IgG4P, produced by GenScript based on recombinant expression of the variable region sequence of 5F9 disclosed in patent US 2015/0183874 A1. "SRF231" is an anti-CD47 antibody in the form of IgG4P, produced by GenScript based on recombinant expression of the 2.3D11 variable region sequence disclosed in US Patent 20180201677 A1.

Invented bispecific antibodies

The present invention provides a bispecific antibody comprising at least one antigen-binding domain capable of specifically binding to CD47 and at least one antigen-binding domain capable of specifically binding to HER2. In some embodiments, the bispecific antibodies provided herein further comprise an Fc domain.

Antigen-binding domain capable of specifically binding CD47

The term "antigen-binding domain capable of specifically binding CD47" refers to a polypeptide that specifically binds CD47. In one aspect, the term "antigen-binding domain capable of specifically binding CD47" refers to a region of a molecule (eg, a bispecific antibody) that contains specific binding to part or the full length of CD47. An antigen-binding domain capable of specifically binding CD47 may be provided, for example, by one or more antibody variable regions. In particular, the antigen-binding domain that specifically binds CD47 includes a light chain variable region (VL) and a heavy chain variable region (VH). In some embodiments, an "antigen-binding domain capable of specifically binding CD47" is a scFv fragment or Fab fragment, particularly a scFv fragment.

In some embodiments, the antigen-binding domain capable of specifically binding CD47 is derived from any anti-CD47 antibody known in the art. "Anti-CD47 antibody" refers to an antibody that can modulate, e.g., inhibit, block, antagonize, neutralize, or otherwise interfere with CD47 expression, activity, and/or signaling. These antibodies may modulate, e.g., inhibit, block, antagonize, neutralize, or otherwise interfere with the interaction between CD47 and SIRPα (signal regulatory protein α) (eg, human CD47 and human SIRPα).

In some embodiments, an antigen-binding domain capable of specifically binding CD47 binds to the same epitope as an anti-CD47 antibody known in the art and described herein. In some embodiments, an antigen-binding domain capable of specifically binding CD47 competes with antibodies known in the art and described herein for binding to CD47. In some embodiments, the antigen-binding domain capable of specifically binding CD47 includes: any anti-CD47 antibody derived from the prior art (e.g., IBI-188, TJC4, SHR-1603, SRF231, IMC-002, CC-90002 , A0-176 or Hu5F9 or a variant thereof) and 3 HCDRs and/or 3 LCDRs described herein.

In some embodiments, the antigen binding domain capable of specifically binding CD47 comprises HCDR1, HCDR2 and HCDR3 of VH and LCDR1, LCDR2 and LCDR3 of VL, wherein VH comprises SEQ ID NO: 1, 3, 5, 6 or 7 The amino acid sequence shown is or consists of, and VL contains or consists of the amino acid sequence shown as SEQ ID NO: 2, 4, 8, 9 or 10.

In some embodiments, the antigen-binding domain capable of specifically binding CD47 comprises HCDR1, HCDR2 and HCDR3 of VH, and comprises LCDR1, LCDR2 and LCDR3 of VL, wherein VH and VL are selected from:

(1) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

(2) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4;

(3) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10;

(4) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 6, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9 or 10; or

(5) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises: HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11, comprising SEQ ID The amino acid sequence shown in NO: 12 or HCDR2 consisting of it, and the HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13 or 17 or 21; and VL includes: comprising SEQ ID NO: 13 or 17 or 21. The amino acid sequence shown in ID NO: 14 or LCDR1 consisting of it, the amino acid sequence shown in SEQ ID NO: 15 or 18 or 22 or the LCDR2 consisting of it, and the LCDR2 including SEQ ID NO: The amino acid sequence shown in 16 or LCDR3 composed thereof.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises: comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11 HCDR1 consisting of, HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12, and HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 17; and VL comprises : LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 18, and LCDR2 comprising or consisting of SEQ ID NO: The amino acid sequence shown in 16 or LCDR3 composed thereof.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises at least 90% similarity to the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 6 or 7 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of the amino acid sequence or consisting of it, VL includes the same as SEQ ID NO. : The amino acid sequence represented by 2, 4, 8, 9 or 10 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 % sequence identity of an amino acid sequence or consisting of it.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises at least 90%, 91%, 92%, 93 similarity to the amino acid sequence set forth in SEQ ID NO: 7 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of an amino acid sequence or consisting of it, VL comprising an amino acid sequence as shown in SEQ ID NO: 8 A sequence has or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the antigen-binding domain capable of specifically binding CD47 comprises VH and VL, wherein VH and VL are selected from:

(1) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

(2) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4;

(3) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10;

(4) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 6, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9 or 10; or

(5) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 1, and VL comprises as set forth in SEQ ID NO: 1 The amino acid sequence shown in 2 may be composed of.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, and VL comprises the amino acid sequence set forth in SEQ ID NO: 3. The amino acid sequence shown in 4 may consist of.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, and VL comprises SEQ ID NO: The amino acid sequence shown in 8 may be composed of.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, and VL comprises SEQ ID NO: The amino acid sequence shown in 9 may be composed of.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, and VL comprises SEQ ID NO: The amino acid sequence shown in 10 may be composed of.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, and VL comprises SEQ ID NO: The amino acid sequence shown in 9 may be composed of.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, and VL comprises SEQ ID NO: The amino acid sequence shown in 10 may be composed of.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, and VL comprises SEQ ID NO: The amino acid sequence shown in 8 may be composed of.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, and VL comprises SEQ ID NO: The amino acid sequence shown in 9 may be composed of.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, and VL comprises SEQ ID NO: The amino acid sequence shown in 10 may be composed of.

Antigen-binding domain capable of specifically binding to HER2

The term "antigen-binding domain capable of specifically binding HER2" refers to a polypeptide that specifically binds HER2. In one aspect, the term "antigen-binding domain capable of specifically binding HER2" refers to a region of a molecule (eg, a bispecific antibody) that contains a portion or full length of a molecule that specifically binds HER2. For example, one or more antibody variable regions can provide an antigen-binding domain that can specifically bind HER2. In particular, the antigen-binding domain that specifically binds HER2 contains the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH). In some embodiments, the "antigen-binding domain capable of specifically binding HER2" may be a scFv fragment or a Fab fragment, especially a Fab fragment.

In some embodiments, the antigen-binding domain capable of specifically binding HER2 is derived from any anti-HER2 antibody known in the art, such as trastuzumab, pertuzumab, and ertumaxomab , or a variant thereof. In some embodiments, the antigen-binding domain capable of specifically binding HER2 binds to the same epitope as any anti-HER2 antibody known in the art (eg, trastuzumab). In some embodiments, an antigen-binding domain capable of specifically binding HER2 competes with any anti-HER2 antibody known in the art (eg, trastuzumab) for binding to HER2. In some embodiments, the antigen-binding domain capable of specifically binding HER2 comprises 3 HCDRs and/or 3 LCDRs from any anti-HER2 antibody known in the art.

In some embodiments, the antigen-binding domain capable of specifically binding HER2 includes HCDR1, HCDR2, and HCDR3 of VH, and includes LCDR1, LCDR2, and LCDR3 of VL, wherein VH includes the amino acid set forth in SEQ ID NO: 29 sequence or consisting of it, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 30.

In some embodiments, the antigen-binding domain capable of specifically binding HER2 comprises VH and VL, wherein VH comprises: HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 23, comprising SEQ ID The amino acid sequence shown in NO: 24 or HCDR2 consisting of it, and the HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 25; and VL includes: comprising SEQ ID NO: 26 The amino acid sequence shown or LCDR1 consisting of it, including as LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 27, and LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 28.

In some embodiments, the antigen-binding domain capable of specifically binding to HER2 comprises VH and VL, wherein VH comprises at least 90%, 91%, 92%, 93 similarity to the amino acid sequence shown in SEQ ID NO: 29 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of an amino acid sequence or consisting of it, VL comprising an amino acid sequence as shown in SEQ ID NO: 30 A sequence has or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In one aspect, bispecific antibodies provided herein include:

(a) The antigen-binding domain capable of specifically binding to CD47 includes HCDR1, HCDR2 and HCDR3 of VH and LCDR1, LCDR2 and LCDR3 of VL, wherein VH includes as shown in SEQ ID NO: 1, 3, 5, 6 or 7 an amino acid sequence or consisting of an amino acid sequence, and VL comprises or consists of an amino acid sequence set forth in SEQ ID NO: 2, 4, 8, 9 or 10; and

(b) The antigen-binding domain capable of specifically binding to CD47 includes HCDR1, HCDR2 and HCDR3 of VH and LCDR1, LCDR2 and LCDR3 of VL, wherein VH includes or consists of the amino acid sequence shown in SEQ ID NO: 29 , and VL contains or consists of the amino acid sequence shown in SEQ ID NO: 30.

In one aspect, bispecific antibodies provided herein include:

(a) The antigen-binding domain capable of specifically binding to CD47 includes HCDR1, HCDR2 and HCDR3 of VH and LCDR1, LCDR2 and LCDR3 of VL, wherein VH includes or consists of the amino acid sequence shown in SEQ ID NO: 7 , and VL contains or consists of the amino acid sequence shown in SEQ ID NO: 8; and

(b) The antigen-binding domain capable of specifically binding to CD47 includes HCDR1, HCDR2 and HCDR3 of VH and LCDR1, LCDR2 and LCDR3 of VL, wherein VH includes or consists of the amino acid sequence shown in SEQ ID NO: 29 , and VL contains or consists of the amino acid sequence shown in SEQ ID NO: 30.

In one aspect, bispecific antibodies provided herein include:

(a) At least one antigen-binding domain capable of specifically binding CD47 comprising VH and VL, wherein VH comprises the amino acid sequence shown in SEQ ID NO: 11 or HCDR1 consisting of it, including SEQ ID NO: The amino acid sequence shown in 12 or HCDR2 consisting of it, and HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 17; and wherein VL includes: comprising as shown in SEQ ID NO: 14 LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18 or consisting of the amino acid sequence shown in SEQ ID NO: 18, and LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 16 or LCDR3 composed of it; and

(b) At least one antigen-binding domain capable of specifically binding HER2 comprising VH and VL, wherein VH comprises the amino acid sequence shown in SEQ ID NO: 23 or HCDR1 consisting of it, including SEQ ID NO: HCDR2 containing or consisting of the amino acid sequence shown in SEQ ID NO: 24, and HCDR3 containing or consisting of the amino acid sequence shown in SEQ ID NO: 25; and wherein VL contains the amino acid sequence shown in SEQ ID NO: 26 LCDR1 containing or consisting of the amino acid sequence shown in SEQ ID NO: 27, LCDR2 containing or consisting of the amino acid sequence shown in SEQ ID NO: 28 Composed of LCDR3.

In one aspect, bispecific antibodies provided herein include:

(a) At least one antigen-binding domain capable of specifically binding CD47 comprising VH and VL, wherein VH comprises at least 90% similarity to the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 6 or 7 , An amino acid sequence with or consisting of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and capable of specifically binding to CD47 antigen The VL of the binding domain contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 2, 4, 8, 9 or 10 , or consisting of an amino acid sequence with 97%, 98%, 99% or 100% sequence identity, and

(b) At least one antigen-binding domain capable of specifically binding HER2 comprising VH and VL, wherein VH comprises at least 90%, 91%, 92%, 93 of the amino acid sequence shown in SEQ ID NO: 29 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to or consisting of an amino acid sequence, and wherein VL comprises an amine as set forth in SEQ ID NO: 30 The amino acid sequence has or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In one aspect, bispecific antibodies provided herein include:

(a) At least one antigen-binding domain capable of specifically binding CD47 comprising VH and VL, wherein VH comprises at least 90%, 91%, 92%, 93 of the amino acid sequence shown in SEQ ID NO: 7 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence or a VH consisting thereof, and comprising an amine as shown in SEQ ID NO: 8 Amino acid sequences with or consisting of amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity VL, and

(b) At least one antigen-binding domain capable of specifically binding HER2 comprising VH and VL, wherein VH comprises at least 90%, 91%, 92%, 93 of the amino acid sequence shown in SEQ ID NO: 29 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence or a VH consisting thereof, and a VL comprising an amino acid sequence as shown in SEQ ID NO: 30 The amino acid sequence has at least or consist of an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In one aspect, bispecific antibodies provided herein include:

(a) At least one antigen-binding domain capable of specifically binding CD47 comprising VH and VL, wherein VH and VL are selected from:

(1) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

(2) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4;

(3) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10;

(4) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 6, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9 or 10; or

(5) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10; as well as

(b) At least one antigen-binding domain capable of specifically binding HER2 comprising VH and VL, wherein VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 29, and VL comprises SEQ ID NO: The amino acid sequence shown in 30 or the VL composed thereof.

In one aspect, bispecific antibodies provided herein include:

(a) at least one antigen-binding domain capable of specifically binding CD47 comprising VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, and wherein VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 : The amino acid sequence shown in 2 or VL consisting of it; and

(b) at least one antigen-binding domain capable of specifically binding HER2 comprising VH and VL, wherein VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 29, and wherein VL comprises as set forth in SEQ ID NO: : The amino acid sequence shown in 30 or consisting of it.

Structure of bispecific antibodies

In one aspect, the present invention provides a bispecific antibody, wherein the antigen-binding domain capable of specifically binding to CD47 and/or the antigen-binding domain capable of specifically binding HER2 comprises: a Fab fragment or a scFv fragment.

In some embodiments, the Fab fragment is a Cross-Fab fragment. In some embodiments, the Fab fragment is Cross-Fab (CL-CH1).

In some embodiments, the scFv fragment includes a peptide linker connecting the C-terminus of VH to the N-terminus of VL (eg, from N-terminus to C-terminus, the structure is "VH-peptide linker-VL"). In some embodiments, the scFv fragment includes: a peptide linker connecting the C-terminus of VL to the N-terminus of VH (eg, from N-terminus to C-terminus, the structure is "VL-peptide linker-VH").

In some embodiments, the peptide linker connecting VH to VL comprises a GS linker. In some embodiments, the GS linker comprises one selected from the group consisting of (G ₄ S) n, (SG ₄ ), or G ₄ (SG ₄ ) _n peptide linkers, where “n” is an integer ranging from 1 to 10, Preferably range from 2 to 4, more preferably 3 or 4. In some embodiments, the GS linker comprises (G ₄ S) ₂ (SEQ ID NO: 32). In some embodiments, the GS linker comprises (G ₄ S) ₃ (SEQ ID NO: 33). In some embodiments, the GS linker comprises (G ₄ S) ₄ (SEQ ID NO: 34). In some embodiments, the GS linker comprises AST(G ₄ S) ₃ (SEQ ID NO: 35).

In some embodiments, the antigen-binding domain capable of specifically binding CD47 comprises an scFv fragment comprising a peptide linker connecting VL and VH. In some embodiments , the antigen-binding domain capable of specifically binding to CD47 includes an scFv fragment that includes a peptide linker connecting the C-terminus of VH to the N-terminus of VL.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises an scFv fragment comprising a peptide linker connecting the C-terminus of VH to the N-terminus of VL, wherein VH comprises a linker with, for example, SEQ ID NO: 1 , the amino acid sequence represented by 3, 5, 6 or 7 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence An amino acid sequence or consisting of an identical amino acid sequence, and VL contains at least 90%, 91%, 92%, 93% identity with an amino acid sequence set forth in SEQ ID NO: 2, 4, 8, 9 or 10 , or consist of an amino acid sequence with 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises an scFv fragment comprising a peptide linker connecting the C-terminus of VH to the N-terminus of VL, wherein VH comprises as set forth in SEQ ID NO: 7 VL contains or consists of the amino acid sequence shown in SEQ ID NO: 8.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises an scFv fragment comprising or consisting of the amino acid sequence shown in SEQ ID NO: 38.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 comprises an scFv fragment comprising or consisting of the amino acid sequence shown in SEQ ID NO: 39.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 and/or the antigen-binding domain capable of specifically binding to CD47 comprises a Fab fragment.

In some embodiments, the antigen-binding domain capable of specifically binding HER2 comprises a Fab fragment, the Fab fragment comprising VH and VL, wherein VH comprises at least 90%, or consisting of an amino acid sequence that has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and VL contains an amino acid sequence identical to, for example, SEQ ID NO. : The amino acid sequence shown in 30 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. sequence or consisting of.

In some embodiments, the antigen-binding domain capable of specifically binding HER2 comprises a Fab fragment comprising VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 30.

In some embodiments, an antigen-binding domain capable of specifically binding CD47 comprises a Fab fragment comprising VH and VL, wherein VH comprises an amine as set forth in SEQ ID NO: 1, 3, 5, 6 or 7 The amino acid sequence has or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and VL comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to the amino acid sequence set forth in SEQ ID NO: 2, 4, 8, 9 or 10 , or consisting of an amino acid sequence with 98%, 99% or 100% sequence identity.

In some embodiments, the antigen-binding domain capable of specifically binding CD47 comprises a Fab fragment comprising VH and VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO:8.

In some embodiments, the antigen-binding domain capable of specifically binding to CD47 is CrossFab (CL-CH1), which CrossFab (CL-CH1) includes VH and VL, wherein VH includes an amine group as shown in SEQ ID NO: 7 acid sequence or consisting of it, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO:8.

In one aspect, the bispecific antibodies provided herein are multivalent, such as bivalent, trivalent or tetravalent.

Bispecific antibodies, (1+1) structure

In one aspect, the bispecific antibodies provided herein are bivalent.

In one aspect, bispecific antibodies provided herein include:

(a) an antigen-binding domain capable of specifically binding CD47, and

(b) An antigen-binding domain capable of specifically binding HER2.

In some embodiments, the bispecific antibodies provided herein further comprise an Fc domain consisting of a first subunit and a second subunit capable of stable association, wherein one antigen binding domain is linked to the The N-terminus of one subunit in the Fc domain, and the other antigen-binding domain is linked to the N-terminus of other Fc domain subunits. In some embodiments, the Fc domain contains "knob-into-hole" modifications. In a specific embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (knob) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (hole ) (according to Kabat EU index number).

In one aspect, bispecific antibodies provided herein include:

(a) The first Fab fragment capable of specifically binding to CD47,

(b) a second Fab fragment capable of specifically binding to HER2, and

(c) An Fc domain consisting of a first subunit and a second subunit capable of stable association.

In some embodiments, the first Fab fragment and the second Fab fragment are each directly linked to the N-terminus of the Fc domain subunit. In some embodiments, one of the Fab fragments is a CrossFab fragment. In some specific embodiments, one of the Fab fragments is CrossFab (CL-CH1).

In some embodiments, bispecific antibodies provided herein comprise:

(a) The first Fab fragment capable of specifically binding to CD47,

(b) a second Fab fragment capable of specifically binding to HER2, and

(c) An Fc domain consisting of a first subunit and a second subunit capable of stable association, wherein the first Fab fragment is CrossFab (CL-CH1) and is directly connected to the N of one subunit in the Fc domain end, and the second Fab fragment is directly linked to the N-terminus of another subunit in the Fc domain.

In some embodiments, bispecific antibodies provided herein comprise:

(i) The first polypeptide chain has the structure: VH (anti-CD47)-CL-Fc domain subunit,

(ii) The second polypeptide chain has the structure: VL (anti-CD47)-CH1,

(iii) The third polypeptide chain has the structure: VH (anti-HER2)-CH1-Fc domain subunit, and

(iv) The fourth polypeptide chain has the structure: VL (anti-HER2)-CL,

One of the Fc domain subunits contains the amino acid substitutions S354C and T366W (knob), while the other Fc domain subunit contains the amino acid substitutions Y349C, T366S, L368A and Y407V (hole) (numbered according to the Kabat EU index).

As used herein, VH (anti-CD47) and VL (anti-CD47) refer to the heavy and light chain variable domains, respectively, that are capable of specifically binding the antigen-binding domain of CD47; VH (anti-HER2) and VL (anti- -HER2) refers to the heavy chain and light chain variable domains of the antigen-binding domain capable of specifically binding to HER2, respectively. In some embodiments, CL in the first polypeptide chain includes an amine group as shown in SEQ ID NO: 61 acid sequence, and CH1 in the fourth polypeptide chain includes or consists of the amino acid sequence shown in SEQ ID NO: 60.

In some embodiments, the Fc domain subunit in the first polypeptide chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57, and/or the Fc domain in the second polypeptide chain The subunits comprise or consist of the amino acid sequence shown in SEQ ID NO: 56.

In some embodiments, bispecific antibodies provided herein comprise:

(i) A first polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity,

(ii) a second polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity,

(iii) A third polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity, and

(iv) a fourth polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consists of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity.

Bispecific antibodies, (2+2) structure

In one aspect, the bispecific antibodies provided herein are tetravalent.

In one aspect, bispecific antibodies provided herein include:

(a) Two antigen-binding domains capable of specifically binding CD47,

(b) two antigen-binding domains capable of specifically binding to HER2, and

(c) Fc domain.

In one aspect, bispecific antibodies provided herein include:

(a) Two scFv fragments capable of specifically binding to CD47, and

(b) Two heavy chains and two light chains of a whole antibody comprising two Fab fragments and an Fc domain capable of specifically binding to HER2.

In some embodiments, each of the two scFv fragments is linked to a terminus, such as the N-termini of both heavy chains, the C-termini of both heavy chains, the N-termini of both light chains, or The C-termini of both light chains. In some embodiments, each of the two scFv fragments is linked to the C-terminus of both heavy chains of the whole antibody.

In some embodiments, each of the two scFv fragments is linked to the C-terminus of both heavy chains of the whole antibody by a peptide linker.

In some embodiments, the peptide linker linking the scFv fragment to the chain of the whole antibody comprises a GS linker. In some embodiments, the GS linker comprises (G4S)3 (SEQ ID NO: 33). In some embodiments, the GS linker comprises (G4S)4 (SEQ ID NO: 34). In some embodiments, the GS linker comprises (G4S)4 (SEQ ID NO: 34). In some embodiments, the GS linker comprises G4SG3S (SEQ ID NO: 36).

In some embodiments, each of the two heavy chains comprises at least 90%, 91%, 92%, 93%, 94 similarity to the amino acid sequence set forth in SEQ ID NO: 40, 41, 42 or 63. %, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of or consisting of an amino acid sequence, and each of the two light chains comprises an amino acid sequence as set forth in SEQ ID NO: 43 The amino acid sequence shown has at least or consist of an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

Variants

In certain cases, amino acid sequence variants of the bispecific antibodies of the invention are contemplated. For example, it is desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, but are not limited to, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any deletions, insertions and substitutions or combinations thereof may be made to obtain the final construct, provided that the final construct has the desired properties (eg, antigen binding).

In some cases, variants with one or more amino acid substitutions are provided. Sites that can be subjected to substitution mutations include HVR, FR, Fc or constant region. Conservative substitutions are shown in Table A under the heading "Better Substitutions". More substantial changes are provided in Table A under the heading "Exemplary Substitutions" and are described further below with reference to the amino acid side chain class. Amino acid substitutions can be introduced into the antibody of interest and target the desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependence Cytotoxicity (CDC)) screening products.

Table A.

Amino acids can be grouped according to common side chain properties:

(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, lie;

(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gin;

(3) Acidic: Asp, Glu;

(4) Alkaline: His, Lys, Arg;

(5) Residues that affect chain orientation: Gly, Pro; or

(6) Aromatic: Trp, Tyr, Phe.

Nonconservative substitutions would require exchanging members of one of these categories for the other.

In some embodiments, the antigen-binding domains of the bispecific antibodies provided herein also encompass an antigen-binding domain in which the three CDRs of the heavy chain variable region contain at least one and no more than the CDRs disclosed herein. 5, 4, 3, 2 or 1 amino acid changes (preferable amino acid substitutions, more preferable conservative substitutions); and/or wherein the light chain variable region is modified compared to the CDRs disclosed herein The three CDRs contain at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions).

In some embodiments, the antigen binding domains of the bispecific antibodies provided herein also encompass an antigen binding domain wherein, as compared to the heavy chain variable region and/or light chain variable region of the antibodies specifically disclosed herein, The heavy chain variable region and/or the light chain variable region contains one or more (preferably no more than 10, more preferably no more than 6, 5, 4, 3, 2 or 1) amine groups Acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), in which the amino acid changes do not occur in the CDR region.

Fc domain variants

In embodiments according to various aspects of the invention, the bispecific antibody further comprises an Fc domain.

In some embodiments, the two subunits of the Fc domain: the first subunit and the second subunit are the same or different.

In certain aspects, one or more amino acid modifications can be introduced into the Fc domain of the antibodies provided herein, thereby creating Fc domain variants. Fc domain variants may comprise a human Fc domain sequence (eg, human IgG1, IgG2, IgG3, or IgG4 Fc domain) with amino acid modifications (eg, substitutions) at one or more amino acid positions.

The present invention provides antibody variants with some, but not all, effector functions that make the antibody variants desirable candidates for applications where, for example, the in vivo half-life of the antibody is important, However, some effector functions (eg, complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/attenuation of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity or antibody cross-linking activity), but retains FcRn binding ability. NK cells (the main cells that mediate ADCC) express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. In Ravetch and Kinet, Annu.Rev.Immunol.. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of 9:457-492 (1991). Binding sites with FcγRI, FcγRII, FcγRIII and FcRn on human IgGl have been described, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591 -6604,2001).

Antibodies with reduced effector function include antibodies with substitutions for one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent Nos. 6,737,056 and 8,219,149). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called " DANA" Fc mutants (U.S. Patent Nos. 7,332,581 and 8,219,149).

This article describes certain antibody variants with improved or reduced binding to FcR. (See, eg, U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).) In some cases, antibody variants contain improved Fc domain with one or more amino acid substitutions of ADCC, for example, substitutions at positions 298, 333 and/or 334 of the Fc domain (EU numbering of residues). In some cases, changes occur in the Fc domain that result in altered (i.e., improved or diminished) C1q binding and/or complement-dependent cytotoxicity (CDC), for example, as in U.S. Patent No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol., 164:4178-4184 (2000).

Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus, as described in US 2005/0014934 A1 (Hinton et al., J. Immunol. 1 17:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Those antibodies contain an Fc domain in which one or more amino acid substitutions improve binding of the Fc domain to FcRn. Such Fc variants include those at Fc domain residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, Variants having substitutions at one or more of 382, 413, 424, 428, or 434 (eg, substitution of Fc domain residue 434 (U.S. Patent No. 7,371,826)). See also Duncan and Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821 and WO 94/29351 for other examples of Fc domain variants.

In one aspect, the antibodies provided herein are modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of antibody glycosylation sites can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites. For example, glycosylation can be altered to increase the affinity of an antibody for the "antigen." Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. This deglycosylation (aglycosylation) can increase the affinity of the antibody for the antigen. Such methods are described, for example, in US Patent No. 5,426,300. When an antibody contains an Fc domain, the carbohydrates attached to it can be changed. In some applications, modifications that remove unwanted glycosylation sites are useful, such as removal of fucose components to improve antibody-dependent cell-mediated cytotoxicity (ADCC) function. In other applications, galactosylation modifications can be performed to modify complement-dependent cytotoxicity (CDC).

Fc domain modifications that promote heterodimerization

Bispecific antibodies can contain different components (e.g., antigen-binding domains) fused to one or other of the two subunits of the Fc domain, so the two subunits of the Fc domain are usually contained in two different in the polypeptide chain. Recombinant coexpression and subsequent dimerization of these polypeptides results in several possible combinations of the two polypeptides. To improve the yield and purity of such antibodies in recombinant production, it would therefore be advantageous to introduce modifications in the Fc domain of the antibody that promote association of the desired polypeptide.

In one aspect, the Fc domain of the bispecific antibodies provided herein comprises a modification that promotes association of the first subunit and the second subunit of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is in the CH3 domain of the Fc domain. Any known method for modification in the CH3 domain of the Fc domain to promote heterodimerization can be used in the present invention, for example in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205 , WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291.

In a specific embodiment, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, contained in both subunits of the Fc domain A "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. Knob-into-hole technology is described, for example, in US 5,731,168, US 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).

In a specific embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (according to Kabat EU index numbering) .

Modifications in the Fab domain

Multispecific antibodies with domain substitutions/exchanges (CrossMabVH-VL or CrossMabCL-CH1) in one binding arm are described in detail in WO 2009/080252 and Schaefer, W. et al., PNAS, 108 (2011) 1 1187-1191 . They significantly reduce by-products caused by erroneous mispairing of the light chain of the first antigen with the heavy chain of the second antigen (compared to methods without such domain swapping).

In one aspect, the invention relates to bispecific antibodies comprising

(a) The first Fab fragment capable of specifically binding to CD47,

(b) a second Fab fragment capable of specifically binding to HER2, and

(c) An Fc domain consisting of a first subunit and a second subunit capable of stable association, wherein the variable domains VH and VL or the constant domains CH1 and CL are exchanged in one of the Fab fragments. Bispecific antibodies are prepared based on Crossmab technology. In one aspect, in one of the Fab fragments, the constant domains CL and CH1 are substituted for each other such that the CH1 domain is part of the light chain and the CL domain is part of the heavy chain. More specifically, in the first Fab fragment, the constant domains CL and CH1 are substituted for each other, so that the CH1 domain is part of the light chain and the CL domain is part of the heavy chain.

On the other hand, and to further improve correct pairing, bispecific antibodies containing Fab fragments can contain different charged amino acid substitutions (so-called "charged residues"). These modifications were introduced into exchanged or non-exchanged CH1 and CL domains. In a specific aspect, the invention relates to a bispecific antibody wherein in one CL domain the amino acid at position 123 (EU numbering) is replaced by arginine (R) and by lysine (K ) replaces the amino acid at position 124 (EU numbering), and wherein in one CH1 domain, the amino acid at position 147 (EU numbering) and position 213 (EU numbering) is replaced by glutamic acid (E) .

Cysteine-engineered antibody variants

In one aspect, it may be desirable to generate cysteine-engineered antibodies, such as "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues.

Antibody derivatives

In one aspect, the antibody portions provided herein can be further modified to include other non-proteinaceous portions known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to: polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly -1,3-dioxane, poly-1,3,6-triane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide/ethylene oxide copolymer, polyoxyethylenated polyol (such as glycerol), polyvinyl alcohol, and mixtures thereof.

Immunoconjugate

The present invention also provides an immunoconjugate comprising an enzyme active in combination with one or more cytotoxic agents (e.g., chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins), bacterial, fungal, plant or animal sources). bispecific antibodies conjugated to toxins or fragments thereof), radioisotopes or biologically active peptides.

In one aspect, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent Nos. EP 0 425 235 B1); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); dolastatin ); calicheamicin or its derivatives (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336- 3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines, such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 (2006); Torgov et al., Bioconj. Chem. 16: 717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al. , J Med. Chem. 45: 4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, texet taxel and otasal; trichothecenes; and CC-1065.

In one aspect, the immunoconjugate comprises a bispecific antibody described herein conjugated to one or more enzymatically active toxins or fragments thereof, including, but not limited to, diphtheria A chain, diphtheria toxin Non-binding active fragment, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, rhizoxin A chain, α-broomotoxin, Tung tung protein, carnation toxin, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, jatropha toxin, croton toxin, sapaonaria officinalis) inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin and tricothecenes .

Phytoweed proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitors, Jatropha curcas toxin, Crotonin, Phytophthora sinensis inhibitor, Polyflora polyphylla protein, Serinomycin, Isolin, Staphylococcus aureus, enomycin, and trichothecin.

In one aspect, the immunoconjugate comprises a bispecific antibody described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ³⁵ S, ³ H, ¹¹¹ In, 112 In, ¹⁴ C, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹⁷⁷ Lu, ^{211 At} , ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi and ³² P, as well as radioactive isotopes of Lu.

In one aspect, an immunoconjugate comprises a bispecific antibody described herein conjugated to one or more polypeptides. In some embodiments, the polypeptide is a ligand, such as a lymphokine and/or cytokine that interacts with a specific cell receptor. Lymphokines are low molecular weight proteins secreted by T cells when antigens or lectins stimulate their growth. Examples of lymphokines include, but are not limited to: interferon-alpha, interferon-gamma, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), tumor necrosis factor (TNF), colony-stimulating factor (such as CSF-1, G-CSF or GM-CSF), chemokines, macrophage migration inhibitory factor (MIF), macrophage activating factor (MAF), NK cell activating factor, T cell replacement factor, leukocyte inhibitory factor (LIF), lymphotoxin, osteoclast activating factor (OAF), soluble immune response suppressor factor (SIRS), growth stimulating factor, monocyte growth factor.

Techniques for conjugating therapeutic moieties to bispecific antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," cited in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. Pages 243-256 (Alan R. Liss, Inc. 1985).

Antibody production and purification

The invention further provides an isolated polynucleotide encoding an antibody of the invention as described herein, or a fragment thereof.

An isolated polynucleotide encoding an antibody of the invention may be expressed as a single polynucleotide encoding the entire antibody or as co-expressed multiple (eg, two or more) polynucleotides. Polypeptides encoded by coexpressed polynucleotides can be associated, for example, by disulfide bonds or other means to form functional antibodies.

In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotides of the invention are RNA, for example in the form of messenger RNA (mRNA). The RNA of the invention can be single-stranded or double-stranded.

According to another aspect of the invention, there is provided an isolated polynucleotide encoding an antibody as defined hereinbefore or a fusion polypeptide as previously described herein. The invention further provides vectors, in particular expression vectors, comprising an isolated polynucleotide of the invention, as well as host cells comprising an isolated polynucleotide or vector of the invention. In one aspect, the host cell is a eukaryotic cell, particularly a mammalian cell.

Antibodies of the invention can be obtained, for example, by solid-state peptide synthesis (eg, Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the antibody or polypeptide fragment thereof, such as described above, are isolated and inserted into one or more vectors for further selection in host cells and/or Express. Such polynucleotides can be readily isolated and sequenced using conventional procedures. In one aspect of the invention, there is provided a vector, preferably an expression vector, comprising one or more polynucleotides of the invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the antibody (fragment) and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo recombination/genetic recombination. The expression vector may be part of a plasmid, a virus, or may be a nucleic acid fragment. Expression vectors include expression cassettes in which a polynucleotide encoding a bispecific antibody or polypeptide fragment thereof (i.e., the coding region) is selected to be operably associated with a promoter and/or other transcriptional or translational control elements. .

In another aspect of the invention, host cells comprising one or more polynucleotides of the invention are provided. In certain embodiments, host cells containing one or more vectors of the invention are provided. The polynucleotide and vector may each, individually or in combination, comprise the polynucleotides and vectors described herein. any characteristics of the carrier. In one aspect, the host cell comprises (eg has been transformed or transfected) a vector containing a polynucleotide encoding (part of) an antibody of the invention. As used herein, the term "host cell" refers to any kind of cellular system that can be engineered to produce the fusion proteins of the invention or fragments thereof. Suitable host cells for replication and support of antibody expression are well known in the art. Such cells can be suitably transfected or transduced with specific expression vectors, and large numbers of vector-containing cells can be cultured for inoculating large-scale fermenters to obtain sufficient amounts of antibodies for clinical applications. Suitable host cells include prokaryotic microorganisms (eg, E. coli) or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), human embryonic kidney (HEK) cells, insect cells, etc.

In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as Chinese hamster ovary (CHO) cells, expi-293 cells, human embryonic kidney (HEK) cells, or lymphoid cells (e.g., Y0, NSO, Sp20 cells). Standard techniques are known in the art for expressing foreign genes in these systems. Cells that express a polypeptide of a heavy or light chain that contains an antigen-binding domain can be engineered to also express another one of the immunoglobulin chains such that the expressed product has the antigen-binding domains of both the heavy and light chains.

In another aspect, a method for producing an antibody of the invention is provided, comprising the steps of: (i) culturing a host cell of the invention under conditions suitable for expression of the antibody, and (ii) isolating from the host cell or host cell culture medium the antibody.

Antibodies of the invention prepared as described herein can be purified by techniques known in the art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those of ordinary skill in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens that bind to the antibody can be used. For example, for the antibody of the present invention For affinity chromatography purification, a matrix with protein A or protein G can be used. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate antibodies essentially as described in the Examples. The purity of the antibody or fragment thereof can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high-pressure liquid chromatography, and the like. For example, reducing and non-reducing SDS-PAGE proved that the expressed antibodies in the examples were complete and correctly assembled.

pharmaceutical composition

The term "pharmaceutical composition" refers to a preparation/formulation that allows the active ingredients contained therein to be biologically active and does not contain unacceptable toxicity to the subject to whom the preparation/formulation is administered. exist in the form of other ingredients.

The term "pharmaceutical excipient" refers to a pharmaceutical carrier, diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)) or vehicle with which a therapeutic agent is administered.

The pharmaceutical composition of the present invention may include the antibody of the present invention and pharmaceutical excipients. These pharmaceutical compositions may be included in kits, such as diagnostic kits.

As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, and the like that are physiologically compatible. Pharmaceutical carriers suitable for use in the present invention may be sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When pharmaceutical compositions are administered intravenously, aqueous solutions are preferred carriers. Physiological saline, aqueous dextran solutions, and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions.

Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicone, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, Glycerin, propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, see also "Handbook of Pharmaceutical Excipients", fifth edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. The compositions may also contain small amounts of wetting or emulsifying agents or pH buffering agents. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin, and the like.

The invention provides pharmaceutical compositions comprising one or more bispecific antibodies of the invention and nucleic acids, vectors or host cells, or immunoconjugates or fusions. It should be understood that the bispecific antibodies, nucleic acids, vectors or host cells thereof, or immunoconjugates, or fusions, or pharmaceutical compositions thereof provided by the present invention can be combined with suitable pharmaceutical carriers and excipients in the preparation. Use in combination with other agents for co-administration to provide improved transfer, delivery, tolerability, etc.

Pharmaceutical preparations/formulations comprising the bispecific antibodies described herein can be prepared by mixing the bispecific antibodies of the invention with the desired purity and one or more optional pharmaceutical excipients, preferably aqueous solutions or in the form of lyophilized preparations. Exemplary lyophilized antibody preparations/formulations are described in US Patent No. 6,267,958. Aqueous antibody preparations/formulations include those described in US Patent No. 6,171,586 and WO 2006/044908, the latter preparation/formulation including histate-acetate buffer.

The pharmaceutical compositions or preparations/formulations of the present invention may also contain one or more other active ingredients required for the treatment of a particular disease, preferably those active ingredients that have complementary activities and do not adversely affect each other. For example, other therapeutic agents may also be included. In one aspect, the other therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, a vaccine, other antibodies, an immunomodulatory agent, or other biological macromolecule drug.

In one aspect, the pharmaceutical composition of the present invention may further comprise a nucleic acid encoding the bispecific antibody.

Instructions

In one aspect, the invention provides a method for preventing, diagnosing or treating a CD47-related and/or HER2-related disease or condition in a subject. The method includes administering to a patient in need thereof an effective amount of a bispecific antibody described herein, or an immunoconjugate or immunofusion comprising the same, or a pharmaceutical composition comprising a bispecific antibody described herein. antibody, or immunoconjugate or immunofusion), or the nucleic acid, vector or host cell described herein.

In one aspect, the invention provides the use of a bispecific antibody in the production or preparation of a medicament for preventing, diagnosing or treating a CD47-related and/or HER2-related disease or condition in a subject.

In one aspect, the bispecific antibodies described herein, or their immunoconjugates or immunofusions, or pharmaceutical compositions (the pharmaceutical compositions comprise the bispecific antibodies, or immunoconjugates provided by the invention, or Immunofusions) can be used as therapeutics to prevent or treat CD47-related and/or HER2-related diseases or conditions in a subject. For CD47-related and/or HER2-related diseases or conditions in a subject identified by using standard methods, the bispecific antibodies, pharmaceutical compositions or immunoconjugates or immunofusions disclosed herein can be administered, or as described herein. The nucleic acid, vector or host cell described above.

In one aspect, the methods and uses described herein further include administering to the individual an effective amount of at least one additional therapeutic agent or treatment modality. In one aspect, the therapeutic agent is, for example, a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, a vaccine, other antibodies, an immunomodulatory agent, or other biological macromolecule drug. In one aspect, treatment modalities include surgery, as well as radiation therapy, localized or focused irradiation, and the like.

The above-mentioned combination therapy includes: combined administration (in which two or more therapeutic agents are contained in the same or separate preparations/formulations) and separate administration, wherein the administration of the bispecific antibody of the invention can be before, simultaneously or in addition to the administration of therapeutic agents and/or adjuvants and/or treatment.

In one aspect, a CD47-related disease or condition of the present invention refers to a disease or condition associated with abnormal CD47 expression, activity and/or signaling in a subject, including but not limited to cancer. on one side In a CD47-related disease or condition, for example, compared to a healthy subject, or compared to adjacent tissue, cells or organs (e.g., non-cancerous tissue, cells or organs) without such disease or condition, encoding The nucleic acid (level or content) of CD47 is increased, or CD47 expression is increased, or CD47 protein level is increased, or activity is increased, or activity signaling is increased.

In one aspect, a HER2-related disease or condition of the present invention refers to a disease or condition associated with abnormal HER2 expression, activity and/or signaling in a subject, including but not limited to cancer. In one aspect, in a HER2-related disease or condition, for example, compared to a healthy subject, or compared to adjacent tissue, cells or organs (e.g., non-cancerous tissue, cells or organs) without such disease or condition. , the nucleic acid (level or content) encoding HER2 is increased, or HER2 expression is increased, or HER2 protein level is increased, or activity is increased, or activity signaling is increased.

In one aspect, treatment of disease would benefit from inhibiting CD47 at the nucleic acid or protein level, or from blocking the binding of CD47 to its ligands or CD47-mediated signaling. In one aspect, treatment of the disease would benefit from inhibiting HER2 at the nucleic acid or protein level, or from blocking the binding of HER2 to its ligands or HER2-mediated signaling. In one aspect, treatment of the disease would benefit from inhibiting CD47 and HER2 at the nucleic acid or protein level, or from blocking the binding of CD47 and HER2 to its ligands or CD47 and HER2-mediated signaling, e.g., as in healthy humans. or compared to adjacent tissues, cells, or organs that do not have these diseases or conditions (e.g., non-cancerous tissues, cells, or organs). .

In one aspect, the subject may be a mammal, such as a primate, preferably a higher primate, such as a human (eg, an individual suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from or is at risk of suffering from a disease (eg, cancer) described herein. In certain embodiments, the subject receives or has received other treatments, such as chemotherapy and/or radiation therapy.

In one aspect, cancer includes various hematological cancers and solid tumors, as well as metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. Cancer can be in early, intermediate or late stages, or metastatic.

In specific embodiments, blood cancers include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), non-Hodgkin lymphoma (eg, Burkitt lymphoma), B-lymphoma cell leukemia/lymphoma, B-cell chronic lymphocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), follicular lymphoma, small lymphocytic leukemia (SLL), central nervous system (CNS) lymphoma tumour, Richter's Syndrome, multiple myeloma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma and anaplastic large cell lymphoma.

In specific embodiments, solid tumors include, but are not limited to, breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, head and neck squamous cell carcinoma, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme Cell tumor, gastric junction adenocarcinoma, esophagogastric junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, melanoma, Ewing sarcoma, rhabdomyosarcoma, or neuroblastoma.

The antibody of the invention may be administered in any suitable manner, including oral, parenteral, intrapulmonary and intranasal administration, and if local treatment is desired, it may be administered intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. A variety of dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion.

The antibodies of the invention will be formulated and administered in a manner consistent with good medical practice. Not necessarily, but optionally, the antibody is formulated with one or more agents currently used to prevent or treat disease. These other The effective amount of other agents will depend on the amount of antibody present in the preparation/formulation, the type of condition or disease being treated, and other factors discussed above. In order to prevent or treat a disease or condition, the antibody or antigen-binding fragment of the invention will be determined based on the type of disease or condition to be treated, the type of antibody, the severity and duration of the disease, whether the antibody is for preventive or therapeutic purposes, and previous treatment. , the patient's clinical history and response to the antibody, and the judgment of the attending physician to administer the appropriate dose. The antibody is appropriately administered to the patient in a single treatment or series of treatments.

In certain embodiments, any of the bispecific antibodies provided herein can be used to detect the presence of CD47 and/or HER2 in a sample. In one aspect, the detection method includes:

(a) contacting the sample with an antibody of the invention, or an antigen-binding fragment or conjugate or fusion thereof; and

(b) Detecting the formation of a complex of the antibody or antigen-binding fragment or conjugate or fusion thereof with CD47 and/or HER2 proteins.

As used herein, the term "detection" includes quantitative or qualitative detection. In certain embodiments, the sample is a blood, serum, or other liquid sample of biological origin. In certain embodiments, the sample includes cells or tissue. In one aspect, the sample is from a hyperproliferative or cancer-related lesion.

In one embodiment, the antibodies of the invention, or antigen-binding fragments thereof, may be used to diagnose a CD47-related and/or HER2-related disease or condition, such as cancer, e.g., to assess (e.g., monitor) response to treatment or a disease or condition described herein. progression, and diagnosis and/or stage in an individual. In certain embodiments, labeled bispecific antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, electron density labels, chemiluminescent labels, and radioactive labels), as well as indirect detection moieties, such as enzymes or ligands, e.g., by Enzymatic reactions or intermolecular interactions. exist In one aspect, provided herein are kits for diagnosing CD47-related and/or HER2-related diseases or conditions, the kit comprising an antibody of the invention or an antigen-binding fragment thereof.

In one aspect provided herein, the sample is obtained prior to treatment with the bispecific antibody. In one aspect, the sample is obtained prior to treatment with other therapies. In one aspect, the sample is obtained during or after treatment with other therapies.

Kits and products

In another aspect of the invention, there is provided a kit or article of manufacture containing materials for the treatment, prevention and/or diagnosis of the conditions described above. A kit or article of manufacture includes a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. Containers can be made from a variety of materials, such as glass or plastic. The container may contain (either by itself or in combination with another composition) a composition effective for treating, preventing, and/or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or have a container that is pierceable by a hypodermic needle) stoppered vial). At least one active agent in the composition is a bispecific antibody of the invention.

The label or package insert indicates that the composition is used to treat the selected condition. Additionally, a kit or article of manufacture may comprise a first container containing a composition, wherein the composition comprises a bispecific antibody of the invention; and (b) a second container containing a composition, wherein the composition comprises other cells Toxic therapeutic agents or other therapeutic agents. Articles of manufacture, in embodiments of the present invention, may further comprise a package insert indicating that the composition may be used to treat a particular condition.

Alternatively or additionally, the kit or article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Lin Grignard solution and dextran solution. It may further include other materials as desired from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

The invention includes any combination of the specific embodiments described herein. It should be understood that while specific details and examples are described to illustrate preferred embodiments of the invention, these are illustrative only and are used as examples only. The present invention also covers modified embodiments based on the preferred embodiments of the present invention, which will be obvious to those with ordinary skill in the art. All publications, patents and patent applications cited herein (including citations) are hereby incorporated by reference in their entirety for all purposes.

Exemplary embodiments

Embodiment 1. A bispecific antibody, the bispecific antibody comprising

(a) at least one antigen-binding domain capable of specifically binding CD47, and

(b) At least one antigen-binding domain capable of specifically binding to HER2, wherein the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises: comprising HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12, and HCDR2 comprising or consisting of SEQ ID NO: 13 or The amino acid sequence shown in SEQ ID NO: 14 or HCDR3 consisting of it; and the VL capable of specifically binding to the antigen-binding domain of CD47 includes: comprising the amino acid sequence shown in SEQ ID NO: 14 or consisting of it LCDR1 composed of, LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 15 or 18 or 22, and LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16 .

Embodiment 2. The bispecific antibody as described in Embodiment 1, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises: comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11 HCDR1, HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12, and HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 17 HCDR3; and the VL capable of specifically binding the antigen-binding domain of CD47 includes: an LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14, and an amine shown in SEQ ID NO: 18 LCDR2 containing or consisting of an amino acid sequence as shown in SEQ ID NO: 16, and LCDR3 containing or consisting of an amino acid sequence as shown in SEQ ID NO: 16.

Embodiment 3. The bispecific antibody of Embodiment 1 or 2, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises a compound as shown in SEQ ID NO: 1, 3, 5, 6 or 7 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or consisting of an amino acid sequence Composed of, and the VL capable of specifically binding the antigen-binding domain of CD47 comprises at least 90%, 91%, 92%, or consisting of an amino acid sequence with 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

Embodiment 4. The bispecific antibody according to any one of embodiments 1 to 3, wherein the VH and VL of the antigen-binding domain capable of specifically binding to CD47 are selected from the group consisting of

(1) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

(2) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4;

(3) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10;

(4) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 6, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9 or 10; or

(5) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10.

Embodiment 5. The bispecific antibody according to any one of embodiments 1 to 4, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises the amino acid sequence shown in SEQ ID NO: 7 Or consisting of it, and the VL capable of specifically binding the antigen-binding domain of CD47 includes or consists of the amino acid sequence shown in SEQ ID NO: 8.

Embodiment 6. The bispecific antibody of any one of embodiments 1 to 5, wherein the antigen-binding domain capable of specifically binding HER2 comprises VH and VL, wherein

The VH of the antigen-binding domain capable of specifically binding to HER2 includes: HCDR1 that includes or consists of the amino acid sequence shown in SEQ ID NO: 23, and includes the amino acid sequence shown in SEQ ID NO: 24 or HCDR2 consisting of it, and HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 25; and the VL capable of specifically binding the antigen-binding domain of HER2 comprises: comprising SEQ ID NO : The amino acid sequence shown in SEQ ID NO: 26 or LCDR1 consisting of it, the amino acid sequence shown in SEQ ID NO: 27 or LCDR2 consisting of it, and the amino group shown in SEQ ID NO: 28 acid sequence or LCDR3 consisting of it.

Embodiment 7. The bispecific antibody of embodiment 6, wherein the VH of the antigen-binding domain capable of specifically binding to HER2 comprises at least 90% of the amino acid sequence shown in SEQ ID NO: 29. or consisting of an amino acid sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and which is capable of specifically binding to HER2 The VL of the antigen-binding domain includes at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or consists of an amino acid sequence with 99% or 100% sequence identity.

Embodiment 8. The bispecific antibody of any one of embodiments 1 to 7, wherein the bispecific antibody comprises

(a) at least one antigen-binding domain comprising VH and VL capable of specifically binding CD47, wherein

The VH of the antigen-binding domain capable of specifically binding to CD47 includes at least 90%, 91%, 92%, 93%, or consisting of an amino acid sequence with 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and the VL capable of specifically binding to the antigen-binding domain of CD47 includes an amino acid sequence with, for example, The amino acid sequence shown in SEQ ID NO: 2, 4, 8, 9 or 10 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% sequence identity to or consisting of an amino acid sequence, and

(b) at least one antigen-binding domain comprising VH and VL capable of specifically binding HER2, wherein

The VH of the antigen-binding domain capable of specifically binding to HER2 contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 29 , or consisting of an amino acid sequence with 97%, 98%, 99% or 100% sequence identity, and the VL of the antigen-binding domain capable of specifically binding to HER2 includes the same as shown in SEQ ID NO: 30 Amino acid sequences having or consisting of amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity .

Embodiment 9. The bispecific antibody of any one of embodiments 1 to 8, wherein the antigen binding domain is chimeric, fully human or humanized.

Embodiment 10. The bispecific antibody of any one of embodiments 1 to 9, wherein the antigen-binding domain comprises a Fab fragment or a scFv fragment.

Embodiment 11. The bispecific antibody of any one of embodiments 1 to 10, wherein the antigen-binding domain capable of specifically binding HER2 comprises a Fab fragment, and the antigen-binding domain capable of specifically binding CD47 Contains scFv fragments or Fab fragments.

Embodiment 12. The bispecific antibody of any one of embodiments 10 to 11, wherein the scFv fragment comprises a peptide linker connecting the C-terminus of VH to the N-terminus of VL.

Embodiment 13. The bispecific antibody of any one of embodiments 1 to 12, wherein the bispecific antibody is bivalent, trivalent or tetravalent.

Embodiment 14. The bispecific antibody of any one of embodiments 1 to 13, wherein the bispecific antibody comprises

(a) The first Fab fragment capable of specifically binding to CD47,

(b) a second Fab fragment capable of specifically binding to HER2, and

(c) An Fc domain consisting of a first subunit and a second subunit capable of stable association.

Embodiment 15. The bispecific antibody of embodiment 14, wherein the first Fab fragment is crossFab (CL-CH1).

Embodiment 16. The bispecific antibody of any one of embodiments 1 or 15, wherein the bispecific antibody comprises

(i) The first polypeptide chain has the structure: VH (anti-CD47)-CL-Fc domain subunit,

(ii) The second polypeptide chain has the structure: VL (anti-CD47)-CH1,

(iii) The third polypeptide chain has the structure: VH (anti-HER2)-CH1-Fc domain subunit, and

(iv) The fourth polypeptide chain has the structure: VL (anti-HER2)-CL.

Embodiment 17. The bispecific antibody of any one of embodiments 1 to 16, wherein the bispecific antibody comprises

(i) A first polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity,

(ii) a second polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity,

(iii) A third polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity, and

(iv) a fourth polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consists of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity.

Embodiment 18. The bispecific antibody of any one of embodiments 1 to 13, wherein the bispecific antibody comprises

(a) Two scFv fragments capable of specifically binding to CD47, and

(b) two heavy chains and two light chains of a whole antibody comprising two Fab fragments and an Fc domain capable of specifically binding to HER2,

wherein the two scFv fragments are each linked to the whole antibody.

Embodiment 19. The bispecific antibody of embodiment 18, wherein each of the two scFv fragments is linked to the whole antibody through a peptide linker.

Embodiment 20. The bispecific antibody of embodiment 18 or 19, wherein the two scFv fragments are each linked to the C-termini of the two heavy chains of the whole antibody via a peptide linker.

Embodiment 21. The bispecific antibody of any one of embodiments 12, 19 to 20, wherein the peptide linker comprises a GS linker.

Embodiment 22. The bispecific antibody of any one of embodiments 12, 19 to 21, wherein the peptide linker comprises SEQ ID NO: 31, 32, 33, 34, 35, 36 or 37 Amino acid sequence.

Embodiment 23. The bispecific antibody of any one of embodiments 18 to 22, wherein the scFv fragment comprises or consists of the amino acid sequence shown in SEQ ID NO: 38 or 39.

Embodiment 24. The bispecific antibody of any one of embodiments 18 to 23, wherein the bispecific antibody comprises two heavy chains and two light chains, each of the heavy chains comprising SEQ. The amino acid sequence shown in ID NO: 40, 41, 42 or 63 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 % sequence identity to or consisting of an amino acid sequence, and each of the light chains comprises at least 90%, 91%, 92%, 93% with the amino acid sequence shown in SEQ ID NO: 43 , or consist of an amino acid sequence with 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

Embodiment 25. The bispecific antibody of any one of embodiments 1 to 13, wherein the bispecific antibody comprises an Fc domain.

Embodiment 26. The bispecific antibody of any one of embodiments 14 to 25, wherein the Fc domain is a human IgG1 Fc domain.

Embodiment 27. The bispecific antibody of any one of embodiments 14 to 26, wherein the Fc domain comprises: one or more that promotes association of the first subunit and the second subunit of the Fc domain. Amino acid modification, and/or one or more amino acid modifications that increase the binding affinity of the antibody to FcRn.

Embodiment 28. The bispecific antibody of any one of embodiments 14-27, wherein the Fc domain comprises one or more amino acid substitutions at positions 428 and 434, preferably, the amino acid Acid substitutions are M428L and N434S (according to Kabat EU index numbers).

Embodiment 29. The bispecific antibody of any one of embodiments 14 to 28, wherein each of the two subunits of the Fc domain comprises SEQ ID NO: 53, 54, 55 or 64 The amino acid sequence shown is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or consists of.

Embodiment 30. An isolated nucleic acid encoding the bispecific antibody of any one of embodiments 1 to 29.

Embodiment 31. A recombinant vector or expression vector comprising one or more nucleic acids as described in embodiment 30, wherein the vector is suitable for the recombination of the bispecific antibody as described in any one of embodiments 1 to 29 production.

Embodiment 32. A host cell comprising one or more recombinant vectors or expression vectors according to embodiment 31.

Embodiment 33. An immunoconjugate comprising the bispecific antibody of any one of embodiments 1 to 29.

Embodiment 34. A pharmaceutical composition comprising the bispecific antibody as described in any one of embodiments 1 to 29, the nucleic acid as described in embodiment 30, the vector as described in embodiment 31, as in the embodiment 32 or the immunoconjugate of embodiment 33, and optionally comprising a pharmaceutically acceptable excipient.

Embodiment 35. A method for treating or preventing a disease or disorder, the method comprising administering to an individual an effective amount of a bispecific antibody of any one of embodiments 1 to 29, such as The nucleic acid described in Embodiment 30, the vector described in Embodiment 31, the host cell described in Embodiment 32, the immunoconjugate described in Embodiment 33, or the pharmaceutical composition described in Embodiment 34 .

Embodiment 36. The method of embodiment 35, wherein the disease or disorder is CD47-related and/or HER2-related.

Embodiment 37. The method of embodiment 35 or 36, wherein the disease or condition is cancer.

Embodiment 38. The method of embodiment 37, wherein the cancer is a blood cancer, optionally selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin lymphoma ( For example, Burkitt lymphoma), B-lymphoblastic leukemia/lymphoma, B-cell chronic lymphocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), follicular lymphoma, small lymphocytic Leukemia (SLL), central nervous system (CNS) lymphoma, Richter's Syndrome, multiple myeloma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, and anaplastic Large cell lymphoma.

Embodiment 39. The method of embodiment 38, wherein the cancer is a solid tumor, optionally selected from the group consisting of breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, head and neck squamous cell carcinoma, ovarian cancer Carcinoma, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, esophagogastric junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing sarcoma, rhabdomyosarcoma, or Neuroblastoma.

Embodiment 40. The bispecific antibody according to any one of embodiments 1 to 29, the nucleic acid according to embodiment 30, the vector according to embodiment 31, the host cell according to embodiment 32, Or the use of the immunoconjugate as described in Embodiment 33, or the pharmaceutical composition as described in Embodiment 34 in the preparation of a medicament for treating a disease or disorder in a subject.

Example 1 Preparation and screening of fusion tumor antibodies

Anti-CD47 antibodies were obtained using fusion tumor technology, and the recombinant protein CD47-Fc (ACROBiosystems, Cat: CD7-H5256) with Fc-tagged human CD47 extracellular domain was used as the antigen to immunize mice. CD47-Fc recombinant protein was mixed and emulsified with complete or incomplete Freund's adjuvant (Sigma-Aldrich), and then immunized to SJL (Beijing Vitong Lever Laboratory Animal Technology Co., Ltd.) and BALB/c (Yangzhou University Medical Center) mice. . After the mice underwent one round of immunization (complete Freund's adjuvant) and two rounds of booster immunization (incomplete Freund's adjuvant), blood was collected after each booster immunization, and ELISA technology was used to detect the post-immune mouse serum and recombinant Human CD47-Fc (ACROBiosystems, Cat: CD7-H5256) protein binding activity, and the binding potency of mouse serum and CHO cells (GenScript construct) overexpressing human CD47 were detected by flow cytometry (FACS). The spleen cells of mice with high serum titers were selected and fused with the myeloma cell line SP2/0 (ATCC). Four days before fusion, the recombinant protein CD47-Fc of human CD47 extracellular domain was intraperitoneally injected into mice to enhance immunity. On the day of fusion, the mice were euthanized, and the mouse spleen cells were homogenized to obtain a single-cell suspension. Mouse spleen cells were fused (3:1) with mouse myeloma cell line SP2/0 using an electrofusion instrument. The fused cells were resuspended in medium containing HAT (hypoxanthine, aminopterin and thymidine, GIBCO, Cat: 21060016) to screen out the fusion tumor cells that successfully fused. The supernatant of fusion tumor cells was collected, and fusion tumor cells secreting antibodies that specifically bind to human CD47 were screened out through two rounds of ELISA. Subsequently, CD47-related functional screening experiments (such as binding specificity to human or cynomolgus CD47; no activity to promote erythrocyte agglutination; promotion of macrophage phagocytosis of tumor cells) were used to identify the activity of the fusion tumor secreted supernatant, and positive fusions Tumor strains undergo single or multiple rounds of sub-selection to obtain individual strains. After screening, 125G4A4 was the final selected fusion tumor strain.

The candidate fusion tumor cells 125G4A4 were expanded and cultured. After 7-10 days of culture, the supernatant was collected, centrifuged and filtered to remove cells and debris. Pass the supernatant through a Protein A purification column (GenScript). The equilibrium was then cleaned with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0), rinsed with 0.1M sodium citrate (pH 3.5), and immediately washed with one-ninth volume of 1M Tris-HCl (pH 9). ) and then dialyzed against PBS buffer. The fusion tumor antibody 125G4A4 was finally obtained for further characterization.

1.1 FACS detection of binding activity of antibodies to CHO-K1 cells overexpressing human CD47 protein

Human CD47 protein (NCBI accession number: NP_001768.1) was overexpressed in the hamster ovary cell line CHO-K1 to establish a CHO-K1 cell line overexpressing human CD47 protein. The cells were incubated with the serially diluted antibody 125G4A4 and the control antibody C0774CK230-C (i.e. Hu5F9) (the highest concentration was 300 nM, three times diluted, a total of 12 concentration points), and incubated at 4°C for 50 minutes. The cells were washed twice with ice-cold PBS, iFluor647-labeled goat anti-mouse IgG (H+L) antibody (Genscript) was added, and incubated at 4°C in the dark for 40 minutes. After the cells were washed twice with ice-cold PBS, the fluorescence signal was detected by a Calibur (BD Biosciences) flow cytometer, and a concentration-dependent curve was fitted with GraphPad based on its mean fluorescence intensity (MFI) and the EC ₅₀ was calculated. As shown in Table 1, the finally obtained fusion tumor antibody 125G4A4 has high binding activity to CHO-K1 overexpressing human CD47 protein, with an EC ₅₀ of 0.22nM.

1.2 FACS detection of antibody binding activity to CD47 on the surface of tumor cell lines

The human Burkitt lymphoma cell line Raji cells endogenously express human CD47 on the surface. The antibody 125G4A4 and the control antibody Hu5F9 were serially diluted into PBS containing 2% fetal bovine serum (FBS, Gibco, Cat: 10100147) (the highest concentration was 46.3nM, Three times dilution, a total of 8 concentration points), mix the diluted antibody with Raji cells (purchased from ATCC) (5*10 ⁵ cells per well), and incubate at 4°C for 1 hour. The cells were then washed three times with PBS containing 2% fetal bovine serum (FBS), PE-labeled mouse anti-human IgG Fc antibody (Biolegend, Cat: 409304) was added, and incubated at 4°C in the dark for 1 hour. After the cells were washed three times with PBS containing 2% fetal bovine serum (FBS), the fluorescence signal was detected by a CantoII (BD Biosciences) flow cytometer, and the concentration dependence was fitted using GraphPad according to its mean fluorescence intensity (MFI). curve and calculate EC ₅₀ . As shown in Table 1, fusion tumor antibody 125G4A4 has binding activity to Raji cells, with an EC ₅₀ of 0.84±0.02nM.

1.3 Anti-CD47 antibody blocks the interaction between human CD47 and SIRPα

The ELISA method was used to detect the ability of 125G4A4 to block the binding of human CD47 to SIRPα. Human CD47 extracellular segment and human IgG Fc-terminal recombinant protein hCD47-Fc (ACROBiosystems, Cat: CD7-H5256) were coated on a 96-well plate and incubated at 4°C overnight. After washing the plate three times with PBST (PBS containing 0.5% Tween-20), add PBST containing 1% BSA to block for 2 hours. After washing the plate three times with PBST, add serially diluted antibody 125G4A4 or control antibody Hu5F9 (the highest concentration is 66.7 nM, three times diluted, a total of 8 concentration points) and SIRPα-His recombinant protein (ACROBiosystems, 2016) with a final concentration of 2.5 μg/ml. Cat: SIA-5225) and incubate at room temperature for 1 hour. Wash the plate three times with PBST, and add horseradish peroxidase-labeled goat anti-His-tag secondary antibody (CWBIO, Cat: CW0285M) to detect SIRPα captured by coated CD47. After incubating the 96-well plate at 37°C for 30 minutes, wash the plate 5 times with PBST, add TMD (Surmodics, Cat: TMBW-1000-01) chromogenic solution, and incubate in the dark for 15 minutes. The color reaction was terminated by adding 2N H ₂ SO ₄ . Read the OD ₄₅₀ on a microplate reader. The absorbance value reflects the amount of SIRPα bound to CD47. Use Graphpad to fit a concentration-dependent curve and calculate the IC ₅₀ of anti-CD47 antibody blocking the binding of CD47 to SIRPα. As shown in Table 1, 125G4A4 can effectively block the interaction between CD47 and SIRPα, with an IC ₅₀ of 3.06nM.

1.4 Detection of anti-CD47 antibody-induced agglutination activity of human red blood cells

It is known in the prior art that most anti-CD47 antibodies have the property of inducing red blood cell agglutination. It is generally believed that this property is closely related to side effects such as anemia that occur during therapeutic anti-CD47 antibody treatment. To this end, we evaluated the anti-CD47 antibodies of the present invention through in vitro erythrocyte agglutination experiments to screen for antibodies without erythrocyte agglutination-promoting properties. The specific method is as follows. Collect fresh human blood from healthy donors. The blood was washed five times with PBS and diluted into a suspension containing 10% human red blood cells. The red blood cell suspension was mixed with experimental antibodies (antibody 125G4A4 and control antibody Hu5F9, the highest concentration was 667nM, three times diluted, a total of 12 concentration points) Then add it to a round-bottom 96-well plate, incubate at room temperature for 16 hours, take pictures, and judge the results based on the performance of the cells in the wells. If agglutination of red blood cells occurs, the cells will be spread out into a network, and a larger sheet-like cell layer will appear in the well, with a diameter larger than that of the negative control well; conversely, if agglutination does not occur, red blood cells will be deposited at the bottom of the well. , smaller dot-like cell clusters will precipitate in the wells. 125G4A4 did not have obvious erythrocyte agglutination-inducing properties in this experiment.

1.5 Detection of the effect of anti-CD47 antibodies on the phagocytosis of tumor cells by human macrophages

The flow cytometry-based assay method detects the ability of the antibody 125G4A4 of the present invention to promote macrophage phagocytosis of tumor cells. Fresh blood from healthy donors was collected, and peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Cat: 17-1440-02). Monocytes were further isolated using human total monocyte isolation kit (Miltenyi Biotec, Cat: 130-096-537), and macrophage colony-stimulating factor (M-CSF, R&D Systems, Cat: 216-MC) was added. The wall culture was continued for 7 days to induce differentiation of monocytes into macrophages. On the day of the phagocytosis test, the differentiated macrophages were starved in serum-free medium for 2 hours. At the same time, target tumor cells Raji were fluorescently labeled according to the labeling procedures recommended by the instructions of CFSE (eBioscience, Cat: 65-0850-85). Mix the labeled tumor cells and macrophages at a ratio of 4:1, and add the experimental antibody at the concentration to be tested and incubate at 37°C for 2 hours. The cells were then washed twice with PBS and digested with trypsin (Gibco, Cat: 25200072). APC-labeled anti-CD14 antibody (Biolegend, Cat: 325608) was added and placed on ice in PBS containing 2% fetal calf serum to protect from light. Incubate for 30 minutes. Cells were washed twice and analyzed by flow cytometry. Calculate the proportion of CFSE-positive cells in the CD14-positive macrophage population. As shown in Table 1, 125G4A4 can effectively promote the phagocytosis of tumor cells by macrophages.

Table 1 Activity and functional identification of fusion tumor antibody 125G4A4

Example 2 Humanization of fusion tumor antibodies

2.1 Determination of fusion tumor antibody variable region sequence

Using the fusion tumor sequencing method, the cells of the fusion tumor strain 125G4A4 were expanded and cultured, and total RNA was extracted using TRIzol (purchased from Ambio), and reverse transcribed into DNA using antibody-specific primers (Takara, PrimerScript 1 ^st Strand cDNA Synthesis Kit), and used antibody-specific primers to amplify the gene fragment encoding the mouse immunoglobulin V-region. Through sequence determination and analysis, the fusion tumor antibody variable region sequence, the heavy chain variable region and light chain variable region of the 125G4A4 antibody were obtained. The amino acid sequences of the chain variable regions are shown in SEQ.ID No.: 1 and 2 respectively, and the nucleotide sequences are shown in SEQ.ID No.: 19 and 20 respectively.

2.2 Construction and expression of chimeric antibodies

According to the mechanism of action of CD47, in specific embodiments of the present invention, the constant region of human IgG4 (S228P) is used as the heavy chain constant region of the antibody, and the human light chain kappa chain constant region is used as the antibody light chain constant region. Mutation of serine at position 228 of the IgG4 core hinge region to proline (S228P) can enhance the disulfide bonding of the core hinge region, reduce IgG4 Fab arm exchange, and greatly reduce the formation of half-molecules. After the heavy chain and light chain constant region genes were synthesized, the heavy chain and light chain variable region genes were digested with EcoRI and BamHI double enzyme digestion vector PTT5, and The source is recombined into the vector. After correct sequencing, the antibody heavy chain and light chain were co-transfected into HEK293 cells at a molar ratio of 1.5:1. After 120 hours of culture, the supernatant was collected by centrifugation and purified to obtain chimeric antibodies.

Before humanized antibody design, some post-translational modification (PTM) sites in the CDR region need to be mutated to avoid affecting the protein conformation and thus its function. After PTM analysis, two PTM sites were identified in the CDR of 125G4A4, including an NSS glycosylation site in the heavy chain and a DG isomerization site in the light chain. The NSS glycosylation site and DG isomerization site were mutated to QSS and EG, respectively. . The mutated chimeric antibody purified according to this example was named Ch-125G4-m35. The amino acid sequences of the heavy chain variable region and light chain variable region of the Ch-125G4-m35 antibody are shown in SEQ. ID No.: 3 and 4 respectively.

2.3 Humanized design of chimeric antibodies

The chimeric antibody 125G4A4m antibody variable region sequence was Blast compared with the PDB_Antibody database, and the human skeleton sequence with the highest similarity was selected for humanization: 125G4A4 m heavy chain variable region and human germline IGHV1-69, and the light chain can be The variable region has high sequence homology with human germline IGKV1-16. The Kabat assignment system is then used to define the amino acid sequence of the variable region CDR and its precise boundaries. Then, the CDR segment of the variable region of the mouse antibody is grafted into the human framework sequence to achieve humanization of the antibody.

In order to maintain the activity of the antibody, computer simulation technology can be used to analyze the variable region and its surrounding framework amino acid sequences using molecular docking to examine its three-dimensional binding mode. By calculating the electrostatic force, van der Waals force, hydrophobicity and entropy value, the key amino acid individuals in each candidate antibody gene sequence that can interact with CD47 and maintain the spatial structure are analyzed, and then grafted back to the selected human antibody gene framework. On this basis, the amino acid sites in the framework region that must be retained are marked to synthesize humanized antibodies. Some key sites in the antibody framework region need to be back mutated to the antibody framework region sequence of the chimeric antibody Ch-125G4-m35. According to reply The number and arrangement of mutations were used to design multiple different humanized heavy chain variable regions (SEQ.ID No.: 5, SEQ.ID No.: 6, SEQ.ID No.: 7) and light chain variable regions. area (SEQ.ID No.: 8, SEQ.ID No.: 9, SEQ.ID No.: 10) (see Table 2). The humanized antibody Hu-125G4A4-48 finally identified in the present invention was named HMA02h14-48 in subsequent experiments. The amino acid sequences of the heavy chain variable region and light chain variable region of the antibody are shown in SEQ. ID No.: 7 and 8 respectively.

Table 2 Number and arrangement of 125G4A4m heavy chain and light chain back mutations

Table 3 Amino acid sequences included in anti-CD47 antibodies

Table 4 CDR amino acid sequences contained in anti-CD47 antibodies (Kabat definition)

2.4 Expression of humanized antibodies

The DNA fragments of the humanized designed heavy chain and light chain variable regions were amplified and cloned into a vector containing the human antibody constant region to construct a plasmid expressing the antibody (pCDNA3.4, purchased from Thermo Cat# A14697) . The heavy chain and light chain expression vectors were co-transfected into Expire293 cells (Thermo Cat#A14525). After culturing at 37°C for 6 days, the supernatant was collected and purified by Protein A affinity according to the aforementioned method to obtain recombinant antibodies for antibody use. further characterization. Humanized antibodies are of the IgG4 S228P (IgG4P) subtype.

Example 3 Screening of humanized antibodies

Highly active humanized antibodies were screened by detecting the binding ability of humanized antibodies to cynomolgus monkey B cells, the ability of human macrophages to phagocytose tumor cells, and the ability to promote erythrocyte agglutination.

Detection of the binding ability of humanized antibodies to cynomolgus monkey B cells: Flow cytometry was used to detect the binding ability of 125G4A4 humanized series antibodies to CD47 on the surface of cynomolgus monkey B cells. The specific method is as follows: from cynomolgus monkey blood (provided by Shanghai Yinuosi Biotechnology Co., Ltd.), Ficoll-Paque PLUS (GE Healthcare, Cat: 17-1440-02) was used to separate peripheral blood by density gradient centrifugation. Peripheral Blood Mononuclear Cells (PBMCs). PBMC were incubated with 125G4A4 humanized series antibodies or isotype (IgG4P) in PBS containing 2% fetal calf serum for 30 minutes at 4°C. The cells were then washed three times and incubated with secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, Cat: 409304) in PBS containing 2% fetal calf serum for 30 minutes at 4°C in the dark. Cells were washed three times and analyzed by flow cytometry. B cells were labeled with an anti-human HER2 antibody cross-reactive with cynomolgus monkey (Brilliant Violet 421 ^TM labeled anti-human HER2 Antibody, Biolegend, Cat: 302330), and flow cytometry was performed on Canto II (BD Biosciences). Detect and obtain its mean fluorescence intensity (MFI).

The ability of macrophages to phagocytose tumor cells and the agglutination ability of red blood cells were detected according to the methods described in Examples 1.5 and 1.4 respectively.

The results are shown in Table 5. The 125G4A4 humanized series of antibodies combined with CD47 expressed on cynomolgus monkey B cells under the test concentration conditions and promoted the phagocytosis of tumor cells Raji by macrophages. Among them, Hu-125G4A4m-48 was at 33nM. The phagocytosis efficiency is the strongest, and other activities are similar to those of the chimeric antibody Ch-125G4m-m35, with a smaller number of back mutations. Therefore, it was selected for further testing, and subsequent testing was named HMA02h14-48.

Table 5 Anti-CD47 humanized antibody series antibody in vitro activity test

Example 4 FACS detection of the binding ability of HMA02h14-48 to tumor cells

Human Burkitt lymphoma cell line Raji cells (Shanghai Institute of Life Sciences, SIBS, CCL-86 ^TM /ATCC), human diffuse large cell lymphoma Toledo cells (ATCC® CRL-2631 ^TM )) and human mantle cell lymphoma REC -1 cells (ATCC® CRL-3004 ^TM ) endogenously express human CD47 on the surface. As described in the detection method described in Example 1.2, flow cytometry was used to detect the interaction between humanized antibody HMA02h14-48 and CD47 on the surface of the above tumor cells. combine. The highest antibody concentration was 667nM, with gradient dilution, and a total of 8 concentrations were tested.

The results are shown in Figure 1-3. Both HMA02h14-48 and Hu5F9 bind to CD47 expressed on the surface of tumor cells Raji, Toledo and REC-1 cells. The maximum fluorescence intensity of HMA02h14-48 when reaching the plateau phase is higher than that of Hu5F9. EC ₅₀ and maximum fluorescence intensity are shown in Table 6.

Table 6 Binding activity of antibody HMA02h14-48 to CD47 on tumor cell surface

The negative isotype control antibody (isotype) used in this example and other examples is human IgG4P, purchased from Shanghai Ruizhi Chemical Research Co., Ltd.

Example 5 Biacore detection of affinity between antibody HMA02h14-48 and human CD47

Biacore determines binding kinetic parameters by measuring Surface Plasmon Resonance (SPR). This technology detects the microscopic rate constants of the binding (ka) and dissociation (kd) of antibodies and antigens, and calculates the affinity values between antibodies and antigens. Biacore instrument (Biacore T200) and reagents were purchased from GE Healthcare. Anti-human Fc antibody was immobilized on sensor chip CM5. The purified antibodies (HMA02H14-48 and Hu5F9) were diluted in mobile phase buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween-20, pH 7.4), and flowed through the CM5 chip plated with anti-human Fc antibodies. Then, the gradient-diluted human CD47-His (ACROBiosystems, Cat: CD7-H5227) fusion protein flows through the detection chip to measure the binding of the antigen and the antibody, and then the mobile phase buffer flows through the chip to detect the dissociation of the antigen and the antibody. Collect the binding and dissociation signal data of antigen and antibody at different concentrations, and calculate the affinity of antigen and antibody through 1:1 Langmuir model fitting.

The results are shown in Table 7. HMA02h14-48 binds to human CD47 with high affinity, and the K _D value is 7.77E-10(M).

Table 7 Kinetic constants for Biacore detection of humanized antibodies binding to human CD47

Example 6 ELISA detection of the activity of HMA02h14-48 in blocking the interaction between human CD47 and SIRPα

As described in the aforementioned detection method in Example 1.3, ELISA was used to detect the ability of HMA02h14-48 to block the interaction between human CD47 and SIRPα. The highest antibody concentration was 67nM, with gradient dilution, and a total of 8 concentrations were tested.

The results are shown in Figure 4. The antibody HMA02h14-48 of the present invention blocks the interaction between human CD47 and SIRPα, with IC ₅₀ =1.58nM.

Example 7 Effect of HMA02h14-48 on the phagocytosis of tumor cells by human macrophages

According to the method described in Example 1.5, HMA02h14-48 was detected to promote the response of human macrophages to human Burkitt lymphoma cell line Raji cells, human diffuse large cell lymphoma Toledo cells, human mantle cell lymphoma REC-1 cells, and human progeroblastic lymphoma cells. Phagocytosis of myeloid leukemia cell line HL-60 cells. The highest antibody concentration was 100 μg/mL, with gradient dilution, and a total of 8 concentrations were tested.

The results show that compared with the control antibodies Hu5F9 and SRF231, HMA02h14-48 can promote the phagocytosis of human Burkitt lymphoma cell line Raji by macrophages, with the highest phagocytosis efficiency reaching 36.5%, at different concentrations of 0.1~100μg/ml. The phagocytosis rates were higher than those of Hu5F9 and SRF231. The highest phagocytosis efficiency of HMA02h14-48 for human mantle cell lymphoma REC-1 can reach 84.6%, and it can maintain the phagocytosis rate at about 70% at a low concentration of 0.1 μg/ml, which is higher than Hu5F9 and SRF231. HMA02h14-48 promotes the phagocytosis of Toledo cells by macrophages, and the phagocytosis rate can reach 94.2%. HMA02h14-48 can promote the phagocytosis of tumor cells HL-60 by macrophages, with the highest phagocytosis efficiency reaching 65%.

Table 8 Antibody HMA02h14-48 enhances the effect of human MΦ on phagocytosis of tumor cells

Example 8 Detection of the effect of HMA02h14-48 on the agglutination activity of red blood cells in vitro

Human red blood cells were diluted to 10% in PBS and incubated with instilled CD47 antibodies in a round-bottom 96-well plate at room temperature for 16 hours. The presence of unprecipitated red blood cells is evidence of blood cell aggregation. Compared with unaggregated red blood cells that precipitate to form white dots, the unprecipitated red blood cells appear in a reticular shape with an area larger than the negative isotype control antibody (see Figure 9). The test results of negative isotype control antibodies (Isotype) were used as normal standards.

The antibody HMA0214-48 was tested for promoting erythrocyte agglutination according to the method described in the aforementioned Example 1.4. The highest antibody concentration was 667nM, with gradient dilution, and a total of 12 concentrations were tested.

The results are shown in Figure 9. The CD47 antibody Hu5F9 shows that red blood cells will significantly agglutinate at a concentration equal to or higher than 0.9nM, while the antibody HMA02h14-48 of the present invention has no obvious agglutination of human red blood cells in vitro at different concentrations of 0.004 to 667nM. of agglutination.

Example 9 FACS detection of the binding ability of HMA02h14-48 to human red blood cells

It is known in the prior art that when therapeutic anti-CD47 antibodies are used clinically, the side effect of anemia often occurs. It is generally believed that anti-CD47 antibodies bind to CD47 on the surface of red blood cells, causing macrophages to engulf red blood cells, which may be another major cause of anemia. In the present invention, we use flow cytometry to detect the binding ability of HMA02h14-48 to human red blood cells to assess the risk of antibodies. Specifically, red blood cells derived from healthy donors were incubated with diluted HMA02h14-48 (the highest concentration was 667 nM, a total of 8 tested concentrations) in PBS containing 2% fetal calf serum for 30 minutes at 4°C. The cells were then washed three times and incubated with secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, Cat: 409304) in PBS containing 2% fetal calf serum for 30 minutes at 4°C in the dark. The cells were washed three times with PBS containing 2% fetal bovine serum (FBS), and the fluorescence signal was detected by a Canto II (BD Biosciences) flow cytometer, and based on its mean fluorescence intensity (MFI), GraphPad was used to Fit the concentration-dependent curves and calculate the _EC50 .

The results are shown in Figure 10. The maximum average fluorescence intensity of HMA02h14-48 binding to CD47 on the surface of human red blood cells is lower than that of the control antibody Hu5F9. Its EC ₅₀ and maximum average fluorescence intensity are shown in Table 9.

Table 9 Binding activity of antibody HMA02h14-48 to CD47 on the surface of human red blood cells

Example 10 Inhibition of Toledo tumor growth by humanized antibody HMA02h14-48

Purpose: To establish a Toledo subcutaneous tumor model on NOD-Scid mice and study the anti-tumor activity of the antibody of the present invention.

Methods: Human diffuse large cell lymphoma cells Toledo (ATCC® CRL-2631 ^TM ) were cultured in RPMI1640 medium containing 10% fetal calf serum. Tumor cells were suspended in RPMI1640 and implanted subcutaneously into the right flank of male NOD-Scid mice (Shanghai Lingchang Biotechnology Co., Ltd.) at a dose of 1×10 ⁷ cells/mouse.

Fifteen days after tumor cell inoculation, the mice were randomly divided into 6 groups according to tumor volume. Hu5F9 and HMA02h14-48 antibodies were diluted in PBS and administered at a dose of 10 mg/kg according to the schedule shown in Table 10. Negative isotype control antibody (isotype) IgG4P was purchased from Shanghai Ruizhi Chemical Research Co., Ltd.

Table 10. Hu5F9 and HMA02h14-48 dosage regimen

Tumor volume (tumor volume = 0.5 × long diameter × short diameter ² ) and mouse body weight were measured regularly. Student t test in Excel software was used to perform statistical analysis on tumor volume changes and body weight. p <0.05 was considered a significant statistical difference. The tumor regression rate of each antibody treatment group after administration was statistically calculated.

The calculation formula for the tumor regression rate in each treatment group is: [(D ₀ average tumor volume - _{D t} average tumor volume)/D ₀ average tumor volume] × 100%.

The relative weight calculation formula of mice is: (mouse weight on the day of measurement/mouse weight at the time of grouping) × 100%.

result:

The experimental results are shown in Table 11 and Figure 11.

The tumors in the Isotype control antibody group grew well, while in the other treatment groups, after administration of the antibodies, the subcutaneous tumors gradually shrank in size from the initial size until they completely disappeared. Among them, each dose group of Hu5F9 and HMA02h14-48 antibodies achieved complete tumor disappearance (regression rate 100%) when measured on the 11th day. Compared with the control antibody control group, the tumor volume changes were extremely significant. And after stopping the drug, the animals were continuously observed until the 67th day, and there was still no sign of tumor re-growth. In addition, the animals in each dose group of HMA02h14-48 were in good condition, and there was no significant difference in the weight of mice on the 21st day compared with before treatment. The body weight of the Hu5F9 high-dose group on day 21 was about 5% lower than that on day 0, but there was no statistical difference compared with the starting weight ( p >0.05); while there was no weight loss in the low-dose group, suggesting that The effect of Hu5F9 on body weight has a certain dose-effect relationship.

Based on the above data, both Hu5F9 and HMA02h14-48 antibody treatments have extremely significant anti-tumor effects. A single dose of 10 mg/kg can completely eliminate tumors and last for a long time.

Table 11 Effects of Hu5F9 and HMA02h014-48 on the growth of Toledo subcutaneous transplanted tumors

Example 11 Inhibition of REC-1 tumor growth by humanized antibody HMA02h14-48

Purpose: To establish a REC-1 subcutaneous tumor model on NOD-Scid mice and study the anti-tumor activity of the antibody of the present invention.

Methods: Human mantle cell lymphoma cells REC-1 (ATCC ^® CRL-3004 ^TM ) were cultured in RPMI1640 medium containing 10% fetal calf serum. Tumor cells were suspended in RPMI1640 and implanted subcutaneously into the right flank of male NOD-Scid mice (Shanghai Lingchang Biotechnology Co., Ltd.) at a dose of 5×10 ⁶ cells/mouse.

Eleven days after tumor cell inoculation, the mice were randomly divided into 5 groups according to tumor volume. Hu5F9 and HMA02h14-48 antibodies were diluted with PBS and administered according to the schedule shown in Table 12. Antibody Hu5F9 was prepared by GenScript, and antibody HMA02h14-48 was prepared according to the method of Example 2. Isotype control antibody (isotype) IgG4p was purchased from Shanghai Ruizhi Chemical Research Co., Ltd.

Table 12 Hu5F9 and HMA02h14-48 dosage regimen

Tumor volume (tumor volume = 0.5 × long diameter × short diameter ² ) and mouse body weight were measured regularly. The tumor inhibition rate and regression rate of the antibody treatment group on the 12th day after administration were calculated.

The calculation formula for the tumor inhibition rate is: [(average tumor volume change in the control group - average tumor volume change in the administration group)/average tumor volume change in the control group] × 100%. Student t test in Excel software was used to perform statistical analysis on tumor volume changes and body weight. p<0.05 was considered a significant statistical difference.

The calculation formula for the tumor regression rate in each treatment group is: [(D ₀ average tumor volume - _{D t} average tumor volume)/D ₀ average tumor volume] × 100%.

The relative weight calculation formula of mice is: (mouse weight on the day of measurement/mouse weight at the time of grouping) × 100%.

result:

The experimental results are shown in Table 13 and Figure 12.

Twelve days after administration, compared with the Isotype group, antibody Hu5F9 inhibited tumor growth by 16.7% at a single dose of 3 mg/kg (p>0.05); antibody HMA02h14-48 inhibited tumor growth at a single dose of 1 mg/kg. The inhibition rates of tumor growth at doses of kg, 3mg/kg and 10mg/kg were 3.8% (p>0.05), 54.7% (p<0.01) and 107.2% (p<0.001) respectively. Among them, the HMA02h14-48 antibody high-dose group achieved complete tumor disappearance (regression rate 100%) when measured on the 10th day. In addition, there was no significant difference in the relative body weight of mice in each antibody treatment group.

Based on the above data, in the REC-1 model, HMA02h14-48 antibody treatment is dose-dependent, and a single administration of 10 mg/kg can completely eliminate the tumor.

Table 13 Effects of Hu5F9 and HMA02h14-48 on the growth of REC-1 subcutaneous transplanted tumors

Example 12. Generation and production of anti-HER2/CD47 bispecific antibodies

Different bispecific antibodies that can specifically bind to both human CD47 and human HER2 antigens were prepared using the IgG-scFv (2+2) structure or the Crossmab (1+1) structure.

Bispecific antibody with IgG-scFv(2+2) structure

In the present invention, BsAb1, BsAb2, BsAb3, BsAb4 and BsAb6 are examples of bispecific antibodies having an IgG-scFv (2+2) structure. Two scFv fragments that can specifically bind to human CD47 are connected to the C-termini of the two heavy chains of the whole antibody that can specifically bind to human HER2 via peptide linkers. Each molecule in the bispecific antibody is composed of four polypeptide chains, two of which are identical and have the structure VH (anti-HER2)-CH1-Fc domain subunit-peptide linker-scFv fragment (anti-CD47 ), the other two polypeptide chains are identical and have the structure VL (anti-HER2)-CL. As used herein, VH (anti-HER2) and VL (anti-HER2) refer to the heavy and light chain variable domains, respectively, of the antigen-binding domain capable of specifically binding human HER2; scFv fragment (anti-CD47) refers to scFv fragment that can specifically bind to the antigen-binding domain of human CD47, CL refers to the light chain constant region; "VL-CL" refers to the light chain (LC), and "VH-CH1-Fc domain subunit" refers to the heavy chain Chain (HC).

Crossmab(1+1) structure bispecific antibody

The crossmab (1+1) structure anti-HER2/CD47 bispecific antibody (eg, BsAb5) is an IgG type with a Fab fragment that can specifically bind hCD47 and a Fab fragment that can specifically bind hHER2.

In order to generate bispecific antibodies with a Crossmab (1+1) structure, knob-into-hole technology was used to achieve heterodimerization. The S354C/T366W mutation was introduced into one heavy chain (Fc knob heavy chain), and the Y349C/T366S/L368A/Y407V mutation was introduced into the other heavy chain (Fc hole heavy chain). Furthermore, CrossFab technology as described in WO 2009/080252 A1 ensures correct light chain pairing. Specifically, the CH1 region of the Fab arm capable of specifically binding hHER2 is replaced with the light chain constant region (CL); and The light chain variable region of the Fab arm is linked to the heavy chain CH1 region. Therefore, the crossmab (1+1) structure bispecific antibody is composed of four different polypeptide chains. Taking BsAb5 as an example, the first polypeptide chain structure is VH (anti-CD47)-CL-Fc domain (Y349C/T366S/L368A/Y407V), and the second polypeptide chain structure is VL (anti-CD47)-CH1. The third polypeptide chain structure is VH (anti-HER2)-CH1-Fc domain (S354C/T366W), and the fourth polypeptide chain structure is VL (anti-HER2)-CL.

In all these constructs, the antigen-binding domain that specifically binds human CD47 is derived from HMA02h14-48 (hereinafter referred to as mAb1), and the antigen-binding domain that specifically binds human HER2 is derived from ^Herceptin® ( ^Herceptin® , Named mAb2, the heavy chain amino acid sequence is shown in SEQ ID NO: 40, and the heavy chain amino acid sequence is shown in SEQ ID NO: 43).

BsAb-ref is the reference antibody of IgG-scFv(2+2) structure. The only difference between BsAb-ref and BsAb1 is the variable region of the Fab fragment. In BsAb-ref, the variable region of the Fab fragment is derived from Idarucizumab, a monoclonal antibody fragment (Fab) that specifically binds to Dabigatran (CAS ID: 211915-06-9) with high affinity. ), INN Recommended 2014,17).

The amino acid sequences of exemplary antibodies are shown in Tables 14-16 below.

Expression and purification

Molecular biology methods were used to construct recombinant expression vectors. Briefly, the DNA sequence encoding each polypeptide chain of the antibody is cloned into the ORF (open reading frame) of a suitable expression vector. Recombinant expression vectors each containing a coding DNA sequence were obtained and used for subsequent expression after verification by sequencing. Antibodies were expressed by transiently transfecting Expi293 cells with recombinant expression vectors encoding different peptide chains of each antibody. After culturing the cells for 5-7 days, refer to the standard protocol and use protein A affinity chromatography to purify the antibody from the cell culture supernatant, followed by a size exclusion chromatography step.

Table 14: Amino acid sequence of the antigen-binding domain of bispecific antibody (BsAb1-6)

Table 15: Amino acid sequence of bispecific antibody (BsAb1-4, 6) with IgG-scFv (2+2) structure

Table 16: Amino acid sequence of bispecific antibody (BsAb5) with Crossmab (1+1) structure

Example 13 Detection of the binding activity of anti-HER2/CD47 bispecific antibodies to CD47 protein by ELISA experiment

The binding activity of anti-HER2/CD47 bispecific antibody to human CD47 was detected by ELISA experiment. His-tagged recombinant human CD47 protein (ACRO Biosystems, Cat: CD47-H5227) was coated onto a 96-well plate and incubated overnight at 4°C. After washing the plate three times with PBST (PBS containing 0.5% Tween-20), add PBST containing 1% BSA to block the plate for 2 hours. After washing the plate three times with PBST, add serially diluted antibodies and incubate at room temperature for 1 hour. Wash the plate three times with PBST, and add horseradish peroxidase-labeled goat anti-human Fc secondary antibody (Sigma, Cat: A0170-1ML) to detect the Antibody coated with human CD47 protein. After incubating the 96-well plate at 37°C for 30 minutes, wash the plate 5 times with PBST, add TMD (Surmodics, Cat: TMBW-1000-01) chromogenic solution, incubate in the dark for 15 minutes to develop color, and add 2N H2SO4 to terminate the development. color reaction. Read OD450 on a microplate reader. The absorbance value reflects the amount of antibody bound to human CD47 protein. As shown in Figure 13, the anti-HER2/CD47 bispecific antibody can effectively bind to human CD47 protein.

Example 14 Detection of binding activity of anti-HER2/CD47 bispecific antibody to HER2 protein by ELISA experiment

The binding activity of anti-HER2/CD47 bispecific antibodies to human HER2 was detected by ELISA assay. His-tagged recombinant HER2/ERBB2 protein (Sino Biological, Cat: 10004-H08H) was coated onto a 96-well plate and incubated at 4°C overnight. After washing the plate three times with PBST (PBS containing 0.5% Tween-20), add PBST containing 1% BSA to block the plate for 2 hours. After washing the plate three times with PBST, add serially diluted antibodies and incubate at room temperature for 1 hour. The plate was washed three times with PBST, and horseradish peroxidase-labeled goat anti-human Fc secondary antibody (Sigma, Cat: A0170-1ML) was added to detect the antibody captured by the coated HER2 protein. After incubating the 96-well plate at 37°C for 30 minutes, wash the plate 5 times with PBST, add TMD (Surmodics, Cat: TMBW-1000-01) chromogenic solution, and incubate in the dark for 15 minutes. 2N H2SO4 was added to terminate the color reaction. Read OD450 on a microplate reader. The absorbance value reflects the amount of antibody bound to human HER2 protein. As shown in Figure 14, the anti-HER2/CD47 bispecific antibody can effectively bind to human HER2 protein.

Example 15 Experiment on the binding activity of anti-HER2/CD47 bispecific antibodies to CHO-K1 cells overexpressing human CD47 by FACS

Human CD47 protein (NCBI accession number: NP_001768.1) was overexpressed in the hamster ovary cell line CHO-K1 to establish a CHO-K1 cell line overexpressing human CD47 protein. Place cells with ladder Antibodies diluted to 66.7 nM (maximum concentration, three times diluted, 8 concentration points in total) were incubated for 60 minutes at 4°C. After washing twice with cold PBS, cells were incubated with anti-human Fc-tagged IgG PE (Biolegend) for 45 min in the dark at 4°C. The cells were washed three times with cold PBS, and then the fluorescent signal was detected by FACS Canto II (BD Biosciences). According to the mean fluorescence intensity (MFI) of the signal, a concentration-dependent curve was fitted with GraphPad, and the EC50 was calculated. The maximum fluorescence intensity and EC50 are shown in Table 17.

Table 17 Binding activity of BsAb4 to CD47 CHO-K1 cells

The negative isotype control antibody (isotype) used in this example was human IgG1 (Cat# 403502), purchased from Biolegend.

Example 16 Experiment on the binding activity of anti-HER2/CD47 bispecific antibodies to tumor cells expressing human CD47 and HER2 by FACS

Human CD47 and HER2 are endogenously expressed on the surface of human esophageal/gastric cancer cell line OE19 cells (96071721/ECACC). According to the detection method described in the aforementioned Example 14, flow cytometry was used to detect the binding activity of BsAb4 and control antibodies to the above tumor cell lines. The antibody was serially diluted with the highest antibody concentration of 100 nM, and a total of 8 concentration points were tested.

As shown in Figure 16, BsAb4, BsAb5 and BsAb 6 have binding activity to OE19 tumor cells. The maximum fluorescence intensity and EC50 are shown in Table 18.

Table 18 Binding activity of BsAbs to OE19 tumor cells

The negative isotype control antibody (isotype) used in this example was human hIgG1 (Biolegend Cat# 403502) and human IgG4P was purchased from Chempartner.

Example 17 Testing the activity of anti-HER2/CD47 bispecific antibodies in blocking the interaction between human CD47 and SIRPα

The PathHunter® Jurkat SIRPα signaling assay was used to verify the activity of anti-HER2/CD47 bispecific antibodies in blocking the interaction between human CD47 and SIRPα. The assay utilizes Jurkat cells engineered to co-express ProLink ^™ (protein kinase PK)-tagged SIRPα (receptor) and enzyme receptor (EA)-tagged SH2 domains. When SIRPα (receptor) binds to its ligand CD47 (ligand), SIRPα is activated and phosphorylated, and then recruits SH2-EA, ultimately promoting the complementation of the two β-galactosidase fragments (EA and PK). And the functional enzyme produced hydrolyzes the substrate to produce a chemiluminescent signal. Following the manufacturer's recommended protocol, OE19 cells expressing high levels of HER2 and CD47, or CHO-K1/CD47 overexpressing cells expressing only high levels of CD47, were incubated with BsAb4 and control antibodies (maximum concentration 100 nM, 3x dilution, total 10 concentration points) in an incubator at 37°C and 5% CO2 for 1 hour. Jurkat SIRPα-expressing cells (Eurofins, Cat: 93-1135C19) were then added and incubated for 5 hours in an incubator at 37°C and 5% CO2. After incubation, a chemiluminescent signal was generated using the Bioassay PathHunter Bioassay Detection Kit (Eurofins, Cat: 93-0933). Read the chemiluminescence signal using Envision. Use GraphPad to fit concentration-dependent curves and calculate IC50. As shown in Figure 17, both BsAb4 and mAbl could effectively block the interaction between CD47 (expressed on OE19) and SIRPα (on Jurkat SIRPα-expressing cells) and demonstrated equivalent efficiency. As shown in Figure 18, BsAb4 showed a significant decrease in blocking activity compared to mAb1 in CHO-K1/CD47 OE cells. The above experiments show that BsAb4 maintains similar blocking activity to anti-CD47 mAb in HER2 ⁺ CD47 ⁺ tumor cells, but loses part of the blocking activity in CD47 ⁺ cells.

Example 18 Anti-HER2/CD47 bispecific antibody promotes the phagocytosis of tumor cells by human macrophages

By using flow cytometry, the effect of anti-HER2/CD47 bispecific antibodies in promoting macrophage phagocytosis of tumor cells was detected. Human fresh blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Cat: 17-1440-02). Human total monocyte isolation kit (Miltenyi biotec, cat: 130-096-537) was used for further isolation to obtain monocytes. In order to induce the differentiation of monocytes into macrophages, macrophage colony-stimulating factor (M-CSF, R&D Systems, Cat: 216-MC) was added to the monocytes and cultured continuously for 7 days. On the day of the cell phagocytosis experiment, the above-mentioned macrophages were first starved in serum-free medium for 2 hours. At the same time, target tumor cells OE19 were fluorescently labeled using CFSE (eBioscience, Cat: 65-0850-85) according to the steps recommended in the instruction manual. Mix CFSE-labeled tumor cells and starved macrophages at a ratio of 4:1, add the test antibody at the detection concentration, and incubate at 37°C for 2 hours. Cells were then washed twice with PBS and then digested with trypsin (Gibco, Cat: 25200072); APC-labeled anti-CD14 antibody (Biolegend, Cat: 325608) was added and kept on ice in PBS containing 2% fetal calf serum. Incubate in the dark for 30 minutes. Cells were washed twice and analyzed by flow cytometry. The percentage of CFSE-positive cells within the CD14-positive macrophage population was calculated. As shown in Figure 19, compared with anti-HER2 (mAb2), BsAb4, BsAb5 and BsAb6 can effectively promote the phagocytosis of OE19 cells by macrophages.

Example 19 Detection of the activity of anti-HER2/CD47 bispecific antibody in inducing agglutination reaction of human red blood cells

As described in Example 1.4, it is generally accepted that anti-CD47 antibodies can cause clinical side effects, such as treatment-emergent anemia. Therefore, we evaluated the bispecific antibodies of the present invention through in vitro erythrocyte agglutination assays to identify antibodies that do not have erythrocyte agglutination-inducing properties. Here’s how: Collect health volunteers Fresh blood of the patient, washed the cells five times with PBS, and then diluted the cells into a suspension containing 10% human red blood cells; the red blood cell suspension was mixed with the antibodies to be tested (BsAb4, BsAb5 and control antibody, Hu5F9) as a positive control, the highest concentration was 100nM, triple dilution, 10 concentration points in total), mix, and then add the mixture into a round-bottom 96-well plate; incubate at room temperature for 16 hours, then take pictures, and determine the results based on the cell phenomenon in the well. If red blood cell agglutination occurs, cells are spread over each well like a net, and a larger sheet-like cell layer will appear in the well, with a diameter larger than that of the negative control well; on the contrary, if hemagglutination does not occur, red blood cells will be deposited at the bottom of the well. , smaller dot-like cell clumps will appear in the well. BsAb4, BsAb5 and BsAb6 did not show obvious induction of red blood cell agglutination in the experiment (Fig. 20).

Example 20 Detection of binding activity of anti-HER2/CD47 bispecific antibodies to human red blood cells by FACS

As a background as described in Example 9, flow cytometry was therefore performed to detect the binding activity of anti-HER2/CD47 bispecific antibodies to human red blood cells. Briefly, red blood cells from healthy volunteers were incubated with serially diluted antibodies (maximum concentration of 100 nM, 8 test concentration points in total) in PBS containing 2% fetal calf serum for 30 min at 4°C. Cells were then washed three times and incubated with secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, Cat: 409304) in PBS containing 2% fetal calf serum for 30 min at 4°C in the dark. The cells were washed three times with PBS containing 2% fetal bovine serum (FBS), and then the fluorescent signal was detected using a Canto II (BD Biosciences) flow cytometer. According to the mean fluorescence intensity (MFI), use GraphPad to fit the concentration-dependent curve and calculate the EC50.

As shown in Figure 21, the maximum average fluorescence intensity of BsAb4 and BsAb6 was as low as the IgG control, indicating that their binding to human red blood cells was undetectable by FACS.

Example 21 Inhibition of OE19 tumor growth by anti-HER2/CD47 bispecific antibodies

Objective 1: To evaluate the anti-tumor activity of BsAb4 in the OE19 subcutaneous tumor model in NOD SCID mice.

Methods: Human gastroesophageal adenocarcinoma cells OE19 (ECACC® 96071721) were cultured and expanded in RPMI 1640 medium containing 10% fetal calf serum. Tumor cells in the exponential growth phase were collected, mixed with an equal volume of Matrigel, and subcutaneously implanted into NOD SCID mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) at 5 × 10 ⁶ cells per mouse. Animal Technology Co,Ltd.))'s right armpit subcutaneous area.

Mice were randomly grouped according to tumor volume. Dilute the human IgG control antibody in physiological saline. The control antibody mAb2 was purchased from Roche Commercial Antibody and diluted with physiological saline. BsAb4 was diluted with 50mM sodium citrate and 150mM NaCl pH 6.1 solution. Mice were injected with antibodies according to the schedule shown in Table 19.

Table 19. Dosage regimens for mAb2 and BsAb4

Data analysis:

Tumor volume (TV)=0.5×long diameter×short diameter ² . Data are expressed as mean ± standard deviation (SD).

Tumor growth inhibition rate (TGI) = [1-(TV _t -TV ₀ ) _{drug treatment} /(TV _t -TV ₀ ) _human IgG control] × 100%. (TVt: tumor volume on day t after treatment; TV _0: initial tumor volume before treatment.)

If the mean tumor volume after antibody treatment is lower than the mean tumor volume before treatment, the tumor reduction rate (TRR) will be calculated.

TRR=(TV ₀ -TV _t )/TV ₀ ×100%

Complete tumor response (CR) was defined as the absence of palpable tumor for measurement after treatment. Tumor complete response rate (CRR) = (number of CR mice on the day of measurement/total number of mice on the day of measurement) × 100%

The Student t-test was used to analyze the comparison of tumor volume changes through Excel software, where p<0.05 indicated a significant statistical difference between the two groups.

result:

On day 22, the tumor growth inhibition rates (TGI) of mAb2 (10 mg/kg) and BsAb4 were 90.7% and 101.5%, respectively. Compared to the human IgG control, mAb2 and BsAb4 showed significant anti-tumor effects (p<0.01, Table 20 and Figure 22). In addition, by day 22, 13.3 mg/kg BsAb4 treatment caused tumor shrinkage in 3/5 mice, with an average TRR of 50% and a CRR of 60%. The three CR mice were observed until day 66 and still maintained CR status.

Therefore, 13.3 mg/kg of BsAb4 showed potent anti-tumor effects in the OE19 subcutaneous tumor model.

Table 20. Effect of BsAb4 on tumor growth of OE19 subcutaneous tumor model in NOD SCID mice

Objective 2: To evaluate the anti-tumor activity of BsAb6 in the OE19 subcutaneous tumor model in nude mice.

Methods: Human gastroesophageal adenocarcinoma cells OE19 (ECACC® 96071721) were cultured and expanded in RPMI 1640 medium containing 10% fetal calf serum. Tumor cells in the exponential growth phase were collected, mixed with an equal volume of Matrigel, and 5 × 10 ⁶ cells per mouse were subcutaneously implanted into nude mice (LC Laboratory Animal Technology Co, Ltd.) ) under the skin of the right armpit.

Mice were randomly grouped according to tumor volume. Human IgG control antibody was diluted in PBS (pH 7.0). The control antibody mAb2 was purchased from Roche Commercial Antibody and diluted with physiological saline. BsAb6 was diluted with 50mM sodium citrate and 150mM NaCl pH 6.1 solution. Mice were injected with antibodies according to the schedule shown in Table 21.

Table 21. Dosage regimens for mAb2 and BsAb6

Data analysis:

Tumor volume (TV)=0.5×long diameter×short diameter ² . Data are expressed as mean ± standard deviation (SD).

Tumor growth inhibition rate (TGI) = [1-(TV _t -TV ₀ ) _{drug treatment} /(TV _t -TV ₀ ) _{human IgG control} ] × 100%.

(TVt: tumor volume on day t after treatment, _TV0 : initial tumor volume before treatment.)

If the mean tumor volume after antibody treatment is lower than the mean tumor volume before treatment, the tumor reduction rate (TRR) will be calculated.

TRR=(TV ₀ -TV _t )/TV ₀ ×100%

Complete tumor response (CR) was defined as the absence of palpable tumor for measurement after treatment. Tumor complete response rate (CRR) = (number of CR mice on the day of measurement/total number of mice on the day of measurement) × 100%

The comparison of tumor volume changes was analyzed using Excel software using Student's t test, where p<0.05 indicated a significant statistical difference between the two groups.

result:

On day 19, the tumor growth inhibition rate (TGI) of mAb2 (10 mg/kg) was 75.8%, and the TGI of BsAb6 was 104.5%, showing the significant anti-tumor activity of BsAb6 (p<0.01, Table 22 and Figure 23). to 19th 13.3 mg/kg BsAb6 per day caused tumor shrinkage in 6/7 mice, with an average TRR of 41.5% and a CRR of 14.3%.

Therefore, 13.3 mg/kg of BsAb6 showed potent anti-tumor effects in the OE19 subcutaneous tumor model.

Table 22. Effect of BsAb6 on tumor growth of OE19 subcutaneous tumor model in nude mice

Sequence of the invention:

TW202330627A_111138266_SEQL.xml

Claims (40)

A bispecific antibody containing (a) at least one antigen-binding domain capable of specifically binding CD47, and (b) At least one antigen-binding domain capable of specifically binding to HER2, wherein the antigen-binding domain capable of specifically binding to CD47 comprises VH and VL, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises: comprising HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11, HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12, and HCDR2 comprising or consisting of SEQ ID NO: 13 or The amino acid sequence shown in SEQ ID NO: 14 or HCDR3 consisting of it; and the VL capable of specifically binding to the antigen-binding domain of CD47 includes: comprising the amino acid sequence shown in SEQ ID NO: 14 or consisting of it LCDR1 composed of, LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 15 or 18 or 22, and LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16 . The bispecific antibody of claim 1, wherein the VH capable of specifically binding the antigen-binding domain of CD47 includes: an HCDR1 that includes or consists of the amino acid sequence shown in SEQ ID NO: 11, including An HCDR2 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 12, and an HCDR3 comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 17; and the HCDR3 capable of specifically binding to CD47 The VL of the antigen-binding domain includes: LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14, LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16. The bispecific antibody of claim 1 or 2, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 comprises an amine as shown in SEQ ID NO: 1, 3, 5, 6 or 7 The amino acid sequence has or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, And the VL capable of specifically binding the antigen-binding domain of CD47 includes at least 90%, 91%, 92%, or 93% of the amino acid sequence shown in SEQ ID NO: 2, 4, 8, 9 or 10. , or consist of an amino acid sequence with 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. The bispecific antibody according to any one of claims 1 to 3, wherein the VH and VL of the antigen-binding domain capable of specifically binding to CD47 are selected from (1) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2; (2) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4; (3) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10; (4) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 6, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9 or 10; or (5) VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, and VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, 9 or 10. The bispecific antibody according to any one of claims 1 to 4, wherein the VH of the antigen-binding domain capable of specifically binding to CD47 includes or consists of the amino acid sequence shown in SEQ ID NO: 7 , and the VL capable of specifically binding the antigen-binding domain of CD47 includes or consists of the amino acid sequence shown in SEQ ID NO: 8. The bispecific antibody according to any one of claims 1 to 5, wherein the antigen-binding domain capable of specifically binding HER2 includes VH and VL, wherein The VH of the antigen-binding domain capable of specifically binding to HER2 includes: HCDR1 that includes or consists of the amino acid sequence shown in SEQ ID NO: 23, and includes the amino acid sequence shown in SEQ ID NO: 24 or HCDR2 consisting of it, and HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 25; and the VL capable of specifically binding the antigen-binding domain of HER2 comprises: comprising SEQ ID NO : The amino acid sequence shown in SEQ ID NO: 26 or LCDR1 consisting of it, the amino acid sequence shown in SEQ ID NO: 27 or LCDR2 consisting of it, and the amino group shown in SEQ ID NO: 28 acid sequence or LCDR3 consisting of it. The bispecific antibody as described in claim 6, wherein the VH of the antigen-binding domain capable of specifically binding to HER2 comprises at least 90%, 91%, and 92 of the amino acid sequence shown in SEQ ID NO: 29 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of the amino acid sequence or consisting of it, and the antigen-binding domain capable of specifically binding to HER2 The VL comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence shown in SEQ ID NO: 30 % sequence identity of an amino acid sequence or consisting of it. The bispecific antibody according to any one of claims 1 to 7, wherein the bispecific antibody comprises (a) at least one antigen-binding domain comprising VH and VL capable of specifically binding CD47, wherein The VH of the antigen-binding domain capable of specifically binding to CD47 includes at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity, and the VL capable of specifically binding the antigen-binding domain of CD47 comprises SEQ ID NO: 2, The amino acid sequence represented by 4, 8, 9 or 10 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or consisting of a sexual amino acid sequence, and (b) at least one antigen-binding domain comprising VH and VL capable of specifically binding HER2, wherein The VH of the antigen-binding domain capable of specifically binding to HER2 contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 29 , or consisting of an amino acid sequence with 97%, 98%, 99% or 100% sequence identity, and the VL of the antigen-binding domain capable of specifically binding to HER2 includes the same as shown in SEQ ID NO: 30 Amino acid sequences having or consisting of amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity . The bispecific antibody according to any one of claims 1 to 8, wherein the antigen-binding domain is chimeric, fully human or humanized. The bispecific antibody according to any one of claims 1 to 9, wherein the antigen-binding domain comprises a Fab fragment or a scFv fragment. The bispecific antibody according to any one of claims 1 to 10, wherein the antigen-binding domain capable of specifically binding HER2 comprises a Fab fragment, and the antigen-binding domain capable of specifically binding CD47 comprises a scFv fragment or Fab fragment. The bispecific antibody of claim 10 or 11, wherein the scFv fragment includes a peptide linker connecting the C-terminus of VH to the N-terminus of VL. The bispecific antibody according to any one of claims 1 to 12, wherein the bispecific antibody is bivalent, trivalent or tetravalent. The bispecific antibody according to any one of claims 1 to 13, wherein the bispecific antibody comprises (a) The first Fab fragment capable of specifically binding to CD47, (b) a second Fab fragment capable of specifically binding to HER2, and (c) An Fc domain consisting of a first subunit and a second subunit capable of stable association. The bispecific antibody of claim 14, wherein the first Fab fragment is crossFab (CL-CH1). The bispecific antibody as described in claim 1 or 15, wherein the bispecific antibody comprises (i) The first polypeptide chain has the structure: VH (anti-CD47)-CL-Fc domain subunit, (ii) The second polypeptide chain has the structure: VL(anti-CD47)-CH1, (iii) The third polypeptide chain has the structure: VH (anti-HER2)-CH1-Fc domain subunit, and (iv) The fourth polypeptide chain has the structure: VL(anti-HER2)-CL. The bispecific antibody according to any one of claims 1 to 16, wherein the bispecific antibody comprises (i) A first polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity, (ii) a second polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity, (iii) A third polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consisting of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity, and (iv) a fourth polypeptide chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, or consists of an amino acid sequence with 96%, 97%, 98%, 99% or 100% sequence identity. The bispecific antibody according to any one of claims 1 to 13, wherein the bispecific antibody comprises (a) Two scFv fragments capable of specifically binding to CD47, and (b) two heavy chains and two light chains of a whole antibody comprising two Fab fragments and an Fc domain capable of specifically binding to HER2, wherein the two scFv fragments are each linked to the whole antibody. The bispecific antibody of claim 18, wherein each of the two scFv fragments is connected to the whole antibody through a peptide linker. The bispecific antibody of claim 18 or 19, wherein the two scFv fragments are each connected to the C-termini of the two heavy chains of the whole antibody through a peptide linker. The bispecific antibody of any one of claims 12, 19 to 20, wherein the peptide linker comprises a GS linker. The bispecific antibody according to any one of claims 12, 19 to 21, wherein the peptide linker comprises the amino acid sequence shown in SEQ ID NO: 31, 32, 33, 34, 35, 36 or 37 . The bispecific antibody according to any one of claims 18 to 22, wherein the scFv fragment comprises or consists of the amino acid sequence shown in SEQ ID NO: 38 or 39. The bispecific antibody of any one of claims 18 to 23, wherein the bispecific antibody comprises two heavy chains and two light chains, each of the heavy chains comprising SEQ ID NO: 40 , the amino acid sequence represented by 41, 42 or 63 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or consisting of an amino acid sequence, and each of the light chains comprises at least 90%, 91%, 92%, 93%, 94%, or consisting of an amino acid sequence with 95%, 96%, 97%, 98%, 99% or 100% sequence identity. The bispecific antibody according to any one of claims 1 to 13, wherein the bispecific antibody comprises an Fc domain. The bispecific antibody of any one of claims 14 to 25, wherein the Fc domain is a human IgG1 Fc domain. The bispecific antibody of any one of claims 14 to 26, wherein the Fc domain comprises: one or more amino acid modifications that promote association of the first subunit and the second subunit of the Fc domain , and/or one or more amino acid modifications that increase the binding affinity of the antibody to FcRn. The bispecific antibody of any one of claims 14 to 27, wherein the Fc domain comprises one or more amino acid substitutions at positions 428 and 434, preferably, the amino acid substitution is M428L and N434S (numbered according to Kabat EU index). The bispecific antibody of any one of claims 14 to 28, wherein each of the two subunits of the Fc domain comprises an amine as shown in SEQ ID NO: 53, 54, 55 or 64 The amino acid sequence has or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. An isolated nucleic acid encoding the bispecific antibody of any one of claims 1 to 29. A recombinant vector or expression vector comprising one or more nucleic acids as described in claim 30, wherein the vector is suitable for the recombinant production of the bispecific antibody as described in any one of claims 1 to 29. A host cell comprising one or more recombinant vectors or expression vectors as described in claim 31. An immunoconjugate comprising the bispecific antibody of any one of claims 1 to 29. A pharmaceutical composition comprising the bispecific antibody as described in any one of claims 1 to 29, the nucleic acid as described in claim 30, the vector as described in claim 31, or the vector as described in claim 32 Host cells or immunoconjugates as described in claim 33, and optionally containing pharmaceutically acceptable excipients. A method for treating or preventing a disease or disorder, the method comprising administering to an individual an effective amount of a bispecific antibody as described in any one of claims 1 to 29, a nucleic acid as described in claim 30, as claimed The vector described in claim 31, the host cell described in claim 32, the immunoconjugate described in claim 33, or the pharmaceutical composition described in claim 34. The method of claim 35, wherein the disease or disorder is CD47-related and/or HER2-related. The method of claim 35 or 36, wherein the disease or condition is cancer. The method of claim 37, wherein the cancer is a blood cancer, optionally selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (e.g., Burkholderia lymphoblastic leukemia/lymphoma, B-cell chronic lymphocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), follicular lymphoma, small lymphocytic leukemia (SLL) , central nervous system (CNS) lymphoma, Richter's Syndrome, multiple myeloma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, and anaplastic large cell lymphoma . The method of claim 38, wherein the cancer is a solid tumor, optionally selected from the group consisting of breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, head and neck squamous cell carcinoma, ovarian cancer, prostate cancer , pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, esophagogastric junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing sarcoma, rhabdomyosarcoma, or neuroblastoma . A bispecific antibody as described in any one of claims 1 to 29, a nucleic acid as described in claim 30, a vector as described in claim 31, a host cell as described in claim 32, or as requested Use of the immunoconjugate described in claim 33 or the pharmaceutical composition described in claim 34 in the preparation of a medicament for treating a disease or disorder in a subject.

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