CN116462764A

CN116462764A - Multispecific antibodies targeting CD47 and HER2 and uses thereof

Info

Publication number: CN116462764A
Application number: CN202310071717.4A
Authority: CN
Inventors: 金磊; 赵然然; 高云娜; 李美含; 胡辉; 谭忠月; 叶翔赟; 王涛
Original assignee: Changchun Genescience Pharmaceutical Co Ltd
Current assignee: Changchun Genescience Pharmaceutical Co Ltd
Priority date: 2022-01-19
Filing date: 2023-01-18
Publication date: 2023-07-21

Abstract

The invention belongs to the field of antibody medicines, and in particular relates to a multi-specific antibody targeting CD47 and HER2, a pharmaceutical composition containing the multi-specific antibody and application of the multi-specific antibody in tumor treatment.

Description

Multispecific antibodies targeting CD47 and HER2 and uses thereof

Technical Field

Background

HER2, also known as ErbB2. It belongs to the HER subfamily of the type I receptor tyrosine kinase family, which also includes 3 members of HER1 (ErbB 1 or EGFR), HER3 (ErbB 3), and HER4 (ErbB 4). HER subfamily members can form homodimers and heterodimers, HER2 being the strongest dimerization partner of other ErbB receptors. HER2 activation leads to receptor phosphorylation, and can trigger downstream signaling cascade amplification through multiple signaling pathways such as MAPK, PI3K/AKT, JAK/STAT, PKC, and the like, thereby playing an important role in regulating proliferation, differentiation, development, adhesion, and migration of cells. HER2 has over-expression in 10% -34% of various cancers such as breast cancer, ovarian cancer, gastrointestinal cancer and lung cancer, and the HER2 gene amplification has correlation with the total survival and recurrence time of breast cancer patients. At present, trastuzumab (Trastuzumab) and Pertuzumab (Pertuzumab) are commercially available primary HER 2-targeted therapeutic antibodies, trastuzumab recognizes the HER2 extracellular IV domain, pertuzumab recognizes the HER2 extracellular domain II heterodimerization site, significantly improving patient survival, while resistance and relapse of HER 2-positive patients remain a major problem.

CD47, also known as integrin-associated protein (IAP), is a transmembrane glycoprotein that is widely expressed on the cell surface and belongs to the immunoglobulin superfamily. CD47 is highly expressed in multiple types of tumors and inhibits phagocytosis by macrophages as a "do-it-yourself" signal, and elevated levels of CD47 expression are associated with invasive disease and poor survival. CD47 is involved in tumor immune escape and treatment. The targeting CD 47-sirpa pathway can be used as a treatment for multiple types of tumors. Studies have shown that CD47 monoclonal antibodies promote phagocytosis of tumor cells by macrophages, inhibit the growth of Acute Myeloid Leukemia (AML) in mice, eliminate AML that has been successfully transplanted in vivo, and also target the elimination of Leukemia Stem Cells (LSC).

CD47 is co-expressed with HER2 in a variety of cancers, so immunotherapy targeting both antigens simultaneously or development of multispecific antibodies against both antigens may exert stronger antitumor effects.

Disclosure of Invention

CD47 is co-expressed with HER2 in a variety of cancers (Candas-Green d., et al, nat. Commun.,2020, 11 (1): 4591.), particularly in patients with relapsed breast cancer with poor prognosis (Honkanen t.j., et al, sci. Rep.2019,9 (1): 10961; tsao l.c., et al, JCI Insight,4 (24): e 131882.), CD47 mediated anti-phagocytosis combined with proliferation of HER2 is prone to cause tumor invasion metastasis.

The inventors of the present application developed multispecific antibodies directed against CD47 as well as HER2 specific epitopes that showed significantly better anti-tumor activity on tumor models than single antigen-targeted antibodies, thereby providing the following aspects.

Multispecific antibodies

The first aspect of the invention provides a multispecific antibody which specifically binds CD47 and HER2 comprising a first antigen-binding domain specific for extracellular domain IV of HER2 and a second antigen-binding domain specific for CD 47.

In certain embodiments, the first antigen binding domain is a Fab fragment and the second antigen binding domain is a scFv.

Herein, the term "specific for HER2 ectodomain IV" includes "specificity for epitope 4D 5". "epitope 4D5" refers to the region in the extracellular domain of HER2 to which antibody 4D5 (ATCC CRL 10463) and trastuzumab bind. To screen for Antibodies that bind essentially to the 4D5 epitope, conventional cross-blocking assays can be performed, such as described in Antibodies, ALaboratory Manual, cold Spring Harbor Laboratory, ed Harlow and David Lane (1988). Alternatively, epitope mapping can be performed to assess whether the antibody substantially binds to the 4D5 epitope of HER 2.

In certain embodiments, the first antigen binding domain is capable of specifically binding to the sequence set forth in SEQ ID NO. 19.

In certain embodiments, the second antigen binding domain is capable of specifically binding to the sequence set forth in SEQ ID NO. 21.

In certain embodiments, the multispecific antibody of the first aspect is a bispecific antibody.

In certain embodiments, the first antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain CDR1 as set forth in SEQ ID NO:39, a heavy chain CDR2 as set forth in SEQ ID NO:40, a heavy chain CDR3 as set forth in SEQ ID NO:41, and a light chain variable region (VL) comprising a light chain CDR1 as set forth in SEQ ID NO:42, a light chain CDR2 as set forth in SEQ ID NO:43, a light chain CDR3 as set forth in SEQ ID NO: 44.

In certain embodiments, the first antigen binding domain comprises a VH as shown in SEQ ID NO. 1 or a variant thereof and a VL as shown in SEQ ID NO. 2 or a variant thereof. The variants have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which they were derived, or have one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) as compared to them. In certain embodiments, the substitutions are conservative substitutions. The variants retain the biological activity of the sequence from which they were derived.

In certain embodiments, the first antigen binding domain is a Fab fragment. In certain embodiments, the Fab fragment consists of a light chain (vl+cl) and a heavy chain fragment (vh+ch1).

In certain exemplary embodiments, the first antigen binding domain is a Fab fragment and comprises the light chain (VL+CL) shown in SEQ ID NO. 7 and the heavy chain fragment (VH+CH1) shown in SEQ ID NO. 26.

In certain embodiments, the second antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain CDR1 as shown in SEQ ID NO:51, a heavy chain CDR2 as shown in SEQ ID NO:52, a heavy chain CDR3 as shown in SEQ ID NO:53, and a light chain variable region (VL) comprising a light chain CDR1 as shown in SEQ ID NO:54, a light chain CDR2 as shown in SEQ ID NO:55, a light chain CDR3 as shown in SEQ ID NO: 56.

In certain embodiments, the second antigen binding domain is an scFv. In certain embodiments, the second antigen binding domain has a structure represented by VL-L1-VH or VH-L1-VL, wherein L1 is a flexible peptide, e.g., a flexible peptide comprising (G4S) n, n being an integer no less than 0, e.g., 1, 2, 3, or 4.

In certain embodiments, the second antigen binding domain comprises a VH as set forth in SEQ ID NO. 5 and a VL as set forth in SEQ ID NO. 6.

In certain exemplary embodiments, the second antigen binding domain comprises the sequence set forth in SEQ ID NO 34 or 35.

In certain embodiments, the multispecific antibody of the first aspect further comprises an Fc domain. In certain embodiments, the Fc domain is an IgG, such as IgG1, igG2, igG3, or IgG4, such as human IgG1 or human IgG4. In certain embodiments, the Fc domain comprises a first monomer and a second monomer. In certain embodiments, the first antigen binding domain and the second antigen binding domain are linked to the N-terminus of the first monomer and the second monomer, respectively, of the Fc domain, optionally through a linker. In certain embodiments, the linker is a flexible peptide comprising one or more (e.g., 1, 2, 3, or 4) alanine (a).

In certain embodiments, the first antigen binding domain is a Fab fragment and the second antigen binding domain is a scFv, and the multispecific antibody comprises:

(i) A first peptide chain comprising VL and light chain constant regions (CL) of the first antigen binding domain; preferably, the CL is a kappa light chain constant region;

(ii) A second peptide chain comprising the VH and heavy chain constant regions (CH) of the first antigen binding domain; preferably, the CH is an IgG heavy chain constant region, such as an IgG1 or IgG4 heavy chain constant region;

And

(iii) A third peptide chain comprising the second antigen binding domain and an Fc domain monomer; preferably, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1 or IgG 4; preferably, the Fc domain monomer comprises a hinge region, CH2, and CH3; preferably, the second antigen binding domain is linked to the N-terminus of the Fc domain monomer by a linker (e.g., a flexible peptide comprising one or more alanine (a));

wherein the Fc domain monomer of the third peptide chain is capable of forming a dimer with the Fc domain monomer of the heavy chain constant region (CH) of the second peptide chain.

In certain embodiments, the third peptide chain comprises an Fc domain monomer comprising a hinge region comprising a sequence set forth in SEQ ID NO. 31 or 32, such as comprising a sequence set forth in SEQ ID NO. 32.

In certain embodiments, the third peptide chain comprises an Fc domain monomer comprising a sequence shown in SEQ ID NO. 29 or 30, for example comprising a sequence shown in SEQ ID NO. 30.

In certain exemplary embodiments, the multispecific antibody comprises: a first peptide chain comprising the sequence shown in SEQ ID NO. 7, a second peptide chain comprising the sequence shown in SEQ ID NO. 8 and a third peptide chain comprising the sequence shown in SEQ ID NO. 9.

In a second aspect the invention provides a multispecific antibody which specifically binds CD47 and HER2, further comprising a third antigen-binding domain specific for extracellular domain II of HER2, in addition to the multispecific antibody provided in the first aspect.

In this context, the term "specific for HER2 ectodomain II" includes "specificity for epitope 2C 4". "epitope 2C4" refers to the region in the extracellular domain of HER2 to which murine antibody 2C4 and pertuzumab bind (see, e.g., WO 2013055874). To screen for Antibodies that bind essentially to the 2C4 epitope, conventional cross-blocking assays can be performed, such as described in Antibodies, A Laboratory Manual, cold SpringHarbor Laboratory, ed Harlow and David Lane (1988). Alternatively, epitope mapping can be performed to assess whether the antibody substantially binds to the 2C4 epitope of HER 2.

In certain embodiments, the third antigen binding domain is capable of specifically binding to the sequence set forth in SEQ ID NO. 20.

In certain embodiments, the multispecific antibody of the second aspect is a trispecific antibody.

In certain embodiments, the third antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain CDR1 as set forth in SEQ ID NO. 45, a heavy chain CDR2 as set forth in SEQ ID NO. 46, a heavy chain CDR3 as set forth in SEQ ID NO. 47, and a light chain variable region (VL) comprising a light chain CDR1 as set forth in SEQ ID NO. 48, a light chain CDR2 as set forth in SEQ ID NO. 49, a light chain CDR3 as set forth in SEQ ID NO. 50.

In certain embodiments, the third antigen binding domain is an scFv. In certain embodiments, the third antigen-binding domain has a structure represented by VL-L1-VH or VH-L1-VL, wherein L1 is a flexible peptide, e.g., a flexible peptide comprising (G4S) n, n being an integer no less than 0, e.g., 1, 2, 3, or 4.

In certain embodiments, the third antigen binding domain comprises a VH as shown in SEQ ID NO. 3 or a variant thereof and a VL as shown in SEQ ID NO. 4 or a variant thereof. The variants have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which they were derived, or have one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) as compared to them. In certain embodiments, the substitutions are conservative substitutions. The variants retain the biological activity of the sequence from which they were derived.

In certain exemplary embodiments, the third antigen binding domain comprises the sequence set forth in SEQ ID NO. 33.

In certain embodiments, the multispecific antibody of the second aspect further comprises an Fc domain. In certain embodiments, the Fc domain is an IgG, such as IgG1, igG2, igG3, or IgG4, preferably human IgG1 or human IgG4. In certain embodiments, the Fc domain comprises a first monomer and a second monomer.

In certain embodiments, the first antigen binding domain is attached to the N-terminus of the first monomer of the Fc domain, optionally through a linker.

In certain embodiments, the first antigen binding domain is optionally linked to the N-terminus of a first monomer of the Fc domain by a linker, one of the second antigen binding domain or third antigen binding domain is optionally linked to the N-terminus of a second monomer of the Fc domain by a linker, and the other is optionally linked to the C-terminus of the first monomer or second monomer, or the light chain C-terminus of the first antigen binding domain by a linker.

In certain exemplary embodiments, the first antigen binding domain is optionally linked to the N-terminus of a first monomer of the Fc domain by a linker, the second antigen binding domain is optionally linked to the N-terminus of a second monomer of the Fc domain by a linker, and the third antigen binding domain is optionally linked to the C-terminus of the first monomer by a linker.

In certain exemplary embodiments, the first antigen binding domain is optionally linked to the N-terminus of a first monomer of the Fc domain by a linker, the third antigen binding domain is optionally linked to the N-terminus of a second monomer of the Fc domain by a linker, and the second antigen binding domain is optionally linked to the light chain C-terminus of the first antigen binding domain by a linker.

In certain embodiments, the linker is a flexible peptide comprising one or more (e.g., 1, 2, 3, or 4) alanine (a), or is a flexible peptide comprising (G4S) n, n being an integer no less than 0, e.g., 1, 2, 3, or 4.

In certain embodiments, the first antigen binding domain is a Fab fragment, the second antigen binding domain and the third antigen binding domain are scFv, and the multispecific antibody comprises:

(i) A first peptide chain comprising VL and light chain constant regions (CL) of the first antigen binding domain and the second antigen binding domain; preferably, the CL is a kappa light chain constant region; preferably, the second antigen binding domain is linked to the C-terminus of the CL by a linker (e.g., a flexible peptide comprising (G4S) n);

and

(iii) A third peptide chain comprising the third antigen binding domain and an Fc domain monomer; preferably, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1 or IgG 4; preferably, the Fc domain monomer comprises a hinge region, CH2, and CH3; preferably, the third antigen binding domain is linked to the N-terminus of the Fc domain monomer by a linker (e.g., a flexible peptide comprising one or more alanine (a));

(ii) A second peptide chain comprising the VH and heavy chain constant regions (CH) of the first antigen binding domain and the third antigen binding domain; preferably, the CH is an IgG heavy chain constant region, such as an IgG1 or IgG4 heavy chain constant region; preferably, the third antigen binding domain is linked to the C-terminus of the CH by a linker (e.g., a flexible peptide comprising (G4S) n);

and

In certain exemplary embodiments, the multispecific antibody comprises:

a first peptide chain comprising the sequence shown in SEQ ID NO. 10, a second peptide chain comprising the sequence shown in SEQ ID NO. 8 and a third peptide chain comprising the sequence shown in SEQ ID NO. 11; or alternatively, the first and second heat exchangers may be,

a first peptide chain comprising the sequence shown in SEQ ID NO. 7, a second peptide chain comprising the sequence shown in SEQ ID NO. 12 and a third peptide chain comprising the sequence shown in SEQ ID NO. 9.

Modified Fc domains

In certain embodiments, the multispecific antibody of the first or second aspect comprises an Fc domain, and the Fc domain comprises a modification to promote dimerization of the first monomer and the second monomer. Whereby (hetero) dimerization occurs between the polypeptide comprising the first monomer and the polypeptide comprising the second monomer to form a complex.

Such modifications are known to those skilled in the art and may include separate modifications to each of the two Fc domain subunits (i.e., the first and second monomers of the Fc domain) that are desired to be joined, wherein the modifications are complementary to each other, thereby facilitating the joining of the two Fc domain subunits. For example, modifications that promote association may alter the structure or charge of one or both Fc domain subunits, thereby sterically or electrostatically promoting their association, respectively. For example, modifications that facilitate association include amino acid mutations (e.g., amino acid substitutions) in the Fc domain.

In certain embodiments, the modification is in the CH3 domain of the Fc domain.

In certain embodiments, the CH3 domains of the two monomers of the Fc domain comprise amino acid substitutions.

In certain embodiments, the modifications comprise a "knob" modification in one of the two monomers of the Fc domain and a "pocket" modification in the other of the two monomers of the Fc domain to form a "knob-in-pocket" modification. The section-into-hole technique 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). Generally, the method involves introducing a protuberance ("nub") at the interface of a first polypeptide and a corresponding cavity ("pocket") in the interface of a second polypeptide, such that the protuberance can be placed in the cavity to promote heterodimer formation and hinder homodimer formation. The bulge is constructed by replacing the small amino acid side chain from the first polypeptide interface with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the bulge is created in the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g. alanine or threonine).

In certain exemplary embodiments, one amino acid in the CH3 domain of the first monomer is substituted with an amino acid residue having a larger side chain volume, thereby forming a protuberance within the CH3 domain of the first monomer, and one amino acid in the CH3 domain of the second monomer is substituted with an amino acid residue having a smaller side chain volume, thereby forming a complementary cavity within the CH3 domain of the second monomer having the same or similar size as the protuberance; alternatively, one amino acid in the CH3 domain of the second monomer is replaced with an amino acid residue having a larger side chain volume, thereby forming a protuberance within the CH3 domain of the second monomer, and one amino acid in the CH3 domain of the first monomer is replaced with an amino acid residue having a smaller side chain volume, thereby forming a complementary cavity within the CH3 domain of the first monomer having the same or similar size as the protuberance.

In certain embodiments, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (P), tyrosine (Y), tryptophan (W).

In certain embodiments, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (a), serine (S), threonine (T), valine (V).

In certain exemplary embodiments, the first monomer and the second monomer of the Fc domain comprise the amino acid sequences as shown in SEQ ID NOs 29 and 30, respectively.

Preparation of antibodies

The multispecific antibodies of the first or second aspects of the invention may be prepared by various methods known in the art, for example by genetic engineering recombinant techniques. For example, DNA molecules encoding them are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then the host cell is transfected. The transfected host cells are then cultured under specific conditions and express the multispecific antibodies of the invention.

The multispecific antibody of the first or second aspect may be produced by coexpression of a plurality of polynucleotides encoding respective polypeptide chains of the multispecific antibody. Polypeptide chains resulting from co-expression may be joined via, for example, disulfide bonds or other means to form functional multispecific antibodies. For example, the light chain portion of a Fab fragment may be encoded by a separate polynucleotide from the portion of the multispecific antibody that comprises the heavy chain portion of the Fab fragment (which portion may further comprise an Fc domain monomer and optionally other antigen binding domains). When co-expressed, polypeptides comprising the heavy chain portion of the Fab fragment will combine with polypeptides comprising the light chain portion of the Fab fragment to form the Fab fragment. For another example, a portion of a multispecific antibody provided herein that comprises one of the two Fc domain monomers (which portion may further comprise an antigen binding domain) may be encoded by a separate polynucleotide from a portion comprising the other of the two Fc domain monomers (which portion may further comprise an antigen binding domain). When co-expressed, the two Fc domain monomers combine to form an Fc domain.

In a third aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a multispecific antibody or at least one peptide chain thereof according to the first or second aspect.

In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody of the first or second aspect, and the nucleotide sequences encoding each peptide chain are present on the same or different isolated nucleic acid molecules.

In a fourth aspect the invention provides a vector (e.g. an expression vector) comprising a nucleic acid molecule encoding an isolated nucleic acid molecule as described above.

In certain embodiments, the vector comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody of the first or second aspect, and the nucleotide sequences encoding each peptide chain are present on the same or different vectors. For example, the vectors of the invention comprise: a first vector comprising a nucleotide sequence encoding a first peptide chain, a second vector comprising a nucleotide sequence encoding a second peptide chain, and a third vector comprising a nucleotide sequence encoding a third peptide chain.

In a fifth aspect the invention provides a host cell comprising a nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells, such as bacterial cells (e.g., E.coli cells), and eukaryotic cells, such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.).

In another aspect, the invention provides a method of producing a multispecific antibody according to the first or second aspect, comprising culturing a host cell as described above under conditions allowing expression of a protein, and recovering the multispecific antibody from the cultured host cell culture.

Composition and method for producing the same

In a sixth aspect the invention provides a composition comprising the multispecific antibody of the first aspect and a monoclonal antibody specific for extracellular domain II of HER 2.

In certain embodiments, the monoclonal antibody comprises a heavy chain variable region (VH) comprising a heavy chain CDR1 as set forth in SEQ ID NO. 45, a heavy chain CDR2 as set forth in SEQ ID NO. 46, a heavy chain CDR3 as set forth in SEQ ID NO. 47, and a light chain variable region (VL) comprising a light chain CDR1 as set forth in SEQ ID NO. 48, a light chain CDR2 as set forth in SEQ ID NO. 49, a light chain CDR3 as set forth in SEQ ID NO. 50.

In certain embodiments, the monoclonal antibodies include a VH as shown in SEQ ID NO. 3 and a VL as shown in SEQ ID NO. 4.

In certain embodiments, the monoclonal antibody is a full length antibody, e.g., an IgG antibody.

In certain embodiments, the multispecific antibody of the first aspect is provided separately from the component of the monoclonal antibody specific for HER2 ectodomain II, or as a mixed component.

Pharmaceutical composition

The seventh aspect of the invention provides a pharmaceutical composition comprising the multispecific antibody of the first or second aspect, the isolated nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect or the composition of the sixth aspect, and a pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical composition of the invention comprises a multispecific antibody of the first or second aspect.

In certain embodiments, the pharmaceutical composition of the present invention comprises the composition of the sixth aspect.

The pharmaceutical compositions of the present invention are formulated into dosage forms compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal. Solutions or suspensions for parenteral, intradermal or subcutaneous administration applications may include the following components: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulphite; chelating agents such as ethylenediamine tetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Formulations for parenteral administration may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (wherein water is soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, polyoxyethylated castor oil ELTM, or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition agents which delay absorption, for example, aluminum monostearate and gelatin.

Therapeutic use

An eighth aspect of the invention relates to a method for treating a tumor comprising administering to a subject in need thereof a multispecific antibody according to the first or second aspect, a composition according to the sixth aspect or a pharmaceutical composition according to the seventh aspect. The invention also relates to a multispecific antibody of the first or second aspect, an isolated nucleic acid molecule of the third aspect, a vector of the fourth aspect, a host cell of the fifth aspect, a composition of the sixth aspect or a pharmaceutical composition of the seventh aspect, for use in the treatment of a tumor, or for use in the manufacture of a medicament for the treatment of a tumor.

In certain embodiments, the tumor is HER2 positive. In certain embodiments, the tumor is CD47 positive. In certain embodiments, the tumor is HER2 positive and CD47 positive.

In certain embodiments, the tumor is selected from breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, colorectal cancer, non-hodgkin lymphoma, chronic lymphoma leukemia, multiple myeloma, acute myelogenous leukemia, acute lymphoma leukemia, glioma, melanoma, or any combination thereof.

In certain embodiments, the method comprises administering to the subject a composition of the sixth aspect of the invention, wherein the multispecific antibody and the monoclonal antibody specific for HER2 extracellular domain II can be administered simultaneously or sequentially.

The multispecific antibodies, compositions, or pharmaceutical compositions comprising them of the present invention can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, injectable sterile powders, and injectable concentrated solutions), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The multispecific antibodies, compositions, or pharmaceutical compositions comprising them of the present invention should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by the following methods: the necessary amount of the active ingredient is incorporated in a suitable solvent, and optionally, other desired ingredients (including, but not limited to, pH modifiers, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof) are incorporated simultaneously, followed by filter sterilization. In addition, the sterile injectable solutions may be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for convenient storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier prior to use, such as water for injection (WFI), water for bacteriostatic injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

The multispecific antibodies, compositions, or pharmaceutical compositions comprising them of the present invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous injection or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, the multispecific antibodies, compositions, or pharmaceutical compositions comprising them of the present invention are administered by intravenous injection or bolus injection.

The multispecific antibodies, compositions, or pharmaceutical compositions comprising them of the present invention may be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier.

The multispecific antibodies, compositions, or pharmaceutical compositions comprising them of the invention may be administered alone or in combination with another pharmaceutically active agent (e.g., an antineoplastic agent) or another therapy (e.g., an antineoplastic therapy).

The subject described herein may be a mammal, such as a human.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the virology, biochemistry, immunology laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

When used herein, the terms "for example," such as, "" including, "" comprising, "or variations thereof, are not to be construed as limiting terms, but rather as meaning" but not limited to "or" not limited to.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The term "antibody" as used herein refers to an immunoglobulin derived molecule capable of specifically binding to a target antigen, which immunoglobulin derived molecule binds to the target antigen through at least one antigen binding site located in its variable region. When referring to the term "antibody", it includes not only whole antibodies, but also antigen-binding fragments capable of specifically binding to a target antigen, unless the context clearly indicates.

An "intact antibody" typically consists of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha or epsilon and define the isotype of antibodies as respectivelyIgM, igD, igG, igA and IgE. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each V is _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. The amino acid assignment in each region or domain can be followed by Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)), or Chothia&Lesk (1987) J.mol.biol.196:901-917; chothia et al (1989) Nature 342:878-883.

As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity for at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody comprises a plurality of antigen-binding domains having binding specificities for different antigens (or epitopes) so as to be capable of binding at least two different binding sites and/or target molecules. The individual antigen binding domains comprised by the multispecific antibody may each be independently selected from a full-length antibody (e.g., an IgG antibody) or an antigen-binding fragment thereof (e.g., an Fv fragment, a Fab fragment, a F (ab') 2 fragment, or an scFv). In some cases, the individual antigen binding domains are linked by a peptide linker.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs, designated CDR1, CDR2 and CDR3, are contained in each of the variable regions of the heavy and light chains. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, e.g., as in the Kabat numbering system (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md, 1991), the Chothia numbering system (Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al, dev. Comparat. Immunol.27:55-77,2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, for example, lefranc et al, dev. Comparat. Immunol.27:55-77,2003).

As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the term "full length antibody" means an antibody consisting of two "full length heavy chains" and two "full length light chains". Wherein "full length heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to C-terminal direction; and, when the full length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-to C-terminal direction. A "full length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to C-terminal direction. The two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of the two full length heavy chains. Full length antibodies comprise two antigen binding sites formed by VH and VL pairs, respectively, that specifically recognize/bind the same antigen.

As used herein, the term "Fab fragment" means an antibody fragment consisting of a light chain comprising VL and CL and a heavy chain fragment comprising VH and CH 1.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH domains are linked by a linker (linker) (see, e.g., bird et al, science 242:423-426 (1988); huston et al, proc. Natl. Acad. Sci. USA85:5879-5883 (1988); and Pluckaphun, the Pharmacology of Monoclonal Antibodies, volume 113, roseburg and Moore, springer-Verlag, new York, pages 269-315 (1994)). Such scFv molecules may have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a polypeptide having an amino acid sequence (GGGGS) can be used ₄ Variants thereof may be used (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). In some cases, disulfide bonds may also exist between VH and VL of scFv.

As used herein, the term "Fc domain" or "Fc region" or "Fc domain" has the meaning commonly understood by those skilled in the art and is used interchangeably, meaning a portion of the heavy chain constant region comprising CH2 and CH3. The Fc region of antibodies has a number of different functions, but is not involved in antigen binding. "effector functions" mediated by the Fc region include Fc receptor binding; clq binding and Complement Dependent Cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation, etc. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. When the Fc region comprises a hinge, the hinge modulates dimerization between two Fc-containing polypeptides. The Fc region can be any antibody heavy chain constant region isotype, e.g., igG1, igG2, igG3, or IgG4.

The Fc domain may include both a native Fc region and a variant Fc region. The native Fc region comprises an amino acid sequence that corresponds to the amino acid sequence of an Fc region found in nature, e.g., the native sequence human Fc region includes the native sequence human IgG1 Fc region (non-a and a allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region, and naturally occurring variants thereof. The variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification. In some embodiments, the variant Fc region may possess altered effector functions (e.g., fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function) compared to the native Fc region. In some embodiments, the variant Fc region may be provided with modifications that promote dimerization. As used herein, a "monomer" of an Fc domain refers to one of two polypeptides forming a dimeric Fc domain, i.e., a polypeptide comprising a C-terminal constant region in an immunoglobulin heavy chain capable of stabilizing self association.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in a first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in a second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions/total number of positions x 100%). In certain embodiments, the two sequences are the same length.

Determination of percent identity between two sequences can also be accomplished using mathematical algorithms. One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, proc.Natl. Acad. Sci.U.S. A.87:2264-2268, as modified in Karlin and Altschul,1993, proc.Natl. Acad. Sci.U.S. A.90:5873-5877. Such an algorithm was integrated into the NBLAST and XBLAST programs of Altschul et al, 1990, J.mol. Biol. 215:403.

As used herein, the term "variant", in the context of polypeptides (including polypeptides), also refers to polypeptides or peptides comprising an amino acid sequence that has been altered by the introduction of amino acid residue substitutions, deletions or additions. In some instances, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not by way of limitation, the polypeptide may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to a cell ligand or other protein, and the like. The derivatized polypeptide or peptide may be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the variants have similar, identical or improved functions as the polypeptide or peptide from which they are derived.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be determined by the equilibrium dissociation constant (K _D ) And (3) representing. In the present invention, the term "K _D "refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen.

The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "binding rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be summed by concentration and associationThe actual rate of dissociation was calculated (see Malmqvist M, nature,1993, 361:186-187). The kdis/kon ratio is equal to the dissociation constant K _D (see Davies et al, annual Rev Biochem,1990; 59:439-473). K can be measured by any effective method _D The dissociation constants can be measured, for example, in Biacore using Surface Plasmon Resonance (SPR), and also by bioluminescence interferometry or Kinexa.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Furthermore, amino acid residues can also be divided into categories defined by alternative physical and functional properties. For example, alcohol group-containing residues (S and T), aliphatic residues (I, L, V and M), cycloalkenyl-related residues (F, H, W and Y), hydrophobic residues (A, C, F, G, H, I, L, M, R, T, V, W and Y), negatively charged residues (D and E), polar residues (C, D, E, H, K, N, Q, R, S and T), positively charged residues (H, K and R), small residues (A, C, D, G, N, P, S, T and V), very small residues (A, G and S), residues involving corner formation (A, C, D, E, G, H, K, N, Q, R, S, P and T), flexible residues (Q, T, K, S, G, P, D, E and R). Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

The twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., immunology-a Synthesis (2nd Edition,E.S.Golub and D.R.Gren,Eds, sinauer Associates, sundland, mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to: pH modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugar, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in a medicament, including but not limited to sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like.

As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and diminishment of symptoms (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the expected survival (if not treated).

As used herein, the term "subject" refers to a mammal, such as a primate mammal, e.g., a human. In certain embodiments, the subject (e.g., human) has a tumor (e.g., HER2 positive and/or CD47 positive tumor).

Advantageous effects of the invention

The invention provides a multi-specific antibody aiming at specific epitopes of CD47 and HER2, which has high expression quantity, and the transient transfection expression quantity in a mammalian cell HEK293E can reach 120-150mg/L; the assembly rate is high; the affinity is high; having an Fc domain facilitates purification. The multispecific antibody has good cytological activity and in-vivo pharmacodynamic activity, and the cytological activity and the in-vivo pharmacodynamic activity are superior to those of the contrast marketed medicines, and have important clinical values.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention.

Drawings

FIGS. 1A-1C are schematic diagrams showing the structure of C033, C051 and C065 bispecific or trispecific antibodies;

FIG. 2 shows SDS-PAGE detection of C033, C051 and C065 antibody proteins transient expression supernatants: m is Maker; lanes 1, 2 are the C033 non-reduced and reduced samples, lanes 3, 4 are the C051 non-reduced and reduced samples, respectively, and lanes 5, 6 are the C065 non-reduced and reduced samples, respectively;

FIG. 3 shows SDS-PAGE detection after purification of the C033, C051 and C065 antibody proteins: m is Maker; lanes 1, 2 are the C033 non-reduced and reduced samples, lanes 3, 4 are the C051 non-reduced and reduced samples, respectively, and lanes 5, 6 are the C065 non-reduced and reduced samples, respectively;

FIG. 4 shows the results of ELISA assays for binding of C033, C051 and C065 to CD 47;

FIGS. 5A-5B show ELISA detection results of different epitopes of C033, C051 and C065 binding to HER 2;

FIG. 6 shows phagocytosis of NCI-N87 cells by C033, C051 and C065, trastuzumab and pertuzu as anti-HER 2 drugs marketed by Fumihan and Roche, respectively, and 059-1.43.1-H1L3 as anti-CD 47 mab self-developed by vinca-siren pharmaceutical industry (see patent CN 110872348A) as positive control;

FIG. 7 shows the ADCC activity of C033, C051 and C065 on SKOV3 cells, trastuzumab and pertuzu as anti-HER 2 drugs marketed by Fumihan and Roche, respectively, as positive controls;

FIG. 8 shows the hemagglutination activity of C033, with 059-1.82.6 being a vinca-race pharmaceutical self-developed anti-CD 47 mab (humanized mab of 059-1.20.1 in CN 106084052A), IMM2902 being a recombinant bifunctional protein of CD47 and HER2 (see patent CN 112533954A) as a control.

FIG. 9 shows the in vivo efficacy of C033 in SKOV3 xenograft model, trastuzumab was the anti-HER 2 drug marketed in Fuhonghan, and 059-1.43.1-H1L3 was vincristine drug self-developed anti-CD 47 mab as a control.

Fig. 10 shows the in vivo efficacy of the C033 drug combination in HCC-1954 xenograft model, trastuzumab and pertuzu as anti-HER 2 drugs marketed by the company complex honghan and roche, respectively, and 059-1.43.1-H1L3 as vincristine pharmaceutical self-developed anti-CD 47 mab as a control.

FIG. 11 shows the in vivo efficacy of C033, C051 and C065, and drug combinations in JIMT-1 xenograft models, trastuzumab and pertuzu as anti-HER 2 drugs marketed by Fuhonghan and Roche, respectively, and 059-1.43.1-H1L3 as vinca-gold pharmaceutical self-developed anti-CD 47 monoclonal antibodies as controls.

Sequence information

A description of the sequences to which the present application relates is provided in the following table.

Table 1: sequence information

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, molecular biology experimental methods and immunoassays used in the present invention are basically described in j.sambrook et al, molecular cloning: laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-compiled guidelines for molecular biology experiments, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the manufacturer of the product. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1: bispecific or trispecific antibodies and antigen production

1. Construction of vectors expressing C033, C051 and C065

1.1 materials

1.1.1C033 bispecific antibodies

The C033 bispecific antibody structure is shown in fig. 1A and table 2, comprising a Fab fragment binding to antigen 1 (HER 2 extracellular domain IV) and scFv (scFv 2-1) binding to antigen 2 (human CD 47), the heavy and light chain variable regions (VH 1, VL 1) of Fab are obtained from Herceptin/Trastuzumab (Trastuzumab) as set forth in SEQ ID NOs: 1 and SEQ ID NO:2, scFv2-1 heavy and light chain variable regions were derived from the monoclonal antibody 059-1.43.1-H1L3 against human CD47 (see patent CN 110872348A), SEQ ID NO:5 and SEQ ID NO:6, scFv2-1 has a structure shown as VL 2-flexible peptide 1-VH2, and flexible peptide 1 is shown as SEQ ID NO. 36.

Table 2: c033 structure

1.1.2C051 and C065 trispecific antibodies

The C051 and C065 trispecific antibody structures are shown in FIGS. 1B-1C and tables 3-4 and comprise a Fab fragment which binds antigen 1 (HER 2 extracellular domain IV), a scFv (scFv 2-1 or scFv 2-2) which binds antigen 2 (human CD 47), and a scFv (scFv 3) which binds antigen 3 (HER 2 extracellular domain II). The heavy and light chain variable regions (VH 1, VL 1) of Fab are obtained from Herceptin/Trastuzumab (Trastuzumab) as set forth in SEQ ID NOs: 1 and SEQ id no:2. the heavy and light chain variable regions of scFv2-1 and scFv2-2 were derived from monoclonal antibody 059-1.43.1-H1L3 against human CD47, SEQ ID NOs: 5 and SEQ ID NO:6, scFv2-1 has a structure shown as VL 2-flexible peptide 1-VH2, scFv2-2 has a structure shown as VH 2-flexible peptide 1-VL2, and flexible peptide 1 is shown as SEQ ID NO: 36. The heavy and light chain variable regions of scFv3 are derived from Perjeta/Pertuzumab (Pertuzumab) as set forth in SEQ ID NOs: 3 and SEQ id no:4, scFv3 has a structure shown as VL 3-flexible peptide 1-VH3, flexible peptide 1 is shown as SEQ ID NO. 36.

Table 3: c051 structure

Table 4: c065 structure

1.2 construction of expression plasmids

Primers were designed based on the coding sequences of the heavy and light chain variable regions specifically binding to the first antigen HER2 ectodomain IV and the third antigen HER2 ectodomain II, and the coding sequences of the monoclonal antibodies against human CD47, the coding nucleotides of the IgG1 heavy chain constant region CH1, the linker, the hinge region and Fc, and the nucleotide sequences of the Kappa chain constant region and the multiple cloning sites in the vectors, and after PCR amplification, 6 expression vectors were constructed by homologous arm recombination or unique restriction site directed cloning into pGS003 (ampR) (gold self-constructed) background vectors, respectively, the encoded amino acid sequences of which are shown in table 5. The recombinant expression vector is transformed into escherichia coli, plasmids are extracted, and whether the genes are successfully inserted is identified through plasmid sequencing.

Table 5: transient transfection expression vector for bi-or tri-specific antibody

Expression vector name	Encoded amino acid sequence
		L1	SEQ ID NO:13
H1	SEQ ID NO:14
		H2	SEQ ID NO:15
L2	SEQ ID NO:16
		H3	SEQ ID NO:17
H4	SEQ ID NO:18

2. Construction of expression antigen vectors

hHER2 ECD (S310A & L317P & H318A) represents HER2 ectodomain IV, and the amino acid sequence is shown in SEQ ID NO. 19; hHER2 ECD (E580A & P594A) represents the extracellular domain II of HER2, the amino acid sequence of which is shown in SEQ ID NO: 20. hCD47 ECD represents the extracellular domain of CD47, and its amino acid sequence is shown in SEQ ID NO. 21. Designing primers according to coding sequences of antigens hHER2 ECD and hCD47 ECD and multiple cloning sites in a vector, and carrying out PCR amplification, and then directionally cloning the primers into pGS003 (ampR) (gold-race self-constructed) background vectors through homologous arm recombination or unique restriction enzyme cutting sites, wherein the primers respectively carry mIgG1Fc and His labels; primers were designed based on the coding sequences of the antigens hHER2 ECD (S310A & L317P & H318A) and hHER2 ECD (E580A & P594A) and the multiple cloning sites in the vector, and after PCR amplification, were cloned into pcdna3.4 (EBV) (ampR) (gold self-constructed) background vectors, each with an mIgG1Fc tag (4 total, as in table 6), either by homologous arm recombination or unique restriction sites. The recombinant expression vector is transformed into escherichia coli, plasmids are extracted, and whether the genes are successfully inserted is identified through plasmid sequencing.

Table 6: antigen transient transfection expression vector

Expression vector name	Encoded amino acid sequence
		hHER2 ECD-mIgG1Fc	SEQ ID NO:22
hHER2 ECD(S310A&L317P&H318A)-mIgG1Fc	SEQ ID NO:23
		hHER2 ECD(E580A&P594A)-mIgG1Fc	SEQ ID NO:24
hCD47ECD-His	SEQ ID NO:25

3. Transfection, expression and detection of bispecific or trispecific antibodies and antigens in mammalian cells 293E

The bi-or tri-specific antibodies were transfected in combination with the 4 heavy chain expression vectors described above and the 2 light chain expression vectors h1+h2+l1 (C033 structure), h1+h3+l2 (C051 structure), h2+h4+l1 (C065 structure), hCD47 ECD-His and hHER2 ECD-mIgG1Fc, hhr 2ECD (S310A & L317P & H318A) -mIgG1Fc and hhr 2ECD (E580A & P594A) -mIgG1Fc antigen expression vectors, respectively, for expression of antibody proteins.

Transient transfection expression of antibodies of the C033, C051 and C065 constructs, as well as hCD47 ECD-His and hHER2 ECD-mIgG1Fc, was performed using 293E in Freestyle medium. The transient transfection expression level of the bi-or tri-specific antibody proteins C033, C051 and C065 in the HEK293E mammalian cells can reach 120-150mg/L, and the detection result of transient transfection expression supernatant is shown in figure 2.

Transient transfection expression of hHER2ECD (S310A & L317P & H318A) -mIgG1F structure specific antigen was performed using Expi293F in Expi293F expression medium. Transient transfection expression of hHER2ECD (E580A & P594A) -mIgG1 Fc-specific antigen was performed using ExpiCHO-S in ExpiCHO expression medium.

Example 2: purification and detection of bispecific or trispecific antibodies and antigens

1. Purification of C033 protein:

the supernatant was collected, filtered through a 0.22. Mu.M filter membrane, and desalted by Protein A (GE) chromatography, butyl FF (GE) chromatography and Protein A (GE) chromatography in this order to obtain a purified sample. Purified samples were subjected to SDS-PAGE using 4-20% gradient gel (Berle Life medicine Co., ltd.) to detect purified protein, and the results are shown in FIG. 3, which shows that the purity of C033 is 98%.

2. Purification of C051 protein:

the supernatant was collected, and the supernatant was filtered with a 0.22. Mu.M filter membrane and desalted by Protein A (GE) chromatography, butyl FF (GE) chromatography, capto S image (GE) ion chromatography and Protein A (GE) chromatography in this order to obtain a purified sample. Purified proteins were detected by SDS-PAGE using 4-20% gradient gel (Berle Life medicine Co., ltd.) and the results are shown in FIG. 3, which shows that the purity of C051 is 96%.

3. C065 protein purification:

the supernatant was collected, and the supernatant was filtered with a 0.22. Mu.M filter membrane and desalted by Protein A (GE) chromatography, butyl FF (GE) chromatography, capto S image (GE) ion chromatography and Protein A (GE) chromatography in this order to obtain a purified sample. Purified samples were subjected to SDS-PAGE using 4-20% gradient gel (Berle Life medicine Co., ltd.) to detect purified protein, and the results are shown in FIG. 3, which shows that C065 has a purity of 97%.

4. hCD47 ECD-His specific antigen purification:

the supernatant was collected, filtered through a 0.22. Mu.M filter membrane, and a purified sample was obtained by Ni (GE) chromatography. Purified proteins were detected by SDS-PAGE using 4-20% gradient gel (Berle Life medicine Co., ltd.).

5. hHER2 ECD-mIgG1Fc, hHER2 ECD (S310A & L317P & H318A) -mIgG1Fc and hHER2 ECD (E580A & P594A) -mIgG1Fc specific antigen purification:

the supernatant was collected, filtered through a 0.22. Mu.M filter membrane, chromatographed on Protein A (GE), eluting with 20mM citric acid-sodium citrate, pH 3.2, and pH-adjusted to neutral with 1M Tris base. Purified samples were subjected to SDS-PAGE using 4-20% gradient gel (Berle Life medicine products Co., ltd.) to detect purified proteins.

Example 3: ELISA detection of bispecific or trispecific antibody binding CD47 or HER2

1. Antigen coating: the gold homemade hCD47 ECD-His (for C033, C051 and C065 assays), gold homemade hcr 2 ECD (S310A & L317P & H318A) -mIgG1Fc (for C033, C051 and C065 assays), gold homemade hcr 2 ECD (E580A & P594A) -mIgG1Fc (for C051 and C065 assays) were diluted to 1 μg/mL with CBS and added to 96 well ELISA plates and incubated overnight at 4 ℃.

2. Closing: PBST plates were washed three times, blocked with 1% BSA, and incubated at 37℃for 2h at 250. Mu.L per well.

3. Adding a candidate antibody: after washing the plate 3 times, 8 concentration gradient candidate antibody samples with a 3-fold dilution of the initial concentration of 100nM were added. mu.L per well, incubated at 25℃for 1h.

4. Adding a secondary antibody: after 3 washes of PBST, goat anti-human (1:5000) was added and incubated at 25℃for 1h at 50. Mu.L per well.

5. Color development: after PBST plate washing for 4 times, TMB color development liquid is added, 50 mu L of each hole is used, and color development is carried out at room temperature in a dark place for 3-5min.

6. And (3) terminating: the reaction was terminated by directly adding 50. Mu.L of a stop solution per well.

7. And (3) detection: immediately after the reaction is terminated, the ELISA plate is placed into an ELISA apparatus, OD value is measured at 450nm, and original data are stored for finishing. The results are shown in Table 7, FIG. 4 and FIGS. 5A-5B. The results show that C033, C051, C065 have higher binding activity (EC 50 on nM scale) to the corresponding antigen.

Table 7: ELISA results for binding antigens by bispecific or trispecific antibodies

Example 4: bispecific or trispecific antibody affinity assays

The affinity of pertuzumab, C051 and C065 to hHER2 ECD (E580A & P594A) was detected using a Biacore 8K instrument. The specific method comprises the following steps: pertuzus, C051 and C065 antibodies were captured with a CM5 chip (Cytiva) coupled to Anti-human IgG (Fc) Anti-antibodies at 30s capture time, 30. Mu.L/min flow rate, hHER2 ECD (E580A & P594A) -mIgG1Fc was diluted from a 20nmol/L initial multiple to 8 concentration spots and flowed through the chip at 30. Mu.L/min flow rate, antigen bound to candidate antibody for 180s and dissociation time 600s, glycine1.5 was regenerated for 30s. Kinetic fitting was performed using Biacore 8K Evaluation software (Cytiva) to obtain the following affinity constants, table 8, with the affinity of pertuzus, C051 and C065 to hHER2 ECD (E580A & P594A) at the same level of 2.52E-10M, 3.02E-10M and 3.20E-10M, respectively.

Table 8: affinity assay with hHER2ECD (E580A & P594A)

Antibody name	Ka(1/Ms)	Kd(1/s)	KD(M)
				Partuzu (al) ball	3.63E+05	9.14E-05	2.52E-10
C051	2.96E+05	8.94E-05	3.02E-10
				C065	2.43E+05	7.77E-05	3.20E-10

Affinity of trastuzumab, C033, C051 and C065 to hHER2ECD (S310A & L317P & H318A) was detected using a Biacore 8K instrument. The specific method comprises the following steps: trastuzumab, C033, C051 and C065 antibodies were captured with CM5 chip (cytova) coupled to Anti-human IgG (Fc) Anti-antibodies for 30S at 30 μl/min, hHER2ECD (S310A & L317P & H318A) -mIgG1Fc was diluted from a 20nmol/L initial multiple to 8 concentration spots and flowed through the chip at 30 μl/min flow rate, antigen bound to candidate antibody for 180S, dissociation time 600S, glycene 1.5 regeneration 30S. Kinetic fitting was performed using Biacore 8K Evaluation software (Cytiva) to obtain affinity constant results as shown in Table 9 below, with affinities of trastuzum, C033, C051 and C065 for hHER2ECD (S310A & L317P & H318A) of 3.20E-10M, 2.83E-10M, 2.07E-10M and 2.70E-10M, respectively.

Table 9: affinity assay with hHER2ECD (S310A & L317P & H318A)

Affinity of 059-1.43.1-H1L3, C033 and C065 to hCD47 was detected using a Biacore 8K instrument. The specific method comprises the following steps: the antibodies 059-1.43.1-H1L3, C033 and C065 were captured with a CM5 chip (Cytiva) coupled to Anti-human IgG (Fc) Anti-antibodies for 30s at a flow rate of 30. Mu.L/min, hCD47 ECD-His was diluted from 800nmol/L starting concentration to 8 concentration spots and flowed through the chip at a flow rate of 30. Mu.L/min for antigen binding with candidate antibody for 60s, dissociation time for 60s, and Glycine1.5 regeneration for 30s. Kinetic fitting was performed using Biacore 8K Evaluation software (Cytiva) to obtain affinity constants with affinities of 4.63E-09M, 7.29E-09M and 8.74E-09M for hCD47 in tables 10, 059-1.43.1-H1L3, C033, C065, respectively, at the same level.

Table 10: affinity assay results with hCD47

Antibody name	Ka(1/Ms)	Kd(1/s)	KD(M)
				059-1.43.1-H1L3	1.53E+06	7.08E-03	4.63E-09
C033	1.64E+06	1.20E-02	7.29E-09
				C065	1.86E+06	1.63E-02	8.74E-09

Example 5: phagocytosis of NCI-N87 cells by bispecific or trispecific antibodies

The ability of the bispecific or trispecific antibodies of the invention to promote phagocytosis of tumor cells by macrophages is measured based on fluorescent labeling.

Peripheral Blood Mononuclear Cells (PBMC) from fresh blood of donor were extracted by density gradient centrifugation, CD14 positive monocytes were purified from the isolated PBMC according to Kit (MojoSort Human CD Selection Kit, biolegend) and 25ng/mL macrophage colony stimulating factor (rhM-CSF, R) was added&D) Continuous culture for 6 days, wherein the culture supernatant was discarded at the third day, fresh RPMI-1640 complete medium containing 25ng/mL rhM-CSF was replaced, adherent cells were collected by pancreatin after the sixth day, fluorescent-labeled according to DiO (cell membrane Green fluorescent staining kit, biyun) dye Specification, plated in 96-well cell culture plates, 1X 10 per well ⁴ Individual cells, RPMI-1640 complete medium containing 25ng/mL M-CSF and 50ng/mL IFN-gamma was added and incubated at 37℃and 5% CO ₂ Culturing for 24 hours. Target cells (NCI-N87, center of the college of Chinese sciences) were labeled with Far red (CellTrace Far Red Cell Proliferation kit, invitrogen) and then mixed with gradient dilutions of C033, C051 and C065 and control 1:1. The antibody and target cell mixture solution was transferred to a plate containing the above-described differentiated macrophages, and the number ratio of macrophages to NCI-N87 cells was 1:1. Culturing the mixture in a cell culture incubator for 2 hours, washing the cells twice, washing the target cells which are not phagocytized, and then analyzing NCI-N87 (Far red) cell quantity in PBMC (DiO) cells by using a high content instrument to obtain the final phagocytosis rate.

As shown in FIG. 6, the bispecific antibodies C033, C051 and C065 of the present invention have a higher NCI-N87 phagocytosis rate than 059-1.43.1-H1L3, qu Tuo beads or pertuzumab.

Example 6: determination of ADCC Activity of bispecific or trispecific antibodies

Construction of NK92-CD16a (gold-racetam) stably expressing FcgammaRIIIa (CD 16 a) as effector cells using NK92 cells (ATCC) as target cells, mixing the mixed cells at a ratio of 1:20, 5% CO at 37 ℃C ₂ Incubate for 30min in incubator, then add gradient diluted C033, C051, C065 or control protein trastuzumab to mixed cells for 6h. After that, the cell culture plate was centrifuged at 1500rpm for 5min, and 50. Mu.L of the supernatant was subjected to LDH assay (Promega, cat#G1780). The percentage of cell lysis mediated by ADCC is calculated based on the following formula: lysis% = (OD _sample _data -OD _target _plus _effector _cells )/(OD _max _release -OD _min _release ) X 100%. As shown in fig. 7, on SKOV3 cells, C033 and C051 had lower EC50 and better ADCC activity than control mab trastuzumab, pertuzumab; the C065 molecule has a higher maximum target cell killing rate than other molecules.

Example 7: bispecific antibody hemagglutination risk assay

Since normal erythrocytes also express CD47, CD47 targeted drugs have the potential to promote the risk of erythrocyte agglutination, which is further investigated by the present invention for bispecific antibodies in this application.

Human fresh blood was collected intravenously, and red blood cells were obtained by centrifugation at Ficoll (GE Healthcare) density gradient, and 50. Mu.l/well of 2% red blood cells (volume ratio, final concentration 1%) were prepared. The final concentration of the antibody is 1000nM, the antibody is diluted 3 times, 12 concentration points are obtained, the incubator is kept stand and incubated for 2 hours at 37 ℃ to observe the agglutination condition, and after the reaction is finished, the result is photographed and judged. If the red blood cells are in a punctiform form and sink at the bottom of the hole, no agglutination occurs; if the plate is spread in a net mist form, an agglutination reaction occurs.

As shown in FIG. 8, C033 did not undergo hemagglutination, whereas control CD47 mab 059-1.82.6 caused hemagglutination at 0.15-1000nM and IMM2902 (a recombinant bifunctional protein of CD47 and HER2, see CN 112533954A) caused hemagglutination at 111.1-1000 nM. It can be seen that the C033 antibody has the advantage of significantly reducing side effects in therapy.

Example 8: anti-tumor drug effect of bispecific antibody in SKOV3 xenograft model

Human ovarian cancer SKOV3 cells (ATCC) were cultured in McCoy' 5A medium containing 10% FBS and maintained at 5% CO ₂ Is placed in a saturated humidity incubator at 37 ℃. The SKOV3 cells in logarithmic growth phase were collected, resuspended in McCoy' 5A basal medium containing 50% Matrigel, and the cell concentration was adjusted to 5X 10 ⁷ And each mL. Under aseptic conditions, 0.2mL of cell suspension was inoculated under the skin of the right back of Balb/cnude mice (Shanghai Ling Biotechnology) at a concentration of 1X 10 ⁷ A/mouse. When the tumor volume reaches 150-200mm ³ At this time, animals were divided into 5 groups according to tumor volume by a random block method, 8 mice per group, and the average tumor volume reached about 195mm ³ . Mice were treated with physiological saline, trastuzumab (3.3 mg/kg), 059-1.43.1-H1L3 (3.3 mg/kg), C033 (5.5 mg/kg) and trastuzumab+059-1.43.1-H1L 3 (3.3+3.3 mg/kg), respectively, for four weeks, 2 times per week. A total of 8 treatments were given. The group diary was Day 0 and dosing was started according to animal body weight. Tumor volumes and body weights were measured twice a week. During the administration period, if the animal body weight is reduced by more than 15% compared to Day 0 (BWL. Gtoreq.15%), the administration is stopped. When the average tumor volume of any group exceeds 2000mm ³ Or at the completion of the experiment, animals were sacrificed. Tumor Volume (TV) is calculated as: 1/2 Xa Xb ² Wherein a and b are the length and width of the tumor measurement, respectively. Calculation of tumor inhibition rate TGI (%): TGI (%) = [ (1- (mean tumor volume at the end of dosing of a treatment group-mean tumor volume at the beginning of dosing of a treatment group))/(mean tumor volume at the end of treatment of a solvent control group-mean tumor volume at the beginning of treatment of a solvent control group)]X 100%. In this study, experimental data are all expressed in mean±sem.

As shown in table 11 and fig. 9, the Tumor Growth Inhibition (TGI) of the C033 bispecific antibody alone was 73.6% better than the Qu Tuo bead single drug group, the 059-1.43.1-H1L3 single drug group, and the trastuzumab+059-1.43.1-H1L 3 combination group.

Table 11: anti-SKOV 3 tumor model effect of bispecific antibodies

Example 9: anti-tumor efficacy of bispecific antibody drug combinations in HCC-1954 xenograft model

Human breast cancer HCC-1954 cells (ATCC) were cultured in RPMI-1640 medium containing 10% FBS and maintained at 5% CO ₂ Is placed in a saturated humidity incubator at 37 ℃. HCC-1954 cells in logarithmic growth phase were collected, resuspended in DPBS containing 50% Matrigel, and the cell concentration was adjusted to 2.5X10 ⁷ And each mL. 0.2mL of the cell suspension was inoculated subcutaneously into the right back of Balb/c nude mice (Vetolihua) until the average tumor volume reached 150-200mm ³ The administration of the packets was started at that time. Animals were divided into 7 groups of 8 mice each with an average tumor volume of up to about 190mm ³ . Mice were treated with physiological saline, trastuzumab (6 mg/kg), pertuzui (6 mg/kg), 059-1.43.1-H1L3 (6 mg/kg), c033+ pertuzui (10+6mg/kg), trastuzui+pertuzui (6+6mg/kg) and trastuzui+pertuzui+059-1.43.1-H1L 3 (6+6+6mg/kg) for four weeks, 1 time per week, respectively. A total of 4 treatments were given. The group diary was Day 0 and dosing was started according to animal body weight. Tumor volumes and body weights were measured twice a week. During the administration period, if the animal body weight is reduced by more than 15% compared to Day 0 (BWL. Gtoreq.15%), the administration is stopped. When the average tumor volume of any group exceeds 3000mm ³ Or at the completion of the experiment, animals were sacrificed. Tumor Volume (TV) is calculated as: 1/2 Xa Xb ² Wherein a and b are the length and width of the tumor measurement, respectively. Calculation of tumor inhibition rate TGI (%): TGI (%) = [ (1- (mean tumor volume at the end of dosing of a treatment group-mean tumor volume at the beginning of dosing of a treatment group))/(mean tumor volume at the end of treatment of a solvent control group-solvent control groupAverage tumor volume at initial treatment]X 100%. In this study, experimental data are all expressed in mean±sem.

As shown in table 12 and fig. 10, the TGI of the c033+ pertuzus combined group was 103% on this xenograft model, significantly superior to the mab group and other combined groups.

Table 12: anti-HCC-1954 tumor model effect of bispecific antibody drug combination

Example 10: anti-tumor efficacy of bispecific or trispecific antibodies in JIMT-1 xenograft models

Human breast cancer JIMT-1 cells (Nanjac Bai Co.) were cultured in DMEM medium containing 10% FBS and maintained at 5% CO ₂ Is placed in a saturated humidity incubator at 37 ℃. Collecting JIMT-1 cells in logarithmic growth phase, re-suspending in DMEM basal medium containing 50% Matrigel, and adjusting cell concentration to 2×10 ⁷ And each mL. Inoculating 0.1mL cell suspension under aseptic condition to the right armpit of Balb/cnude mice (JieXtensikang) at a concentration of 2×10 ⁶ A/mouse. When the tumor volume reaches 150-200mm ³ At this time, animals were divided into 9 groups of 8 mice each according to tumor volume by a random block method, and the average tumor volume was about 180mm ³ . Mice were treated with physiological saline, trastuzumab (10 mg/kg), pertuzui (10 mg/kg), 059-1.43.1-H1L3 (10 mg/kg), c033+ pertuzui (16.67+10 mg/kg), C051 (20 mg/kg), C065 (20 mg/kg), trastuzui+pertuzui (10+10 mg/kg) and trastuzui+pertuzui+059-1.43.1-H1L 3 (10+10+10 mg/kg), respectively, for four weeks, 2 times per week. A total of 8 treatments were given. The group diary was Day 0 and dosing was started according to animal body weight. Tumor volumes and body weights were measured twice a week. During the administration period, if the animal body weight is reduced by more than 15% compared to Day 0 (BWL. Gtoreq.15%), the administration is stopped. When any oneAverage tumor volume of the group was over 2000mm ³ Or at the completion of the experiment, animals were sacrificed. Tumor Volume (TV) is calculated as: 1/2 Xa Xb ² Wherein a and b are the length and width of the tumor measurement, respectively. Calculation of tumor inhibition rate TGI (%): TGI (%) = [ (1- (mean tumor volume at the end of dosing of a treatment group-mean tumor volume at the beginning of dosing of a treatment group))/(mean tumor volume at the end of treatment of a solvent control group-mean tumor volume at the beginning of treatment of a solvent control group) ]X 100%. In this study, experimental data are all expressed in mean±sem.

As shown in table 13 and fig. 11, the Tumor Growth Inhibition (TGI) of the C051 and C065 trispecific antibodies was 111.9% and 115.0% superior to trastuzumab+pertuzu+059-1.43.1-H1L 3 mab combination group. 131.2% of the C033+ pertuzu combined group, significantly higher than all other groups, indicated that the efficacy of the C033 and pertuzu drug combination was superior to that of the single antigen-targeted antibody.

Table 13: anti-JIMT-1 tumor model Effect of bispecific or trispecific antibodies

Group of	Test article	Dosage (mg/kg)	Treatment mode	TGI
					1	Physiological saline control group	N/A	IP,BIW×4	N/A
2	059-1.43.1-H1L3	10	IP,BIW×4	-8.3％
					3	Trastump bead	10	IP,BIW×4	37.9％
4	Partuzu (al) ball	10	IP,BIW×4	54.3％
					5	C033+ pertuzhu	16.67+10	IP,BIW×4	131.2％
6	C051	20	IP,BIW×4	111.9％
					7	C065	20	IP,BIW×4	115.0％
8	Trastuzumab and pertuzhu	10+10	IP,BIW×4	44.4％
					9	trastuzumab+pertuzhb+059-1.43.1-H1L 3	10+10+10	IP,BIW×4	101.1％

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. A multispecific antibody that specifically binds CD47 and HER2 comprising a first antigen-binding domain specific for HER2 extracellular domain IV, which is a Fab fragment, and a second antigen-binding domain specific for CD47, which is a scFv.

2. The multi-specific antibody of claim 1, wherein,

a) The first antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain CDR1 as shown in SEQ ID NO 39, a heavy chain CDR2 as shown in SEQ ID NO 40, a heavy chain CDR3 as shown in SEQ ID NO 41, a light chain variable region (VL) comprising a light chain CDR1 as shown in SEQ ID NO 42, a light chain CDR2 as shown in SEQ ID NO 43, a light chain CDR3 as shown in SEQ ID NO 44; and/or the number of the groups of groups,

b) The second antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain CDR1 as shown in SEQ ID NO:51, a heavy chain CDR2 as shown in SEQ ID NO:52, a heavy chain CDR3 as shown in SEQ ID NO:53, and a light chain variable region (VL) comprising a light chain CDR1 as shown in SEQ ID NO:54, a light chain CDR2 as shown in SEQ ID NO:55, a light chain CDR3 as shown in SEQ ID NO: 56.

3. The multispecific antibody of claim 1 or 2, wherein the first antigen-binding domain comprises a VH as set forth in SEQ ID No. 1 or variant thereof and a VL as set forth in SEQ ID No. 2 or variant thereof; and/or the second antigen binding domain comprises a VH as set forth in SEQ ID No. 5 or a variant thereof and a VL as set forth in SEQ ID No. 6 or a variant thereof; the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it was derived, or has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to it; preferably, the substitutions are conservative substitutions.

4. The multispecific antibody of any one of claims 1-3, further comprising an Fc domain comprising a first monomer and a second monomer;

preferably, the first and second antigen binding domains are linked to the N-terminus of the first and second monomers of the Fc domain, respectively, optionally through a linker.

5. The multispecific antibody of any one of claims 1-4, wherein the first antigen-binding domain is a Fab fragment and the second antigen-binding domain is a scFv, the multispecific antibody comprising:

and

Preferably, the Fc domain monomer of the third peptide chain is capable of forming a dimer with the Fc domain monomer of the heavy chain constant region (CH) of the second peptide chain.

6. The multispecific antibody of any one of claims 1-5, further comprising a third antigen-binding domain specific for HER2 ectodomain II.

7. The multispecific antibody of claim 6, wherein the third antigen-binding domain comprises a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID No. 45, heavy chain CDR2 shown in SEQ ID No. 46, heavy chain CDR3 shown in SEQ ID No. 47, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID No. 48, light chain CDR2 shown in SEQ ID No. 49, light chain CDR3 shown in SEQ ID No. 50;

preferably, the third antigen binding domain is an scFv.

8. The multispecific antibody of claim 6 or 7, wherein the third antigen-binding domain comprises a VH as set forth in SEQ ID No. 3 or variant thereof and a VL as set forth in SEQ ID No. 4 or variant thereof; the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it was derived, or has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to it; preferably, the substitutions are conservative substitutions.

9. The multispecific antibody of any one of claims 6-8, further comprising an Fc domain comprising a first monomer and a second monomer;

preferably, the first antigen binding domain is attached to the N-terminus of the first monomer of the Fc domain, optionally through a linker;

preferably, one of the second or third antigen binding domain is optionally linked to the N-terminus of the second monomer of the Fc domain by a linker, the other of which is optionally linked to the C-terminus of the first or second monomer, or the light chain C-terminus of the first antigen binding domain by a linker;

preferably, the second antigen binding domain is linked to the N-terminus of the second monomer of the Fc domain, optionally via a linker, and the third antigen binding domain is linked to the C-terminus of the first monomer, optionally via a linker;

preferably, the third antigen binding domain is attached to the N-terminus of the second monomer of the Fc domain, optionally via a linker, and the second antigen binding domain is attached to the light chain C-terminus of the first antigen binding domain, optionally via a linker.

10. The multispecific antibody of any one of claims 6-9, wherein the first antigen-binding domain is a Fab fragment, the second antigen-binding domain and the third antigen-binding domain are scFv, the multispecific antibody comprising:

and

11. The multispecific antibody of any one of claims 6-9, wherein the first antigen-binding domain is a Fab fragment, the second antigen-binding domain and the third antigen-binding domain are scFv, the multispecific antibody comprising:

and

12. The multispecific antibody of any one of claims 1-11, wherein the Fc domain comprises a modification to promote dimerization of the first monomer and the second monomer;

Preferably, the CH3 domain of the Fc domain comprises an amino acid substitution;

preferably, the modifications comprise a "knob" modification in one of the two monomers of the Fc domain and a "pocket" modification in the other of the two monomers of the Fc domain to form a "knob-in-pocket" modification;

preferably, the first and second monomers of the Fc domain comprise the amino acid sequences as shown in SEQ ID NOS 29 and 30, respectively.

13. The multispecific antibody of any one of claims 1-12, wherein the multispecific antibody comprises:

(1) A first peptide chain comprising the sequence shown in SEQ ID NO. 7, a second peptide chain comprising the sequence shown in SEQ ID NO. 8 and a third peptide chain comprising the sequence shown in SEQ ID NO. 9;

(2) A first peptide chain comprising the sequence shown in SEQ ID NO. 10, a second peptide chain comprising the sequence shown in SEQ ID NO. 8 and a third peptide chain comprising the sequence shown in SEQ ID NO. 11; or alternatively, the first and second heat exchangers may be,

(3) A first peptide chain comprising the sequence shown in SEQ ID NO. 7, a second peptide chain comprising the sequence shown in SEQ ID NO. 12 and a third peptide chain comprising the sequence shown in SEQ ID NO. 9.

14. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the multispecific antibody or at least one peptide chain thereof of any one of claims 1-13.

15. A vector comprising the isolated nucleic acid molecule of claim 14;

preferably, the vector comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody of any one of claims 1-13, and the nucleotide sequences encoding each peptide chain are present on the same or different vectors.

16. A host cell comprising the isolated nucleic acid molecule of claim 14 or the vector of claim 15.

17. A composition comprising the multispecific antibody of any one of claims 1-5 and a monoclonal antibody specific for HER2 ectodomain II;

preferably, the monoclonal antibody comprises a heavy chain variable region (VH) comprising a heavy chain CDR1 as shown in SEQ ID NO. 45, a heavy chain CDR2 as shown in SEQ ID NO. 46, a heavy chain CDR3 as shown in SEQ ID NO. 47, and a light chain variable region (VL) comprising a light chain CDR1 as shown in SEQ ID NO. 48, a light chain CDR2 as shown in SEQ ID NO. 49, a light chain CDR3 as shown in SEQ ID NO. 50;

preferably, the monoclonal antibody comprises a VH as shown in SEQ ID NO. 3 and a VL as shown in SEQ ID NO. 4.

18. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1-13, the isolated nucleic acid molecule of claim 14, the vector of claim 15, the host cell of claim 16 or the composition of claim 17, and a pharmaceutically acceptable carrier and/or excipient.

19. Use of the multispecific antibody of any one of claims 1-13, the isolated nucleic acid molecule of claim 14, the vector of claim 15, the host cell of claim 16, the composition of claim 17 or the pharmaceutical composition of claim 18 in the manufacture of a medicament for the treatment of a tumor;

preferably, the tumor is HER2 positive and/or CD47 positive;

preferably, the tumor is selected from breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, colorectal cancer, non-hodgkin's lymphoma, chronic lymphoma leukemia, multiple myeloma, acute myelogenous leukemia, acute lymphoma leukemia, glioma, melanoma, or any combination thereof.