Anti-CD47-CLDN18.2 bispecific antibody and its use

Abstract

The present invention relates to the field of biopharmaceuticals. In particular, the present invention relates to an anti-CD47-CLDN18.2 bispecific antibody and its use.

Description

[Technical Field]

[0001] This application claims priority to Chinese Patent Application No. 202111214360.8, titled "Anti-CD47-CLDN18.2 Bispecific Antibody and Use thereof," filed on 19 October 2021, the entirety of which is incorporated herein by reference.

[0002] This invention belongs to the field of biopharmaceuticals. Specifically, this invention relates to an anti-CD47-CLDN18.2 bispecific antibody and its use. [Background technology]

[0003] CD47 is a transmembrane glycoprotein widely expressed on the cell surface, belonging to the immunoglobulin superfamily. It interacts with signal regulatory protein α (SIRPα), thrombospondin-1 (TSP1), and integrins to mediate a range of cellular responses, including apoptosis, proliferation, and immunity. Blocking the CD47-SIRPα pathway with anti-CD47 antibodies has been shown to effectively mediate phagocytosis of tumor cells, thereby inhibiting the proliferation of various hematological and solid tumors in vivo. However, CD47 is not only highly expressed in tumor cells, but also in large quantities in normal cells, such as red blood cells, meaning that CD47-targeted therapies may cause undesirable side effects. Some anti-CD47 antibodies disclosed in the prior art (see, for example, US20160304609) not only bind to red blood cells and cause severe anemia, but also require doses of up to 30 mg / kg for administration. These characteristics pose a significant challenge to the clinical application of anti-CD47 antibodies.

[0004] CLDN18 belongs to the Claudins protein family and was discovered by Shoichiro Tsukita et al. in 1998. It is an important molecule that constitutes the tight junction of epithelial cells, and it also plays a role in determining the permeability of epithelial cells and inhibiting the diffusion of cell membrane surface proteins and lipids (Gunzel, D. and AS Yu (2013) Physiol Rev 932: 525-569). The human CLDN18 gene has two different exon 1s, and after transcription, variable splicing ultimately produces two protein subtypes, CLDN18.1 and CLDN18.2, which have different sequences only at the N-terminus. Due to its expression specificity in tumor cells and normal tissues, CLDN18.2 is currently a highly promising target for antitumor drugs. WO 2016165762A1 discloses the anti-CLDN18.2 antibody IMAB362 (Zolbetuximab), which demonstrated a significant improvement in survival (13.2 vs. 8.4 months) compared to standard chemotherapy in a phase 2 clinical trial for gastric cancer, with the advantage being more pronounced in patients with high CLDN18.2 expression.

[0005] WO 2021003082A1 discloses a bispecific antibody against CLDN18.2 and CD47 that substantially does not bind to human erythrocytes. However, the development of novel bispecific antibodies targeting CLDN18.2 and CD47 remains necessary to offer further potential for cancer treatment. [Overview of the project]

[0006] In one embodiment, the present invention provides a bispecific antibody comprising a first antigen-binding moiety that binds to CD47 and a second antigen-binding moiety that binds to CLDN18.2, wherein the first antigen-binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising 1) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, 2) HCDR2 containing the amino acid sequence of SEQ ID NO: 5, and 3) HCDR3 containing the amino acid sequence of SEQ ID NO: 6, and the light chain variable region comprising 1) LCDR1 containing the amino acid sequence of SEQ ID NO: 7, 2) LCDR2 containing the amino acid sequence of SEQ ID NO: 8, and 3) LCDR3 containing the amino acid sequence of SEQ ID NO: 9.

[0007] In some embodiments, the second antigen-binding moiety includes an immunoglobulin monovariate domain (VHH) that binds to CLDN18.2. Preferably, the immunoglobulin monovariate domain includes CDR1 containing the amino acid sequence of SEQ ID NO:13, CDR2 containing the amino acid sequence of SEQ ID NO:14, and CDR3 containing the amino acid sequence of SEQ ID NO:15.

[0008] In a further embodiment, the present invention provides an isolated polynucleotide which encodes the bispecific antibody of the present invention.

[0009] The present invention further provides an expression vector comprising the polynucleotide of the present invention.

[0010] In another embodiment, the present invention provides a host cell comprising the polynucleotide or expression vector of the present invention.

[0011] The present invention further relates to antibody conjugates, which include the bispecific antibody of the present invention conjugates with at least one therapeutic agent.

[0012] In a further embodiment, the present invention relates to a pharmaceutical composition comprising the bispecific antibody or antibody conjugate of the present invention and a pharmaceutically acceptable carrier agent.

[0013] The present invention further relates to the use of the bispecific antibodies, antibody conjugates, or pharmaceutical compositions of the present invention in the manufacture of pharmaceuticals for the treatment of cancer. [Brief explanation of the drawing]

[0014] [Figure 1] This figure shows the schematic structure of the anti-CD47-CLDN18.2 bispecific antibody. [Figure 2ab] This figure shows the binding activity of an anti-CD47-CLDN18.2 bispecific antibody to CD47 in red blood cells. [Figure 3ab] Figure 3 shows the binding activity of anti-CD47-CLDN18.2 bispecific antibodies to tumor cells expressing a single target and a dual target. Figure 3a shows the binding activity of anti-CD47-CLDN18.2 bispecific antibodies to NUGC-4 cells, and Figure 3b shows the binding activity of anti-CD47-CLDN18.2 bispecific antibodies to hCLDN18.2-NUGC-4 cells. [Figure 4abc] Figures 4a and 4c show the ability of anti-CD47-CLDN18.2 bispecific antibodies to block the binding of human CD47 to its receptor SIRPα in tumor cells expressing single and dual targets. Figure 4a shows the ability of anti-CD47-CLDN18.2 bispecific antibodies to block the binding of human CD47 to its receptor SIRPα in NUGC-4 cells, while Figures 4b and 4c show the ability of anti-CD47-CLDN18.2 bispecific antibodies to block the binding of human CD47 to its receptor SIRPα in hCLDN18.2-NUGC-4 cells. [Figure 5ab] This figure shows the inhibitory effect of an anti-CD47-CLDN18.2 bispecific antibody on tumor growth in mice. [Figure 6] This figure shows the ADCP activity of the anti-CD47-CLDN18.2 bispecific antibody as measured by a bioluminescent reporter gene.

Mode for Carrying Out the Invention

[0015] Definition In the present invention, unless otherwise specified, scientific and technical terms used in this specification have meanings generally understood by those skilled in the art. And the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used in this specification are terms widely used in the corresponding fields and normal procedures. At the same time, in order to better understand the present invention, the definitions and interpretations of related terms are provided below.

[0016] As used in this specification, the expressions "comprising", "including", "containing" and "having" are non-limiting and indicate that the recited elements, steps or components are included, but do not exclude other elements, steps or components not recited. The expression "consisting of" does not include any unspecified element, step or component. The expression "substantially consisting of" means that the scope is limited to optionally present elements, steps or components that do not significantly affect the basic and novel features of the claimed subject matter in addition to the specific elements, steps or components. It should be understood that the expressions "substantially consisting of" and "consisting of" are covered by the meaning of the expression "comprising".

[0017] As used herein, "antibody" refers to an immunoglobulin or a fragment thereof, which specifically binds to an antigen epitope via at least one antigen-binding site. As used herein, the definition of antibody covers antigen-binding fragments. The term "antibody" includes multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single-domain antibodies, and antigen-binding fragments. Antibodies may be synthesized (e.g., produced by chemical conjugation or bioconjugation), obtained by enzymatic treatment, or produced recombinantly. Antibodies according to the present specification include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b). Antibodies may be "monovalent", "bivalent", "trivalent", or "tetravalent" or higher, referring to the fact that they contain 1, 2, 3, 4 or more antigen-binding sites.

[0018] As used herein, a "full-length antibody" typically includes four polypeptides, two heavy chains (HC) and two light chains (LC). Each light chain contains a "variable light region (VL)" and a "constant light region (CL)" from the N-terminus (amino acid terminus) to the C-terminus (carboxyl group terminus). Each heavy chain contains a "variable heavy region (VH)" and a "constant heavy region (CH)" from the N-terminus to the C-terminus. Generally, the constant heavy region of a full-length antibody may include CH1-hinge region-CH2-CH3 from the N-terminus to the C-terminus. In some immunoglobulin types (e.g., IgM and IgE), the constant heavy region may include CH1-hinge region-CH2-CH3-CH4 from the N-terminus to the C-terminus.

[0019] The light chain variable region and the heavy chain variable region may each include three highly variable "complementarity-determining regions (CDRs)" and four relatively conservative "framework regions (FRs)," and are linked from the N-terminus to the C-terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In this specification, the CDRs of the light chain variable region (CDRL or LCDR) may be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain variable region (CDRH or HCDR) may be referred to as HCDR1, HCDR2, and HCDR3.

[0020] In this invention, the amino acid sequences of the CDRs are all represented according to the AbM definition rules (the sequences in the claims of this invention are also represented according to the AbM definition rules). However, as is well known to those skilled in the art, the CDR of an antibody can be defined by various methods in this art, for example, Chothia (see, e.g., Chothia, C. et al., Nature, 342, 877-883 (1989), and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)), which is based on the three-dimensional structure of the antibody and the topology of the CDR loop; Kabat (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), which is based on antibody sequence variability; and AbM (Martin, ACR and J. Allen (2007) “Bioinformatics tools for antibody engineering,” in S. Dubel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH) This is a North CDR definition based on affinity propagation clustering, utilizing numerous crystal structures, as described in Verlag (pp. 95-118), Contact (MacCallum, RM et al., (1996) J. Mol. Biol. 262:732-745), IMGT (Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc., and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)).As those skilled in the art will understand, unless otherwise specified, the terms “CDR” and “complementarity-determining region” for a given antibody or region thereof (e.g., variable region) should be understood to cover the complementarity-determining region as defined by any one of the known schemes described in the present invention. While the scope claimed in the claims of the present invention is a sequence shown according to the AbM definition rules, corresponding amino acid sequences according to other CDR definition rules also fall within the scope of protection of the present invention.

[0021] Therefore, when limiting antibodies by a specific CDR sequence as defined in the present invention, the range of the antibody further covers antibodies whose variable region sequence includes the specific CDR sequence, but whose described CDR boundary differs from the specific CDR boundary defined in the present invention due to the application of a different scheme (e.g., a different assignment system rule or combination).

[0022] As used herein, the terms “framework region” and “framework area” are interchangeable. As used herein, the terms “framework region,” “framework area,” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR sequence defined above.

[0023] As used herein, “single-domain antibody (sdAb)” or “nano-antibody” refers to an antibody containing a single immunoglobulin variable domain (single variable domain) as a functional antigen-binding fragment. Similar to the variable region of a full-length antibody, the single variable domain typically includes CDR1, CDR2, and CDR3 that form the antigen-binding site, as well as a supporting framework region. The single variable domain may be, for example, the variable domain of a heavy-chain antibody (VHH), a shark IgNAR variable domain, a human light-chain antibody variable domain, and a heavy-chain antibody variable domain.

[0024] As used herein, “antibody-dependent cell-mediated cytophagocytosis” or “ADCP” refers to a cell-mediated process in which nonspecific cytotoxic cells expressing the Fcγ receptor (FcγR) (e.g., monocytes, macrophages, neutrophils, and dendritic cells) recognize antibodies that bind to target cells (e.g., tumor cells) and subsequently phagocytose the target cells (e.g., tumor cells) as effector cells. In some embodiments, the anti-CD47-CLDN18.2 bispecific antibody of the present invention mediates ADCP against cancer cells expressing CLDN18.2 (particularly those expressing both CD47 and CLDN18.2).

[0025] As used herein, “percent (%) sequence identity” and “sequence identity” of amino acid sequences have definitions well known in the art and refer to the percentage of identity between two polypeptide sequences determined by sequence alignment (e.g., manual inspection or a well known algorithm). This can be determined using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0026] In this specification, an amino acid sequence "from" or "derived from" a reference amino acid sequence is identical or homologous to part or all of the reference amino acid sequence. For example, an amino acid sequence derived from the heavy chain constant region of human immunoglobulin may 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 with the wild-type sequence of the heavy chain constant region from which it is derived.

[0027] Non-essential regions in a polypeptide (e.g., the CDR region of an antibody, non-essential amino acids in the framework region, amino acids in the constant region) can be modified without altering the polypeptide's function, for example, by substituting, adding, and / or deleting one or more amino acids. As those skilled in the art will understand, amino acids in non-essential regions of a polypeptide may be substituted with suitable conserved amino acids and generally do not alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. co., p.224). Suitable conserved substitutions are well known to those skilled in the art. In some cases, amino acid substitutions are non-conservative substitutions. As those skilled in the art will understand, amino acid mutations or modifications can alter the properties of an antibody or antibody fragment, for example, by changing the type of antibody glycosylation modification, changing its ability to form interchain disulfide bonds, or providing active groups for the production of antibody conjugates. Antibodies or antigen-binding fragments containing such amino acid mutations or modifications also fall within the scope of the bispecific antibodies of the present invention.

[0028] The anti-CD47-CLDN18.2 bispecific antibody of the present invention or the polynucleotide encoding it may be isolated. As used herein, the expression “isolated” means that the substance (e.g., polynucleotide or polypeptide) is isolated from the source or environment in which it exists, i.e., substantially free from any other components.

[0029] In this specification, the terms “polynucleotide” and “nucleic acid” are interchangeable and are used to describe oligomers or polymers comprising at least two linked nucleotides or nucleotide derivatives, which may typically include deoxyribose nucleic acids (DNA) and levosic nucleic acids (RNA).

[0030] In this specification, “vector” is a medium for introducing exogenous polynucleotides into host cells, and if the vector is transformed into a suitable host cell, the exogenous polynucleotides may be amplified or expressed. Vectors are usually left free, but may be designed to integrate a gene or a portion thereof into a genomic chromosome. As used herein, the definition of a vector covers plasmids, linear plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, poxvirus vectors, and baculovirus vectors.

[0031] As used herein, the term “expression” refers to the production of RNA and / or polypeptides.

[0032] As used herein, “expression vector” refers to a vector capable of expressing a polynucleotide (including DNA and RNA) of interest. For example, an expression vector can manipulate a polynucleotide sequence (including DNA and RNA) encoding a polypeptide of interest with regulatory sequences (e.g., promoters and ribosome-binding sites) that can influence the expression of the polynucleotide sequence. The regulatory sequences may include promoter and terminator sequences and may optionally include replication origins, selection markers, enhancers, adenylation signals, etc. The expression vector may be a plasmid, phage vector, recombinant virus, or other vector, which, when introduced into a suitable host cell, induces the expression of the polynucleotide of interest. Suitable expression vectors are well known to those skilled in the art. Those skilled in the art may, if necessary, prepare expression vectors as vectors that are replicable in host cells, remain free in host cells, or are integrated into the host cell genome.

[0033] As used herein, “host cell” is a cell that receives, maintains, replicates, or amplifies a vector. The host cell may be used to express a polynucleotide or polypeptide encoded by the vector. The host cell may be a eukaryotic cell or a prokaryotic cell. Examples include prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus species, insect cells such as S2 Drosophila cells or Sf9 cells, and animal cells such as fibroblasts, CHO cells, COS cells, HeLa cells, NSO cells, or HEK293 cells.

[0034] As used herein, the term “treatment” refers to improvement of a disease / symptom, for example, reducing or eliminating the disease / symptom, or preventing or slowing the onset, progression, and / or worsening of the disease / symptom. Therefore, treatment includes prevention, cure, and / or remission.

[0035] As used herein, the term “pharmaceutically acceptable carrier” means a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure-maintaining reagents, absorption-delaying reagents, and 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, for example, Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, a variety of antibacterial and antifungal reagents, for example, parabens, trichlorobutanol, phenol, and sorbic acid. Reagents that maintain osmotic pressure include, but are not limited to, sugars, sodium chloride, and their analogues. Reagents that slow down absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), ols, and polyols (e.g., glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal reagents, such as thimerosal, 2-phenoxyethanol, parabens, trichlorobutanol, phenol, and sorbic acid. Stabilizers have a meaning generally understood by those skilled in the art and can stabilize the desired activity of the active ingredient in a pharmaceutical product. They include, but are not limited to, monosodium glutamate, gelatin, SPGA (Sucrose-Phosphate-Glutamate-Albumin), sugars (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 their degradation products (e.g., lactalbumin hydrolysate).

[0036] As used herein, examples of mammals include, but are not limited to, humans, non-human primates, rats, mice, cattle, horses, pigs, sheep, alpacas, dogs, and cats. In this specification, the term “subject” refers to a mammal, such as a human. In some embodiments, the subject is a human. In some embodiments, the subject is a cancer patient, a human or animal suspected of having cancer or at risk of having cancer.

[0037] Anti-CD47-CLDN18.2 bispecific antibody

[0038] The present invention provides an anti-CD47-CLDN18.2 bispecific antibody comprising a first antigen-binding moiety that binds to CD47 and a second antigen-binding moiety that binds to CLDN18.2, wherein the first antigen-binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL). The aforementioned heavy chain variable region is 1) HCDR1 containing the amino acid sequence of SEQ ID NO:4 or a variant thereof, 2) HCDR2 containing the amino acid sequence of SEQ ID NO:5 or its variant, 3) comprising HCDR3 containing the amino acid sequence of SEQ ID NO:6 or a variant thereof, and The aforementioned light chain variable region is 1) LCDR1 containing the amino acid sequence of SEQ ID NO:7 or a variant thereof, 2) LCDR2 containing the amino acid sequence of SEQ ID NO:8 or its variant, 3) LCDR3 containing the amino acid sequence of SEQ ID NO:9 or its variant, Here, the variant has one or two amino acid substitutions, additions, and / or deletions compared to the sequence from which it is derived.

[0039] In one specific embodiment, the first antigen-binding portion includes a first antigen-binding portion that specifically binds to CD47, and the first antigen-binding portion includes a heavy chain variable region (VH) and a light chain variable region (VL). The aforementioned heavy chain variable region is 1) HCDR1 containing the amino acid sequence of SEQ ID NO:4, 2) HCDR2 containing the amino acid sequence of SEQ ID NO:5, 3) Contains HCDR3 containing the amino acid sequence of SEQ ID NO:6, and, The aforementioned light chain variable region is 1) LCDR1 containing the amino acid sequence of SEQ ID NO:7, 2) LCDR2 containing the amino acid sequence of SEQ ID NO:8, 3) Contains LCDR3 containing the amino acid sequence of SEQ ID NO:9.

[0040] In some embodiments, the VH comprises 1) the amino acid sequence of SEQ ID NO:10, or 2) an amino acid sequence having 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%, and / or The VL includes 1) the amino acid sequence of SEQ ID NO:11, or 2) an amino acid sequence having 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%, or at least 99% sequence identity with SEQ ID NO:11.

[0041] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:10, and VL comprises the amino acid sequence of SEQ ID NO:11.

[0042] The first antigen-binding moiety may include any form of antigen-binding fragment, such as scFv, dsFv, scdsFv, Fab, Fab', or F(ab')2. According to the present invention, the first antigen-binding moiety specifically binds to CD47 on the surface of cancer cells but does not bind to or substantially binds to CD47 on red blood cells, thereby preventing hemagglutination of the bispecific antibody of the present invention.

[0043] The second antigen-binding moiety may include any form of antigen-binding fragment, including, but not limited to, scFv, dsFv, scdsFv, Fab, Fab', F(ab')2, and a single variable domain. In some embodiments, the second antigen-binding moiety includes an immunoglobulin single variable domain that specifically binds to CLD18.2. An immunoglobulin single variable domain that specifically binds to CLD18.2 is described, for example, in CN112480248A and WO 2020238730A1, the entirety of which is incorporated herein by reference. In some embodiments, the immunoglobulin single variable domain includes CDR1 containing the amino acid sequence of SEQ ID NO:13 or a variant thereof, CDR2 containing the amino acid sequence of SEQ ID NO:14 or a variant thereof, and CDR3 containing the amino acid sequence of SEQ ID NO:15 or a variant thereof, wherein the variant has one or two amino acid substitutions, additions and / or deletions compared to the sequence from which it is derived. In one specific embodiment, the immunoglobulin monovariable domain includes CDR1 containing the amino acid sequence of SEQ ID NO:13, CDR2 containing the amino acid sequence of SEQ ID NO:14, and CDR3 containing the amino acid sequence of SEQ ID NO:15. In some embodiments, the immunoglobulin monovariable domain includes the amino acid sequence of SEQ ID NO:12. In further embodiments, the immunoglobulin monovariable domain includes an amino acid sequence having 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%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:12.

[0044] In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety are linked via a linker. The linker may be a peptide linker or a chemical bond, and is preferably a linker. Exemplary peptide linkers include polyglycine (G), polyalanine (A), polyserine (S), or combinations thereof, e.g., GGAS, GGGS, GGGSG, or (G4S). n This may include, but is not limited to, the above, where n is an integer from 1 to 20. Preferably, n is an integer from 1 to 5. In one specific embodiment, the peptide linker contains the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:23.

[0045] In some embodiments, the anti-CD47-CLDN18.2 bispecific antibody of the present invention further comprises an immunoglobulin constant region. The immunoglobulin constant region may be the heavy chain constant region (CH) and light chain constant region (CL) of any species of immunoglobulin. The heavy chain constant region may be derived from the heavy chain constant region of any subtype (e.g., IgA, IgD, IgE, IgG, and IgM), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b) immunoglobulin, or a combination thereof. In preferred embodiments, the heavy chain constant region may include at least an Fc region, for example, the heavy chain constant region of IgG1 may include all or part of the hinge region -CH2-CH3 or CH1-hinge region-CH2-CH3. The light chain constant region may be derived from a λ (Lambda) light chain or a κ (Kappa) light chain constant region. In one preferred embodiment, the heavy chain constant region is the heavy chain constant region of human IgG1. In some embodiments, the heavy chain constant region includes the amino acid sequence of SEQ ID NO:2. In one preferred embodiment, the light chain constant region is the human κ light chain constant region. In some embodiments, the light chain constant region includes the amino acid sequence of SEQ ID NO:3.

[0046] In some embodiments, the VH and VL of the first antigen-binding moiety are fused to the N-terminus of the heavy chain constant region and the light chain constant region, respectively, and the single variable domain of the second antigen-binding moiety is optionally fused via a linker to the N-terminus of VH, the N-terminus of VL, the C-terminus of the heavy chain constant region, or the C-terminus of the light chain constant region.

[0047] In some embodiments, the anti-CD47-CLDN18.2 bispecific antibody comprises a first polypeptide and a second polypeptide, the first polypeptide comprising a first antigen-binding moiety VH and a heavy chain constant region, and the second polypeptide comprising a first antigen-binding moiety VL and a light chain constant region, wherein a single variable domain of the second antigen-binding moiety is optionally fused via a linker to the N-terminus of the VH or VL or the C-terminus of the heavy chain constant region or light chain constant region. In some embodiments, the single variable domain is optionally fused via a linker to the N-terminus of the VH or the C-terminus of the heavy chain constant region, and the first polypeptide may also be called a fused heavy chain. In some embodiments, the fused heavy chain has the structure of formula (I) or formula (III) described below. In another embodiment, the single variable domain of the second antigen-binding moiety is optionally fused via a linker to the N-terminus of the VL or the C-terminus of the light chain constant region, and the second polypeptide may also be called a fused light chain. In some embodiments, the fused light chain has the structure of formula (IV) or formula (VI) described below.

[0048] In some embodiments, the first polypeptide has the structure of formula (I), VH-CH-Linker-VHH formula (I), The second polypeptide has the structure of (II), VL-CL formula (II), Here, VH and VL are the heavy chain variable region and light chain variable region of the first antigen-binding region described above, respectively. VHH is the immunoglobulin monovariate domain described above. CH and CL are the heavy chain steady region and light chain steady region described above, respectively. Linker is a linker.

[0049] In some embodiments, the first polypeptide has the structure of formula (I) and comprises the amino acid sequence of SEQ ID NO: 20, and the second polypeptide has the structure of formula (II) and comprises the amino acid sequence of SEQ ID NO: 17.

[0050] In some embodiments, the first polypeptide has the structure of formula (III), VHH-Linker-VH-CH formula (III), The second polypeptide has the structure of formula (II), Here, VHH is the immunoglobulin monovariate domain described above. VH is the heavy chain variable region of the first antigen-binding region described above, CH is the heavy chain constant region described above. Equation (II) is as described above, Linker is a linker.

[0051] In some embodiments, the first polypeptide has the structure of formula (III) and comprises the amino acid sequence of SEQ ID NO: 16, and the second polypeptide has the structure of formula (II) and comprises the amino acid sequence of SEQ ID NO: 17.

[0052] In some embodiments, the first polypeptide has the structure of formula (III) and comprises the amino acid sequence of SEQ ID NO: 21, and the second polypeptide has the structure of formula (II) and comprises the amino acid sequence of SEQ ID NO: 17.

[0053] In further embodiments, the first polypeptide has the structure of formula (III), and the second polypeptide has the structure of formula (IV), VHH-Linker-VL-CL formula (IV), Here, Equation (III) is as described above, VHH is the immunoglobulin monovariate domain described above. VL is the light chain variable region of the first antigen-binding portion described above, CL is the light chain constant region described above. Linker is a linker.

[0054] In some embodiments, the first polypeptide has the structure of formula (III) and comprises the amino acid sequence of SEQ ID NO: 16, and the second polypeptide has the structure of formula (IV) and comprises the amino acid sequence of SEQ ID NO: 19.

[0055] In another embodiment, the first polypeptide has the structure of formula (V), VH-CH type (V), The second polypeptide has the structure of formula (IV), Here, Equation (IV) is as described above, VH is the heavy chain variable region of the first antigen-binding region described above, CH is the heavy chain constant region described above. Linker is a linker.

[0056] In some embodiments, the first polypeptide has the structure of formula (V) and comprises the amino acid sequence of SEQ ID NO: 18, and the second polypeptide has the structure of formula (IV) and comprises the amino acid sequence of SEQ ID NO: 19.

[0057] In another embodiment, the first polypeptide has the structure of formula (V), and the second polypeptide has the structure of formula (VI), VL-CL-Linker-VHH Equation (VI), Here, Equation (V) is as described above, VHH is the immunoglobulin monovariate domain described above. VL is the light chain variable region of the first antigen-binding portion described above, CL is the light chain constant region described above. Linker is a linker.

[0058] In some embodiments, the anti-CD47-CLDN18.2 bispecific antibody of the present invention is 1) Block the binding of CD47 on the surface of cancer cells to SIRPα, 2) Induce macrophage phagocytosis of cancer cells expressing CD47 and CLDN18.2, and / or 3) It may bind to cancer cells expressing CD47 and CLDN18.2, but may not bind to red blood cells or may not bind substantially.

[0059] Polynucleotides, vectors, and host cells

[0060] In another embodiment, the present invention provides an isolated polynucleotide comprising a polynucleotide sequence encoding the anti-CD47-CLDN18.2 bispecific antibody of the present invention.

[0061] The polynucleotides of the present invention can be obtained by methods known in the art, such as isolation or chemical synthesis from phage display libraries, yeast display libraries, immunized animals, or immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells). The polynucleotides of the present invention can be codon-optimized for expression in host cells.

[0062] In further embodiments, the present invention further comprises a vector containing the polynucleotide of the present invention. In some embodiments, the polynucleotide of the present invention is cloned into an expression vector. The expression vector may further contain additional polynucleotide sequences, e.g., regulatory sequences and antibiotic resistance genes. The expression vector may further contain polynucleotide sequences encoding additional polypeptides. The additional polypeptides may be polypeptides that facilitate the detection and / or isolation of antibody or antigen-binding fragments, and include, but are not limited to, affinity tags (e.g., polyhistidine tags (His6) or glutathione S-transferase (GST) tags), polypeptides containing protease cleavage sites, and reporter proteins (e.g., fluorescent proteins).

[0063] In some embodiments, the polynucleotides of the present invention are prepared as recombinant nucleic acids. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis and DNA recombination techniques (e.g., polymerase chain reaction (PCR) techniques) (see Sambrook, J., EF Fritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0064] The polynucleotides of the present invention may be present in one or more expression vectors. In some embodiments, the expression vector is a DNA plasmid, for example, a DNA plasmid for expression in bacterial, yeast, or mammalian cells. In some other embodiments, the expression vector is a viral vector. In other embodiments, the expression vector is a phage vector or phagemide vector.

[0065] The present invention further comprises a host cell, which comprises at least one polynucleotide or vector described above. The polynucleotide or expression vector of the present invention can be introduced into a suitable host cell using a variety of methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection.

[0066] In a preferred embodiment, the host cell is for expressing the anti-CD47-CLDN18.2 bispecific antibody of the present invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, e.g., Escherichia coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells).

[0067] Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK cells (e.g., HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for antibody expression.

[0068] The present invention further provides a method for producing the anti-CD47-CLDN18.2 bispecific antibody, which is, (I) The step of culturing host cells of the present invention under appropriate conditions in order to express an anti-CD47-CLDN18.2 bispecific antibody, (II) The step of isolating the antibody from host cells or a culture thereof.

[0069] In some embodiments, a single vector is used, which contains a polynucleotide sequence encoding the heavy and light chains. In some embodiments, two vectors are used, one encoding the light chain of the antibody and the other encoding the heavy chain. In some embodiments, the host cell further contains a partner plasmid, which helps to improve the solubility, stability, and / or folding of the antibody. Techniques for isolating and purifying antibodies from host cells or their culture media are well known to those skilled in the art.

[0070] Antibody conjugate

[0071] The present invention further provides an antibody conjugate comprising the anti-CD47-CLDN18.2 bispecific antibody of the present invention conjugate with at least one therapeutic agent. The antibody-drug conjugate (ADC) is a typical antibody conjugate, where the therapeutic agent may be, for example, a cytotoxic agent.

[0072] As used herein, “conjugation” refers to the linking of two or more parts together by covalent or non-covalent action. In a preferred embodiment, the conjugation is a covalent conjugation.

[0073] The therapeutic agent may be selected from cytotoxic agents, therapeutic antibodies (e.g., antibodies or antigen-binding fragments thereof that specifically bind to another antigen), radioisotopes, oligonucleotides and their analogues (e.g., interfering RNA), bioactive peptides, protein toxins (e.g., diphtheria toxin, lysine), and enzymes (e.g., urease).

[0074] Cytotoxic drugs are substances that inhibit or reduce the activity or function of cells and / or kill them. Examples of cytotoxic drugs include maytansinoids (e.g., maytansine), auristatins (e.g., MMAF, MMAE, MMAD), duostatin, cryptophycin, vinca alkaloids (e.g., vinblastine, vincristine), colchicines, drostatins, taxanes, paclitaxel, docetaxel, cabazitaxel, engine antibiotics, cytochalasins, camptothecins, anthracycline antibiotics (e.g., donomycin, dihydroxyanthracindione, doxorubicin), and cytotoxic antibiotics (e.g., mitomycin, actinomycin, duocal). This includes, but is not limited to, duocarmycin (e.g., CC-1065), auromycin, duomycin, calicheamicin, endomycin, phenomycin, doxorubicin, daunorubicin, calicheamicin, cisplatin, ethidium bromide, zeosin, mitomycin, mitramycin, prazienolide, podophyllotoxin, etoposide, mitoxantrone, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nerarabine, carmustine, lomustine, methotrexate, melphalan, teniposide, and glucocorticoids.

[0075] Radioactive isotopes are, for example, 212 Bi, 213 Bi, 131 I, 125 I, 111 In, 177 Lu, 186 Re,188 Re, 153 Sm, 90 It may also be selected from Y. An antibody labeled with a radioisotope may also be called a radioimmunoconjugate.

[0076] In a preferred embodiment, the bioactive polypeptide is a polypeptide or protein having therapeutic activity, binding activity or enzymatic activity. Non-limiting examples of bioactive polypeptides include, but are not limited to, protein toxins (e.g., diphtheria toxin, ricin), enzymes (e.g., urease, horseradish peroxidase), cytokines.

[0077] In some embodiments, the therapeutic agent is a molecule having biological anti-tumor activity. Molecules having biological anti-tumor activity include, but are not limited to, cytotoxic agents, chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, antibodies targeting tumor-specific antigens, and other anti-tumor drugs. In one preferred embodiment, the therapeutic agent is a cytotoxic agent. In a further preferred embodiment, the therapeutic agent is a radioisotope.

[0078] Using any technique known in the art, a therapeutic agent can be conjugated to the anti-CD47-CLDN18.2 bispecific antibody of the present invention via a linker. The linker may include active groups for covalent conjugation, such as amine, hydroxylamine, maleimide group, carboxyl group, phenyl group, thiol, sulfhydryl group or hydroxy group. The linker may be cleavable or non-cleavable. Cleavable linkers are, for example, enzyme-cleavable linkers (e.g., peptides containing protease cleavage sites), pH-sensitive linkers (e.g., hydrazone linkers) or reducible linkers (e.g., disulfide bonds).

[0079] In some embodiments, the linker comprises an active group selected from amines, hydroxylamines, maleimide groups, carboxyl groups, phenyl groups, thiols, sulfhydryl groups, and hydroxyl groups. In some embodiments, the linker is a chemical bond. In some embodiments, the linker comprises an amino acid or a peptide consisting of 2 to 10 amino acids. The amino acids may be natural or unnatural.

[0080] In some embodiments, the therapeutic agent is conjugated with a linker to form an intermediate before conjugating with the anti-CD47-CLDN18.2 bispecific antibody of the present invention. In some embodiments, the intermediate is linked by forming a thioether bond with the sulfhydryl group of the anti-CD47-CLDN18.2 bispecific antibody of the present invention. The structure and method of producing such intermediates, as well as a method for producing an antibody conjugate using them, are described, for example, in International Patent Application Publication No. WO 2019114666, all of which are incorporated herein by reference.

[0081] Drug composition

[0082] The present invention further provides a pharmaceutical composition comprising the anti-CD47-CLDN18.2 bispecific antibody or antibody conjugate of the present invention and a pharmaceutically acceptable carrier agent.

[0083] Pharmacopoecially acceptable carriers include diluents, adhesives and linseeds, lubricants, disintegrants, preservatives, vehicles, dispersants, fluidizers, sweeteners, coatings, excipients, preservatives, and antioxidants (e.g., ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium pyrosulfite, sodium sulfite, ascorbic acid palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, The product may contain, but is not limited to, tartaric acid, phosphoric acid, etc., solubility enhancers, gelling agents, softening agents, solvents (e.g., water, alcohol, acetic acid, and syrup), buffers (e.g., phosphate buffers, histidine buffers, and acetate buffers), surfactants (e.g., nonionic surfactants, e.g., polysorbate 80, polysorbate 20, poloxamer, or polyethylene glycol), antibacterial agents, antifungal agents, isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption retarders, chelating agents, and emulsifiers. For compositions containing antibodies or antibody conjugates, suitable carriers may be selected from buffers (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine salt buffer), isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer), or combinations thereof.

[0084] The pharmaceutical compositions described herein may be in various dosage forms, including but not limited to solid, semi-solid, liquid, powder, or lyophilized forms. Preferably, the pharmaceutical compositions are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial administration (e.g., by injection or infusion). For compositions containing antibodies or antibody conjugates, preferred dosage forms are usually, for example, injectable solutions and lyophilized powders.

[0085] The pharmaceutical compositions according to this specification may be administered to a subject by any method known in the art, for example, by systemic or topical administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, intradermal, or intracavitary), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or topical). As those skilled in the art will understand, the exact dosage depends on various factors, such as the metabolic properties of the pharmaceutical composition, the duration of treatment, the excretion rate of a particular compound, the therapeutic purpose, the route of administration, and the subject's condition, such as the patient's age, health status, weight, sex, diet, medical history, and other factors known in the medical field. Methods of administration may include, for example, injection or infusion.

[0086] As a general guideline, the dosage range for the anti-CD47-CLDN18.2 bispecific antibody of the present invention may be approximately 0.0001 to 100 mg / kg, and more generally, 0.01 to 20 mg / kg of subject body weight. For example, the dosage may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, or 20 mg / kg body weight, or within the range of 1 to 20 mg / kg. An exemplary treatment scheme may involve administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months, or the initial dosing interval may be short and the later dosing intervals may be long. The method of administration may be intravenous infusion.

[0087] treatment

[0088] In a further embodiment, the present invention relates to the use of the anti-CD47-CLDN18.2 bispecific antibody, antibody conjugate, or pharmaceutical composition of the present invention in the manufacture of a pharmaceutical product for treating a disease in a subject.

[0089] The present invention further relates to the anti-CD47-CLDN18.2 bispecific antibody, antibody conjugate, or pharmaceutical composition, which is used to treat a disease.

[0090] The present invention further provides a method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the anti-CD47-CLDN18.2 bispecific antibody, antibody conjugate, or pharmaceutical composition of the present invention.

[0091] In some embodiments, the disease described above is cancer. As used herein, “cancer” includes, but is not limited to, hematological cancers and solid tumors. Cancer may further be metastatic cancer. “Metastasis” refers to the spread of cancer cells from their original site to other parts of the body. For example, an anti-CD47-CLDN18.2 bispecific antibody may be used to treat CLDN18.2-positive cancer. In a preferred embodiment, the anti-CD47-CLDN18.2 bispecific antibody is for treating CD47 and CLDN18.2-positive cancer. In some embodiments, the cancer is gastric cancer.

[0092] kit

[0093] The present invention further provides a kit comprising the anti-CD47-CLDN18.2 bispecific antibody, antibody conjugate, or pharmaceutical composition of the present invention, and instructions for use. The kit may further include a suitable container. In some embodiments, the kit further includes a dispensing device. Typically, the kit further includes a label, which is intended to indicate the intended use and / or method of use of the kit contents. The term “label” includes any written or recorded material that is on the kit, provided with the kit, or otherwise provided with the kit.

[0094] Effects of the invention The anti-CD47-CLDN18.2 bispecific antibody of the present invention can achieve at least one of the following beneficial effects: 1) Block the binding of CD47 on the surface of cancer cells to SIRPα, 2) Induce macrophage phagocytosis of cancer cells expressing CD47 and CLDN18.2, 3) By mediating ADCP against cancer cells expressing CLDN18.2, 4) It binds to cancer cells expressing CD47 and CLDN18.2, but does not bind to red blood cells or does not bind substantially.

[0095] Furthermore, compared to monoclonal antibodies that target only CD47, the anti-CD47-CLDN18.2 bispecific antibody of the present invention exhibits higher binding activity to CD47 and CLDN18.2-positive tumor cells, and a stronger blocking effect against the interaction between CD47 and SIRPα. Examples

[0096] The following examples are intended to illustrate the invention merely illustratively and should not be considered to limit the invention in any way. Materials and methods 1. Construction of antibody expression plasmids

[0097] DNA fragments encoding the antibody heavy chain and light chain were prepared using DNA recombination technology, and then cloned into the expression vector pcDNA3.4-TOPO (Invitrogen) to obtain antibody heavy chain and light chain expression plasmids. These plasmids were amplified in E. coli DH5α, and subsequently extracted and purified. 2. Antibody expression and purification

[0098] All antibodies were expressed using the ExpiCHO transient expression system (Thermo Fisher, A29133) (see WO 2020238730A1), and affinity purification was performed using MabSelect SuRe LX (GE, 17547403). 3. Identification of antibody purity by SDS-PAGE

[0099] Preparation of the reducing solution: 2 μg of the target antibody or IPI (Ipilimlumab, see reference) was added to 5× SDS sampling buffer (containing DTT with a final concentration of 5 mM), heated in a 100°C dry bath for 10 min, cooled to room temperature, and then centrifuged at 12000 rpm for 5 min. The supernatant was collected. The supernatant was added to a Bis-tris 4-15% gradient gel (Kimssey), and protein gel electrophoresis was performed. Subsequently, the protein bands were stained with Coomassie brilliant blue to develop color, and after destaining, the gel was scanned with an EPSON V550 color scanner, and the purity of the protein bands was calculated using the peak area normalization method in ImageJ. 4. Identification of antibody purity by SEC-HPLC

[0100] An Agilent HPLC 1100 chromatography column (XBridge BEH SEC 3.5 μm, 7.8 mm ID × 30 cm, Waters) was set to a flow rate of 0.8 mL / min, with a sample injection volume of 20 μL. The VWD detector wavelengths were 280 nm and 214 nm. The mobile phase was 150 mmol / L phosphate buffer with a pH of 7.4. All samples were diluted to 0.5 mg / mL in the mobile phase, and then blank and sample solutions were sampled sequentially. The proportions of high molecular weight polymers, antibody monomers, and low molecular weight substances in the samples were calculated according to the area normalization method. 5. Identification of antibody stability by differential scanning fluorescence (SIFluorescence)

[0101] Differential scanning fluorescence (DSF) provides information about the structural stability of proteins based on fluorescence change processes in protein maps, can detect changes in protein configuration, and can determine the melting temperature (Tm) of proteins.

[0102] A 0.2 mg / mL antibody sample solution was prepared, using PBS and IPI (Ipilimlumab; 0.2 mg / mL) as reference. The test samples were tripled and added to a 96-well plate (Nunc) at 19 μL / well. 1 μL of 100×SYPRO orange dye was added to each well, and the mixture was homogeneously mixed by pipetting to prepare the plate for on-machine testing. For thermal stability testing of the samples, an ABI 7500 FAST RT-PCR instrument was used, with the melting curve test type selected, continuous mode used, a scan temperature range of 25-95°C, a heating rate of 1%, equilibration at 25°C for 5 minutes, data collected during heating, the reporter group selected as "ROX", the quenching group as "None", and the reaction volume as 20 μL. The temperature corresponding to the first peak and dip of the first derivative of the melting curve was determined as the antibody melting temperature Tm. Example 1: Design of a bispecific antibody against CD47-CLDN18.2

[0103] This embodiment describes an exemplary anti-CD47-CLDN18.2 bispecific antibody, where the CLDN18.2-binding arm is an anti-CLDN18.2 single-domain antibody NA3S-H1 (amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 13, 14, and 15 respectively, and the amino acid sequence of VHH is shown in SEQ ID NO: 12), and the CD47-binding arm is the antigen-binding domain of the anti-CD47 humanized antibody A7H3L3 (amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 4, 5, and 6 respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 7, 8, and 9 respectively, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID The antibody A7H3L3 was used (as shown in NO:11). The heavy chain variable region of antibody A7H3L3 was fused to the human IgG1 heavy chain constant region (SEQ ID NO: 2) to form the heavy chain of antibody A7H3L3, and the light chain variable region was fused to the human κ light chain constant region (SEQ ID NO: 3) to form the light chain of antibody A7H3L3. The anti-CLDN18.2 single-domain antibody NA3S-H1 is disclosed in WO 2020238730A1, and its in vitro ADCC and CDC cell-killing effects, as well as its tumor inhibition studies in a human CLDN18.2-HEK29T-SCID tumor transplantation model, all showed excellent efficacy. The humanized antibody A7H3L3 exhibits weak binding to CD47 on red blood cells, making it an ideal candidate antibody for bispecificity. Furthermore, through the design of the bispecificity antibody, it binds to CLDN18.2, allowing the anti-CD47 antibody A7H3L3 binding arm to better bind to tumor cells expressing the dual target and to better block the SIRPα inhibitory signal.

[0104] Bispecific antibodies were designed based on the valence, position, and linker length of the anti-CLDN18.2 single-domain antibody (VHH) NA3S-H1. Five types of bispecific antibodies (B8-B12, respectively) were designed, and the structures of exemplary bispecific antibodies are shown in Table 1 and Figure 1. The corresponding amino acid sequences are provided in Table 2, where the sequence of Linker1 is GGGGSGGGGS (SEQ ID NO: 22) and the sequence of Linker2 is GGGGS (SEQ ID NO: 23).

[0105] [Table 1]

[0106] [Table 2] Example 2: Binding activity of anti-CD47-CLDN18.2 bispecific antibody to CD47 in red blood cells

[0107] Flow cytometry was used to measure whether the bispecific antibody bound to red blood cells. Another anti-CD47 antibody developed by the applicant, F4AM4-IgG1 (abbreviated as F4AM4 in the drawings, with the heavy chain amino acid sequence being SEQ ID NO:24 and the light chain amino acid sequence being SEQ ID NO:25), was used as a positive control antibody. F4AM4-IgG1 was selected as the positive control in this example because it showed strong binding to CD47 on red blood cells in a series of functional validation experiments (conducted before the start of the experiments in this example).

[0108] The specific method is as follows: Red blood cells were separated from 1 mL of anticoagulated human blood, centrifuged, the supernatant was aspirated and discarded, rinsed twice with PBS, and then resuspended in 1 mL of PBS. Red blood cells were separated into 1 × 10⁶ cells using PBS. 7The antibody was diluted to 1 / mL, and 50 μL of erythrocytes were aspirated per well and added to a 96-well round-bottom cell culture plate. Equivolute amounts of the gradient-diluted antibody were then added, thoroughly mixed, and incubated at 4°C for 1 hour. Next, the cells were rinsed three times with FACS buffer, 0.5 μg of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596) was added, and the cells were incubated at 4°C for 1 hour. Subsequently, the cells were rinsed three times with FACS buffer, resuspended with 200 μL of FACS buffer, and finally, the amount of antibody bound to erythrocytes (expressed as mean fluorescence intensity (MFI)) was detected using a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0109] The binding activity of the antibodies to red blood cells is shown in Figures 2a and 2b. As shown in Figures 2a and 2b, even at very high concentrations (150 μg / mL), all anti-CD47-CLDN18.2 bispecific antibodies hardly bound to red blood cells or had very low binding activity compared to the strong binding of F4AM4-IgG1 to red blood cells. At high concentrations (15 μg / mL), there was no significant difference in the binding of B9, B10, and B11 to red blood cells and the negative control IgG1, and the specific numbers are shown in Table 3. Thus, the bispecific antibodies of the present invention have low binding activity to CD47 on red blood cells and substantially do not cause hemagglutination.

[0110] [Table 3] Example 3: Binding activity of anti-CD47-CLDN18.2 bispecific antibody to tumor cells expressing single and dual targets.

[0111] The binding activity of the anti-CD47-CLDN18.2 bispecific antibody B10 to NUGC-4 cells (expressing endogenous CD47, purchased from the BNCC bacterial species bank, with the number BNCC341962) and hCLDN18.2-NUGC-4 cells (constructed using lentiviral transfection to create a gastric cancer cell line NUGC-4 that overexpresses exogenous human CLDN18.2 (amino acid sequence SEQ ID NO:1) while also expressing endogenous CD47) was measured by flow cytometry. For comparison, the binding activity of antibody 1F8 (1F8 antibody in WO 2018075857A1) and F4AM4-IgG1 to these two cell lines was further measured. Human IgG1 was used as an isotype-negative control.

[0112] The specific method is as follows: 1 × 10 5 Individual NUGC-4 cells or hCLDN18.2-NUGC-4 cells were taken, centrifuged at low speed (300 g), the supernatant was removed, and the cells at the bottom of the centrifuge tube were rinsed once with a formulated FACS buffer (1×PBS buffer containing 2% volume of FBS). The rinsed cells were then given gradient-diluted target antibody and incubated at 4°C for 1 hour. Next, the cells were rinsed three times with the same FACS buffer, 0.5 μg of PE-labeled goat anti-H-IgG Fc antibody (Abcam, ab98596) was added, and the cells were incubated at 4°C for 1 hour. Finally, the cells were rinsed three times with FACS buffer and resuspended in 200 μL of FACS buffer. The amount of antibody bound to erythrocytes (expressed as mean fluorescence intensity (MFI)) was detected using a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0113] The binding activity of the antibodies to NUGC-4 cells and hCLDN18.2-NUGC-4 cells is shown in Figures 3a and 3b, respectively. As shown in Figure 3a, the bispecific antibody B10 showed weak binding activity to tumor cells NUGC-4, slightly weaker than that of antibody 1F8. As shown in Figure 3b, the bispecific antibody B10 showed significantly superior binding activity to antibody 1F8 in hCLDN18.2-NUGC-4 cells expressing both CD47 and CLDN18.2. Based on this, the bispecific antibody B10 showed weaker binding than 1F8 to cells expressing a single target of CD47, but superior binding to cells expressing dual targets of CD47 and CLDN18.2. These results demonstrate that the bispecific antibody B10 can simultaneously bind to CLDN18.2 and CD47 in tumor cells, thus enhancing its ability to bind to tumor cells. Example 4 Ability of anti-CD47-CLDN18.2 bispecific antibody to block the binding of CD47 to SIRPα in tumor cells expressing single and dual targets.

[0114] Flow cytometry was used to measure the ability of anti-CD47-CLDN18.2 bispecific antibodies to block the binding of CD47 to SIRPα in NUGC-4 and hCLDN18.2-NUGC-4 cells. For comparison, the ability of antibodies 1F8, F4AM4-IgG1, and A7H3L3 to block the binding of CD47 to SIRPα in NUGC-4 and hCLDN18.2-NUGC-4 tumor cells was further measured.

[0115] The specific method is as follows: 1 × 10 5Individual NUGC-4 cells or hCLDN18.2-NUGC-4 cells were isolated, centrifuged at low speed (300 g), and the supernatant was removed. Cells at the bottom of the centrifuge tube were rinsed once with a formulated FACS buffer (1×PBS buffer containing 2% FBS), and the rinsed cells were then treated with a gradient-diluted antibody for detection. The cells were incubated for 1 hour, rinsed twice with FACS buffer, and then 100 μL of 1 μg / mL SIRPα-mFc (ACRO, SIA-H52A8) was added. The cells were incubated at 4°C for 1 hour, rinsed three times with FACS buffer, and 100 μL of PE-labeled goat anti-mouse Fc secondary antibody (Abcam, ab98742) diluted 1:200 was added. The cells were incubated at 4°C for 1 hour, then centrifuged to remove the supernatant. The cells were resuspended with 200 μL of FACS buffer, and finally, the amount of SIRPα-mFc bound to the cells (expressed as mean fluorescence intensity (MFI)) was detected using a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0116] The results in NUGC-4 cells are shown in Figure 4a, and the antibody F4AM4-IgG1 can effectively block the binding of CD47 to SIRPα, resulting in IC 50 The concentration was 0.033 μg / mL (0.226 nM), and antibody 1F8 had a weak blocking effect, IC 50 The concentration was 15.36 μg / mL (105.6 nM), and the bispecific antibody B10 had almost no blocking ability.

[0117] The results in hCLDN18.2-NUGC-4 cells are shown in Figure 4b, and the antibody F4AM4-IgG1 still exhibits strong blocking ability in these tumor cells. 50 The concentration was 0.056 μg / mL (0.383 nM), and the blocking ability of bispecific antibody B10 in hCLDN18.2-NUGC-4 cells was clearly improved compared to its blocking ability in NUGC-4 cells, and was superior to antibody 1F8. B10 and 1F8 blocked the binding of CD47 to SIRPα in hCLDN18.2-NUGC-4 cells. 50The concentrations were 0.765 μg / mL (4.476 nM) and 11.98 μg / mL (82.34 nM), respectively. Furthermore, the anti-CLDN18.2 single-domain antibody NA3S-H1 bound only to CLDN18.2 and therefore exhibited no blocking effect. Based on this, the bispecific antibody B10 showed weaker blocking activity than antibody 1F8 in cells expressing the single target of CD47, but clearly superior blocking activity in cells expressing the dual targets of CD47 and CLDN18.2, exhibiting a stronger ability to block the binding of CD47 to the receptor SIRPα.

[0118] The blocking activity of other bispecific antibodies in hCLDN18.2-NUGC-4 cells was further measured, and the results are shown in Figure 4c. Among the bispecific antibodies B8-B12, bispecific antibody B10 exhibited the strongest blocking activity. Example 5: In vivo tumor inhibition experiment with anti-CD47-CLDN18.2 bispecific antibody 5.1 In vivo tumor inhibition experiment 1

[0119] Six-to-seven-week-old female nude mice (16-18 g) were reared in a separate, temperature- and humidity-controlled ventilated box, with the temperature of the rearing room at 21-24°C and the humidity at 30-53%. 3×10 6 When 100 hCLDN18.2-NUGC-4 cells were subcutaneously injected into the left axilla of nude mice (day 0), the subcutaneous tumor volume of the mice was 300-400 mm². 3When the tumor size reached a certain level (day 20), mouse samples with large differences in tumor volume were removed, and the mice were randomly divided into groups according to tumor volume (8 mice per group). These groups were: PBS treatment group, NA3S-H1 monoclonal antibody administration group, A7H3L3 monoclonal antibody administration group, NA3S-H1 + A7H3L3 combined administration group, and bispecific antibody B10 administration group. NA3S-H1 monoclonal antibody was administered at a baseline of 5 mg / kg, with all other drugs administered at equimolar doses: A7H3L3 monoclonal antibody at 9.4 mg / kg, NA3S-H1 + A7H3L3 combined at 5 mg / kg + 9.4 mg / kg, and bispecific antibody B10 at 10.6 mg / kg. Administration was performed twice a week, alternating between intraperitoneal injection (ip) and intravenous injection (iv). The length (mm) and width (mm) of the tumor are observed and recorded at any time, and its tumor volume (V) is calculated. The calculation method is as follows:

number

number

[0120] The results of tumor inhibition by antibodies are shown in Figure 5a and Table 4. As can be seen from the results, at this equimolar dose, the NA3S-H1 monoclonal antibody administration group showed almost no tumor inhibitory effect, while the other groups all showed some degree of tumor inhibitory effect. Here, the bispecific antibody B10 showed the best effect, reaching an antitumor rate of almost 54.54% before day 39, and the tumor size approached the initial tumor volume at day 20. This was superior to the results of combination therapy (A7H3L3 + NA3S-H1 (9.4 + 5 mpk)), indicating that the binding of the bispecific antibody B10 to CLDN18.2 can enhance its blocking effect against the binding of CD47 to SIRPα.

[0121] [Table 4] 5.2 In vivo tumor inhibition experiment 2

[0122] The mice were randomly divided into groups (8 mice per group): a PBS treatment group, a monoclonal antibody NA3S-H1 administration group, and a bispecific antibody B10 administration group. The first dose was administered on the day of inoculation (day 0), and subsequent doses were given twice a week. The NA3S-H1 monoclonal antibody was used as the baseline at 2.5 mg / kg, and the bispecific antibody B10 was administered at an equimolar dose, i.e., 5.3 mg / kg. Other details were consistent with Example 5.1.

[0123] The results are shown in Figure 5b. At this equimolar dose, the NA3S-H1 monoclonal antibody group showed a certain tumor inhibitory effect, with an antitumor rate of approximately 54.99% (day 34), while all mice in the bispecific antibody B10 group showed complete tumor inhibition, with an antitumor rate of approximately 100% (day 34). These results demonstrate that the bispecific antibody B10 has a clearly superior tumor inhibitory effect compared to the anti-CLDN18.2 monoclonal antibody NA3S-H1. Example 6: Measurement of the physicochemical properties of the anti-CD47-CLDN18.2 bispecific antibody 6.1 Purity Identification by SDS-PAGE

[0124] The purity of bispecific antibody B10 was determined using reduced SDS-PAGE. The apparent relative molecular weights of the heavy chain and light chain main bands of bispecific antibody B10 were approximately 65 kD and 25 kD, respectively, which were consistent with the intended size, and the purity reached 90%. 6.2 Purity Identification by SEC-HPLC

[0125] The monomer purity of bispecific antibody B10 was detected using SEC-HPLC. The monomer purity of bispecific antibody B10 measured by SEC-HPLC was greater than 94%. 6.3 Detection of DSF thermal stability of bispecific antibody B10

[0126] The thermal stability of bispecific antibody B10 was evaluated by detecting its Tm value using the DSF method. The results showed that bispecific antibody B10 had two melting peaks, with the Tm value of the first peak being 69.85±0.06℃ and the Tm value of the second peak being 80.60±0.16℃, indicating that bispecific antibody B10 has good thermal stability. Example 7 ADCP activity of anti-CD47-CLDN18.2 bispecific antibody

[0127] Antibody-dependent cell-mediated phagocytosis (ADCP) is an important mechanism of action for therapeutic antibodies against viral infections or tumor cells. This experiment evaluated the ADCP activity of the bispecific antibody of the present invention using a bioluminescent reporter gene. The method used genetically engineered Jurkat cells (BPS Bioscience Inc., 71273) as effector cells, which stably expressed luciferase (Int Immunopharmacol. 2021 Nov;100:108-112.) whose expression is driven by the FcγRIIa receptor and an NFAT response element. After recognizing target cells, the antibody bound to FcγRIIa on the effector cell surface, stimulating the intracellular NFAT response element, which then drove luciferase expression. Luciferase activity can be quantified by bioluminescence.

[0128] The target samples (B10, A7H3L3, NA3S-H1) were each diluted to an initial reaction concentration of 200 nM and then gradient-diluted 2-fold to obtain 10 gradients. The diluted samples were added to a 96-well white bottom plate, with 50 μL per well, and three triplicate wells were placed for each concentration gradient. 150 μL / well of PBS solution was added to the edge wells of the 96-well white bottom plate. Subsequently, 5 × 10⁶ samples were added to the 96-well white bottom plate. 5 hCLDN18.2-NUGC-4 cells (target cells) at a concentration of 1.5 × 10¹⁶ cells / mL were added to 50 μL / well and incubated at room temperature for 30 minutes. Then, the 1.5 × 10¹⁶ cells described above were added. 6Effector cells at a concentration of 1 / mL were added at 50 μL / well. The 96-well white plate was incubated in a 37°C 5% CO2 incubator for 6 hours. Detection: The 96-well white plate was removed and allowed to stand at room temperature for 30 minutes. The detection reagent, luciferase substrate (Bio-Glo® Luciferase Assay System Promega G7940), was added at 50 μL / well and allowed to react at room temperature in the dark for 5-10 minutes. The luminescence intensity (expressed as relative light units (RLU)) was detected using a microplate reader. The logarithm of the antibody concentration was plotted against the RLU at the corresponding concentration, and the data was analyzed using Graphpad Prism software, and the EC (Emission Control Value) was also analyzed. 50 The value was calculated.

[0129] Experimental results: As can be seen from Figure 6 and Table 5, when the target cells were hCLDN18.2-NUGC-4 cells, the bispecific antibody B10 of the present invention showed higher ADCP activity compared to the anti-CD47 monoclonal antibody A7H3L3 and the anti-CLDN18.2 monoclonal antibody NA3S-H1.

[0130] [Table 5]

[0131] While embodiments for carrying out the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings disclosed herein, and all such changes fall within the scope of protection of the present invention. The scope of protection of the present invention is given by the appended claims and any equivalent thereof. Sequence List SEQ ID NO:1 Human CLDN18.2 MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV SEQ ID NO:2 Human IgG1 heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:3 Human Kappa light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:4 A7H3L3 - HCDR1 GFNIKDIYIY SEQ ID NO:5 A7H3L3 - HCDR2 KIDPANGNTK SEQ ID NO:6 A7H3L3 - HCDR3 GYGSGFAY SEQ ID NO:7 A7H3L3-LCDR1 RASQDISNHLN SEQ ID NO:8 A7H3L3-LCDR2 YTSRIHS SEQ ID NO:9 A7H3L3-LCDR3 QQGYTLPFT SEQ ID NO:10 A7H3L3-VH QVQLVQSGAEVKKPGASVKVSCKASGFNIKDIYIYWVRQAPGQGLEWIGKIDPANGNTKYDQKFQGRATITADTSTNTAYLELSSLRSEDTAVYYCARGYGSGFAYWGQGTLVTVSS SEQ ID NO:11 A7H3L3-VL DIQMTQSPSSLSASVGDRVTITCRASQDISNHLNWYQQKPGKAPKLLIYYTSRIHSGVPSSFRGSGSGTDYTLTISSLQPEDIATYFCQQGYTLPFTFGSGTKLEIK SEQ ID NO:12 NA3S-H1 QVQLVESGGGLVQPGGSLRLSCAASGSIFNIPVMGWYRQAPGKQRELVAGISTGGTTNYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVLVVSGIGSTLEVWGQGTLVTVSS SEQ ID NO:13 NA3S-H1 CDR1 GSIFNIPV SEQ ID NO:14 NA3S-H1 CDR2 ISTGGTT SEQ ID NO:15 NA3S-H1 CDR3 NVLVVSGIGSTLEV SEQ ID NO:16 B8 / B11 heavy chain QVQLVESGGGLVQPGGSLRLSCAASGSIFNIPVMGWYRQAPGKQRELVAGISTGGTTNYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVLVVSGIGSTLEVWGQGTLVTVSSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDIYIYWVRQAPGQGLEWIGKIDPANGNTKYDQKFQGRATITADTSTNTAYLELSSLRSEDTAVYYCARGYGSGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:17 Light chain of B8 / B10 / B12 DIQMTQSPSSLSASVGDRVTITCRASQDISNHLNWYQQKPGKAPKLLIYYTSRIHSGVPSSFRGSGSGTDYTLTISSLQPEDIATYFCQQGYTLPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:18 Heavy chain of B9 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDIYIYWVRQAPGQGLEWIGKIDPANGNTKYDQKFQGRATITADTSTNTAYLELSSLRSEDTAVYYCARGYGSGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:19 Light chain of B9 / B11 QVQLVESGGGLVQPGGSLRLSCAASGSIFNIPVMGWYRQAPGKQRELVAGISTGGTTNYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVLVVSGIGSTLEVWGQGTLVTVSSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNHLNWYQQKPGKAPKLLIYYTSRIHSGVPSSFRGSGSGTDYTLTISSLQPEDIATYFCQQGYTLPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:20 Heavy chain of B10 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDIYIYWVRQAPGQGLEWIGKIDPANGNTKYDQKFQGRATITADTSTNTAYLELSSLRSEDTAVYYCARGYGSGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGSIFNIPVMGWYRQAPGKQRELVAGISTGGTTNYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVLVVSGIGSTLEVWGQGTLVTVSS SEQ ID NO:21 B12 heavy chain QVQLVESGGGLVQPGGSLRLSCAASGSIFNIPVMGWYRQAPGKQRELVAGISTGGTTNYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVLVVSGIGSTLEVWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDIYIYWVRQAPGQGLEWIGKIDPANGNTKYDQKFQGRATITADTSTNTAYLELSSLRSEDTAVYYCARGYGSGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:22 Linker1 GGGGSGGGGGS SEQ ID NO:23 Linker2 GGGGS SEQ ID NO:24 F4AM4-IgG1 heavy chain QVQLVQSGAEVKKPGASVKMSCKASGYTFTSSVMHWVRQAPGQGLEWIGYINPYTDGTKYAQKFQGRATLTSDKSTSTAYMEFSSLRSEDTAVYYCGRPYYGTRYGSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:25 F4AM4-IgG1 light chain DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISNLQPEDIATYYCQQGKNYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A bispecific antibody comprising a first antigen-binding moiety that binds to CD47 and a second antigen-binding moiety that binds to CLDN18.2, wherein the first antigen-binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL). The aforementioned heavy chain variable region is 1) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, 2) HCDR2 containing the amino acid sequence of SEQ ID NO: 5, 3) Containing HCDR3 with the amino acid sequence of SEQ ID NO: 6, The aforementioned light chain variable region is 1) LCDR1 containing the amino acid sequence of SEQ ID NO: 7, 2) LCDR2 containing the amino acid sequence of SEQ ID NO: 8, 3) A bispecific antibody containing LCDR3 containing the amino acid sequence of SEQ ID NO:

9.

2. The heavy chain variable region includes 1) the amino acid sequence of SEQ ID NO: 10, or 2) an amino acid sequence having 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%, or at least 99% sequence identity with SEQ ID NO: 10, and / or The bispecific antibody according to claim 1, wherein the light chain variable region comprises 1) the amino acid sequence of SEQ ID NO: 11, or 2) an amino acid sequence having 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%, or at least 99% sequence identity with SEQ ID NO:

11.

3. The bispecific antibody according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

11.

4. The bispecific antibody according to any one of claims 1 to 3, wherein the second antigen-binding portion comprises an immunoglobulin single variable domain (VHH) that binds to CLDN18.

2.

5. The immunoglobulin monovariate domain is CDR1 containing the amino acid sequence of SEQ ID NO: 13, CDR2 containing the amino acid sequence of SEQ ID NO: 14, Includes CDR3 containing the amino acid sequence of SEQ ID NO: 15, The bispecific antibody according to claim 4.

6. The bispecific antibody according to claim 4, wherein the immunoglobulin monovariate domain comprises 1) the amino acid sequence of SEQ ID NO: 12, or 2) an amino acid sequence having 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%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:

12.

7. The bispecific antibody according to any one of claims 1 to 6, wherein the first antigen-binding portion and the second antigen-binding portion are linked via a linker.

8. A bispecific antibody according to any one of claims 1 to 7, further comprising a heavy chain constant region (CH) and a light chain constant region (CL) of an immunoglobulin.

9. The bispecific antibody according to claim 8, wherein the heavy chain constant region is the heavy chain constant region of human IgG1 and / or the light chain constant region is the human κ light chain constant region.

10. The bispecific antibody according to claim 8, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 2, and / or the light chain constant region comprises the amino acid sequence of SEQ ID NO:

3.

11. comprising a first polypeptide and a second polypeptide, Here, The first polypeptide described above, from the N-terminus to the C-terminus, It has a structure called VH-CH-Linker-VHH, The second polypeptide described above, from the N-terminus to the C-terminus, It has a VL-CL structure, Here, VH and VL are the heavy chain variable region and light chain variable region of the first antigen-binding moiety that binds to CD47, respectively. VHH is a single variable immunoglobulin domain that binds to CLDN18.

2. CH and CL are the heavy chain steady region and the light chain steady region, respectively. Linker is Linker. A bispecific antibody according to any one of claims 1 to 10.

12. The bispecific antibody according to claim 11, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 20, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

17.

13. An isolated polynucleotide encoding a bispecific antibody according to any one of claims 1 to 12.

14. An expression vector comprising the polynucleotide described in claim 13.

15. A host cell comprising the polynucleotide described in claim 13 or the expression vector described in claim 14.

16. An antibody conjugate comprising a bispecific antibody according to any one of claims 1 to 12, wherein the bispecific antibody is conjugated with at least one therapeutic agent.

17. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 12 or an antibody conjugate according to claim 16, and a pharmaceutically acceptable carrier agent.

18. Use of a bispecific antibody according to any one of claims 1 to 12, an antibody conjugate according to claim 16, or a pharmaceutical composition according to claim 17 in the manufacture of a pharmaceutical for treating cancer.

19. The use according to claim 18, wherein the cancer is gastric cancer.

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