HK40092489B - Anti-cd47-cldn18.2 bispecific antibodies and uses thereof - Google Patents

Description

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

This application claims priority to Chinese Patent Application No. 202111214360.8, filed on October 19, 2021, entitled “Anti-CD47-CLDN18.2 Bispecific Antibody and Its Use Thereof,” the entire contents of which are incorporated herein by reference.

Technical Field

This invention belongs to the field of biomedicine. Specifically, this invention relates to an anti-CD47-CLDN18.2 bispecific antibody and its uses.

Background Technology

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, mediating a series of responses including apoptosis, proliferation, and immunity. It has been demonstrated that blocking the CD47-SIRPα pathway with anti-CD47 antibodies can effectively mediate phagocytosis of tumor cells, thereby inhibiting the growth of various hematologic malignancies and solid tumors in vivo. However, CD47 is not only highly expressed on tumor cells but also in normal cells, such as erythrocytes. Targeting CD47 therapies may induce undesirable side effects. Some existing anti-CD47 antibodies (see, for example, US20160304609) bind to erythrocytes, which not only induces severe anemia but also requires dosages as high as 30 mg/kg. These characteristics pose significant challenges to the clinical application of anti-CD47 antibodies.

CLDN18, a member of the Claudins protein family, was discovered by Shoichiro Tsukita et al. in 1998. It is a crucial molecule constituting the tight junctions of epithelial cells, determining their permeability and blocking the diffusion of cell membrane surface proteins and lipids (Gunzel, D. and A.S. Yu (2013) Physiol Rev 932:525-569). The human CLDN18 gene has two distinct exons 1, which, after transcription, undergo alternative splicing to generate two protein isoforms, CLDN18.1 and CLDN18.2, with different sequences only at the N-terminus. Due to its specific expression in tumor cells and normal tissues, CLDN18.2 has become a highly promising target for anti-tumor drugs. WO2016165762A1 disclosed the anti-CLDN18.2 antibody IMAB362 (Zolbetuximab), which showed significantly longer survival than standard chemotherapy in a phase II clinical trial of gastric cancer (13.2 vs. 8.4 months), with a more pronounced advantage in patients with high CLDN18.2 expression.

WO2021003082A1 discloses a bispecific antibody against CLDN18.2 and CD47 that does not bind to human erythrocytes. However, there is still a need to develop new bispecific antibodies targeting CLDN18.2 and CD47 to provide more possibilities for cancer treatment.

Summary of the Invention

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

In one embodiment, the second antigen-binding portion comprises an immunoglobulin single variable domain (VHH) that binds CLDN18.2. Preferably, the immunoglobulin single variable domain comprises: CDR1, which comprises the amino acid sequence of SEQ ID NO:13; CDR2, which comprises the amino acid sequence of SEQ ID NO:14; and CDR3, which comprises the amino acid sequence of SEQ ID NO:15.

In another aspect, the present invention provides an isolated polynucleotide encoding the bispecific antibody of the present invention.

The present invention also provides an expression vector comprising the polynucleotides of the present invention.

In another aspect, the present invention provides a host cell containing the polynucleotide or expression vector of the present invention.

The present invention also relates to antibody conjugates comprising the bispecific antibody of the present invention conjugated to at least one therapeutic agent.

In another aspect, the present invention relates to a pharmaceutical composition comprising the bispecific antibody or antibody conjugate of the present invention, and a pharmaceutically acceptable carrier.

The present invention also relates to the use of the bispecific antibody, antibody conjugate or pharmaceutical composition of the present invention in the preparation of a medicament for treating cancer.

Attached Figure Description

Figure 1 shows a schematic structure of the anti-CD47-CLDN18.2 bispecific antibody.

Figures 2a and 2b show the binding activity of the anti-CD47-CLDN18.2 bispecific antibody to CD47 on erythrocytes.

Figures 3a and 3b show the binding activity of the anti-CD47-CLDN18.2 bispecific antibody on tumor cells expressing single and dual targets. Figure 3a shows the binding activity of the anti-CD47-CLDN18.2 bispecific antibody on NUGC-4 cells, and Figure 3b shows the binding activity of the anti-CD47-CLDN18.2 bispecific antibody on hCLDN18.2-NUGC-4 cells.

Figures 4a, 4b, and 4c show the ability of the anti-CD47-CLDN18.2 bispecific antibody to block the binding of human CD47 to receptor SIRPα on tumor cells expressing single and dual targets. Specifically, Figure 4a shows the ability of the anti-CD47-CLDN18.2 bispecific antibody to block the binding of human CD47 to receptor SIRPα on NUGC-4 cells, and Figures 4b and 4c show the ability of the anti-CD47-CLDN18.2 bispecific antibody to block the binding of human CD47 to receptor SIRPα on hCLDN18.2-NUGC-4 cells.

Figures 5a and 5b show the inhibitory effect of the anti-CD47-CLDN18.2 bispecific antibody on tumor growth in mice.

Figure 6 shows the ADCP activity of the anti-CD47-CLDN18.2 bispecific antibody as determined by bioluminescent reporter gene assay.

Detailed Implementation

definition

In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

As used herein, the expressions “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components. The expression “composed of” excludes any unspecified elements, steps, or components. The expression “essentially composed of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the essential and novel nature of the claimed subject matter. It should be understood that the expressions “essentially composed of” and “composed of” are encompassed within the meaning of the expression “comprising.”

As used herein, “antibody” refers to an immunoglobulin or a fragment thereof that specifically binds to an antigenic epitope through at least one antigen-binding site. In this document, the definition of antibody encompasses 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 can be synthetic (e.g., produced by chemical or biological conjugation), enzymatically derived, or recombinant. Antibodies as used herein 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 can be “monovalent,” “bivalent,” “trivalent,” or “quadrivalent” or more, meaning they contain one, two, three, four, or more antigen-binding sites.

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

Both the light chain and heavy chain variable regions can contain three highly variable complementary determinant regions (CDRs) and four relatively conservative frame regions (FRs), connected from the N-end to the C-end in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In this paper, the CDRs (CDRL or LCDR) of the light chain variable region can be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs (CDRH or HCDR) of the heavy chain variable region can be referred to as HCDR1, HCDR2, and HCDR3.

In this invention, the amino acid sequence of CDR is shown according to the AbM definition rule (the sequence in the claims of this invention is also shown according to the AbM definition rule). However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia (see, for example, Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)) based on antibody sequence variability, Kabat (see, for example, Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and AbM (Martin, A. C. R. and J. Allen (2007) "Bioinformatics tools for..."). antibody engineering,” in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp. 95–118), Contact (MacCallum, R.M. et al., (1996) J.Mol.Biol. 262: 732-745), IMGT (Lefranc, M.-P., 2011(6), IMGT, the International ImMunoGeneTics Information System ColdSpring Harb Protoc.; and Lefranc, M.-P. et al., Dev.Comp.Immunol., 27, 55-77 (2003)), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described in any of the known schemes described herein. While the scope of protection claimed in the claims of this invention is based on the sequence indicated by the AbM definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

As used herein, the terms “frame region” and “architecture region” are used interchangeably. As used herein, the terms “frame region,” “architecture region,” or “FR” residues refer to the amino acid residues in the antibody variable region other than the CDR sequence as defined above.

As used herein, "single-domain antibody (sdAb)" or "nanobody" refers to an antibody that contains a single immunoglobulin variable domain (single variable domain) as a functional antigen-binding fragment. Similar to the variable region of a full-length antibody, a single variable domain typically contains CDR1, CDR2, and CDR3 that form the antigen-binding site, as well as a supporting framework region. Examples of single variable domains include, for instance, the variable domain of a heavy-chain antibody (VHH), the variable domain of shark IgNAR, the variable domain of a human light-chain antibody, and the variable domain of a heavy-chain antibody.

As used herein, “antibody-dependent cell-mediated phagocytosis” 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 CD47 and CLDN18.2).

As used herein, “percentage (%) sequence identity” or “sequence identity” has a generally accepted definition in the art, referring to the percentage of identical amino acid sequences between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a known algorithm). This can be determined using methods known to those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, Clustal Omega, and FASTA software.

In this document, amino acid sequences “derived from” or “from” a reference amino acid sequence are partially or entirely identical or homologous to 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 from which the heavy chain constant region of human immunoglobulin from which it is derived.

Non-critical regions of peptides (e.g., CDR regions of antibodies, non-critical amino acids in framework regions, amino acids in constant regions) can be modified, for example by substituting, adding, and/or deleting one or more amino acids, without altering the peptide's function. Those skilled in the art will understand that amino acids in non-critical regions of peptides can be substituted with suitable conserved amino acids, and generally do not alter their 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-conserved substitutions. Those skilled in the art will understand that amino acid mutations or modifications can be made to antibodies or antibody fragments to alter their properties, for example, by changing the type of antibody glycosylation modification, altering the ability to form interchain disulfide bonds, or providing active groups for the preparation of antibody conjugates. Antibodies or antigen-binding fragments containing such amino acid mutations or modifications are also covered within the scope of the bispecific antibodies of this invention.

The anti-CD47-CLDN18.2 bispecific antibody of the present invention, or the polynucleotide encoding it, may be isolated. As used herein, the term "isolated" means that the substance (e.g., polynucleotide or polypeptide) is isolated from its source or environment of existence, i.e., it substantially does not contain any other components.

In this document, the terms “polynucleotide” and “nucleic acid” are used interchangeably to refer to oligomers or polymers containing at least two linked nucleotides or nucleotide derivatives, which may typically include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

In this article, "vector" is a medium used to introduce exogenous polynucleotides into host cells, whereby the exogenous polynucleotides are amplified or expressed when the vector is transformed into a suitable host cell. Vectors typically remain free, but can be designed to integrate genes or portions thereof into the chromosome of the genome. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, granules, phage vectors, phage particles, 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, etc.

As used in this article, the term “expression” refers to the production of RNA and/or polypeptides.

As used herein, "expression vector" refers to a vector capable of expressing a polynucleotide of interest (including DNA and RNA). For example, in an expression vector, a polynucleotide sequence (including DNA and RNA) encoding a polypeptide of interest can be operatively linked to a regulatory sequence (such as a promoter and ribosome binding site) capable of influencing the expression of the polynucleotide sequence. The regulatory sequence may include promoter and terminator sequences, and optionally may include origin of replication, selection markers, enhancers, polyadenylation signals, etc. The expression vector may be a plasmid, phage vector, recombinant virus, or other vector that, when introduced into a suitable host cell, results in the expression of the polynucleotide of interest. Suitable expression vectors are well known to those skilled in the art. Those skilled in the art can prepare expression vectors as needed to be replicable in host cells, remain free in host cells, or integrate into the host cell genome.

As used herein, a “host cell” is a cell used to receive, maintain, replicate, or amplify a vector. Host cells can also be used to express polynucleotides or polypeptides encoded by the vector. Host cells can be eukaryotic or prokaryotic cells. Examples of prokaryotic cells include E. coli or Bacillus subtilis; fungal cells include yeast or Aspergillus; insect cells (such as S2 Drosophila cells or Sf9); and animal cells (such as fibroblasts, CHO cells, COS cells, HeLa cells, NSO cells, or HEK293 cells).

As used herein, the term "treatment" refers to the improvement of a disease/symptom, such as reducing or eliminating the disease/symptom, preventing or slowing its occurrence, progression, and/or worsening. Therefore, treatment includes prevention, treatment, and/or cure.

As used herein, the term "pharmaceuticalally acceptable carrier" means a carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well 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 maintainers, absorption delayers, 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, 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, etc. Osmotic pressure maintainers include, but are not limited to, sugars, sodium chloride, and the like. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, and sorbic acid. Stabilizers have the meaning commonly understood by those skilled in the art, which stabilize the desired activity of the active ingredient in the drug, including, but not limited to, monosodium glutamate, gelatin, SPGA (Sucrose-Phosphate-Glutamate-Albumin), sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid and glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate).

As used herein, examples of mammals include, but are not limited to, humans, non-human primates, rats, mice, cattle, horses, pigs, sheep, alpacas, dogs, cats, etc. In this document, 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 person or animal suspected of having cancer or at risk of developing cancer.

Anti-CD47-CLDN18.2 bispecific antibody

This invention provides a bispecific antibody against CD47-CLDN18.2, comprising a first antigen-binding moiety binding to CD47 and a second antigen-binding moiety binding 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 includes:

1) HCDR1, which contains the amino acid sequence of SEQ ID NO:4 or a variant thereof;

2) HCDR2, which contains the amino acid sequence of SEQ ID NO:5 or a variant thereof; and

3) HCDR3, comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof; and

The light chain variable region includes:

1) LCDR1, which contains the amino acid sequence of SEQ ID NO:7 or a variant thereof;

2) LCDR2, which contains the amino acid sequence of SEQ ID NO:8 or a variant thereof; and

3) LCDR3, which contains the amino acid sequence of SEQ ID NO:9 or a variant thereof;

The variant has one or two amino acid substitutions, additions, and/or deletions compared to the sequence from which it originates.

In one specific embodiment, the first antigen-binding portion includes a first antigen-binding portion that specifically binds to CD47, the first antigen-binding portion including a heavy chain variable region (VH) and a light chain variable region (VL).

The heavy chain variable region includes:

1) HCDR1, which contains the amino acid sequence of SEQ ID NO:4;

2) HCDR2, which contains the amino acid sequence of SEQ ID NO:5; and

3) HCDR3, which contains the amino acid sequence of SEQ ID NO:6; and

The light chain variable region includes:

1) LCDR1, which contains the amino acid sequence of SEQ ID NO:7;

2) LCDR2, which contains the amino acid sequence of SEQ ID NO:8; and

3) LCDR3, which contains the amino acid sequence of SEQ ID NO:9.

In one embodiment, 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%, or at least 99% sequence identity with SEQ ID NO:10; and/or

The VL comprises: 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.

In one embodiment, the VH comprises the amino acid sequence of SEQ ID NO:10, and the VL comprises the amino acid sequence of SEQ ID NO:11.

The first antigen-binding portion may contain 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 portion specifically binds to CD47 on the surface of cancer cells but does not bind to or substantially does not bind to CD47 on erythrocytes, thus preventing the bispecific antibody of the present invention from causing erythrocyte agglutination.

The second antigen-binding moiety may comprise 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 comprises an immunoglobulin single variable domain that specifically binds to CLD18.2. Immunoglobulin single variable domains that specifically bind to CLD18.2 are described, for example, in CN112480248A and WO2020238730A1, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the immunoglobulin single variable domain comprises: CDR1, which comprises the amino acid sequence of SEQ ID NO:13 or a variant thereof; CDR2, which comprises the amino acid sequence of SEQ ID NO:14 or a variant thereof; and CDR3, which comprises 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 embodiment, the immunoglobulin single variable domain comprises: CDR1, which comprises the amino acid sequence of SEQ ID NO:13; CDR2, which comprises the amino acid sequence of SEQ ID NO:14; and CDR3, which comprises the amino acid sequence of SEQ ID NO:15. In one embodiment, the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO:12. In yet another embodiment, the immunoglobulin single variable domain comprises 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.

In some embodiments, the first antigen-binding portion and the second antigen-binding portion are connected by a linker. The linker can be a peptide linker or a chemical bond, preferably a peptide linker. Exemplary peptide linkers may include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S), or combinations thereof, such as GGAS, GGGS, GGGSG, or (G ₄ S) _n , 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 comprises the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:23.

In some embodiments, the anti-CD47-CLDN18.2 bispecific antibody of the present invention further comprises an immunoglobulin constant region. The immunoglobulin constant region can be the heavy chain constant region (CH) and light chain constant region (CL) of an immunoglobulin of any species. The heavy chain constant region can 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) of an immunoglobulin, or a combination thereof. In a preferred embodiment, the heavy chain constant region comprises 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 can be derived from the λ (Lambda) light chain or the κ (Kappa) light chain constant region. In a preferred embodiment, the heavy chain constant region is the heavy chain constant region of human IgG1. In one embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, the light chain constant region is the human κ light chain constant region. In one embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO:3.

In one embodiment, the VH and VL of the first antigen-binding portion 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 portion is optionally fused to the N-terminus of the VH, the N-terminus of the VL, the C-terminus of the heavy chain constant region, or the C-terminus of the light chain constant region via a linker.

In some embodiments, the anti-CD47-CLDN18.2 bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide comprises a VH and a heavy chain constant region of a first antigen-binding moiety, and the second polypeptide comprises a VL and a light chain constant region of the first antigen-binding moiety. Optionally, a single variable domain of the second antigen-binding moiety is fused via a linker to the N-terminus of the VH or VL, or to the C-terminus of the heavy chain constant region or the light chain constant region. In one embodiment, 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 referred to as a fused heavy chain. In one embodiment, the fused heavy chain has a structure of formula (I) or formula (III) as 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 referred to as a fused light chain. In one embodiment, the fused light chain has a structure of formula (IV) or formula (VI) as described below.

In one embodiment, the first polypeptide has the structure of formula (I):

VH-CH-Linker-VHH Equation (I),

The second polypeptide has the structure of formula (II):

VL-CL Formula (II),

in

VH and VL are the heavy chain variable region and light chain variable region of the first antigen-binding region as described above;

VHH is a single variable domain of immunoglobulin as described above;

CH and CL are the heavy chain constant region and the light chain constant region, respectively, as described above.

Linker is a connector.

In one embodiment, the first polypeptide has the structure of formula (I) and contains the amino acid sequence of SEQ ID NO:20; the second polypeptide has the structure of formula (II) and contains the amino acid sequence of SEQ ID NO:17.

In one embodiment, the first polypeptide has the structure of formula (Ⅲ):

VHH-Linker-VH-CH Formula (III),

The second polypeptide has the structure of formula (II).

in

VHH is a single variable domain of immunoglobulin as described above;

VH is the heavy chain variable region of the first antigen-binding moiety as described above;

CH represents the heavy chain constant region as described above;

Equation (II) is as defined above;

Linker is a connector.

In one embodiment, the first polypeptide has the structure of formula (III) and contains the amino acid sequence of SEQ ID NO:16; the second polypeptide has the structure of formula (II) and contains the amino acid sequence of SEQ ID NO:17.

In one embodiment, the first polypeptide has the structure of formula (III) and contains the amino acid sequence of SEQ ID NO:21; the second polypeptide has the structure of formula (II) and contains the amino acid sequence of SEQ ID NO:17.

In yet another embodiment, 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),

in

Equation (Ⅲ) is as defined above;

VHH is a single variable domain of immunoglobulin as described above;

VL is the light chain variable region of the first antigen-binding region as described above;

CL is the constant region of the light chain as described above;

Linker is a connector.

In one embodiment, the first polypeptide has the structure of formula (Ⅲ) containing the amino acid sequence of SEQ ID NO:16; and the second polypeptide has the structure of formula (Ⅳ) containing the amino acid sequence of SEQ ID NO:19.

In another embodiment, the first polypeptide has the structure of formula (V):

VH-CH formula (V),

The second polypeptide has the structure of formula (Ⅳ).

in

Equation (Ⅳ) is as defined above;

VH is the heavy chain variable region of the first antigen-binding moiety as described above;

CH represents the heavy chain constant region as described above;

Linker is a connector.

In one embodiment, the first polypeptide has the structure of formula (V) containing the amino acid sequence of SEQ ID NO:18; and the second polypeptide has the structure of formula (Ⅳ) containing the amino acid sequence of SEQ ID NO:19.

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 Formula (VI),

in

Equation (V) is as defined above;

VHH is a single variable domain of immunoglobulin as described above;

VL is the light chain variable region of the first antigen-binding region as described above;

CL is the constant region of the light chain as described above;

Linker is a connector.

In some embodiments, the anti-CD47-CLDN18.2 bispecific antibody of the present invention is capable of:

1) Blocking the binding of CD47 on the surface of cancer cells to SIRPα;

2) Inducing macrophage phagocytosis of cancer cells expressing CD47 and CLDN18.2; and/or

3) It binds to cancer cells expressing CD47 and CLDN18.2 but does not bind to or barely binds to red blood cells.

Polynucleotides, vectors and host cells

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

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

In another aspect, the present invention also provides vectors comprising the polynucleotides of the present invention. In some embodiments, the polynucleotides of the present invention are cloned into an expression vector. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The expression vector may also comprise polynucleotide sequences encoding additional polypeptides. The additional polypeptides may be, for example, polypeptides that facilitate the detection and/or separation of antibody or antigen-binding fragments, including but 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).

In one embodiment, the polynucleotide preparation of the present invention is a recombinant nucleic acid. Recombinant nucleic acids can be prepared using techniques well-known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase chain reaction (PCR) techniques), etc. (see Sambrook, J., E.F. Fritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The polynucleotides of the present invention can be present in one or more expression vectors. In some embodiments, the expression vector is a DNA plasmid, such as a DNA plasmid for expression in bacterial, yeast, or mammalian cells. In other embodiments, the expression vector is a viral vector. In still other embodiments, the expression vector is a bacteriophage vector or a phage particle vector.

The present invention also provides a host cell comprising at least one polynucleotide or vector as described above. The polynucleotide or expression vector of the present invention can be introduced into a suitable host cell using various methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection.

In a preferred embodiment, the host cell is used to express 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, such as Escherichia coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells).

Suitable mammalian host cells 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.

The present invention also provides a method for generating the anti-CD47-CLDN18.2 bispecific antibody of the present invention, comprising the following steps:

(I) The host cells of the present invention are cultured under suitable conditions to express the anti-CD47-CLDN18.2 bispecific antibody, and

(II) Isolate the antibody from the host cell or its culture.

In some embodiments, a single vector is used, containing a polynucleotide sequence encoding both 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 also contains a chaperone plasmid, which can help 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.

Antibody conjugates

The present invention also provides an antibody conjugate comprising the anti-CD47-CLDN18.2 bispecific antibody of the present invention conjugated to at least one therapeutic agent. Antibody-drug conjugates (ADCs) are a typical type of antibody conjugate, wherein the therapeutic agent can be, for example, a cytotoxic agent.

As used herein, “joint” refers to two or more parts being connected to each other by covalent or non-covalent interactions. In a preferred embodiment, the join is a covalent join.

Therapeutic agents may be selected from cytotoxic agents, therapeutic antibodies (e.g., antibodies that specifically bind to other antigens or their antigen-binding fragments), radioactive isotopes, oligonucleotides and their analogues (e.g., interfering RNA), bioactive peptides, protein toxins (e.g., diphtheria toxin, ricin toxin), and enzymes (e.g., urease).

Cytotoxic agents are substances that inhibit or reduce the activity, function, and/or kill cells. Examples of cytotoxic agents may include, but are not limited to: maytansinoids (e.g., maytansine), orlistatins (e.g., MMAF, MMAE, MMAD), duostatin, cryptophycin, vinca alkaloids (e.g., vincristine), colchicine, salicylate toxins, taxanes, paclitaxel, docetaxel, carbazide, enediyne antibiotics, cytochalasin derivatives, camptothecin derivatives, anthracycline antibiotics (e.g., daunorubicin, dihydroxyanthracindione, doxorubicin), and cytotoxic antibiotics (e.g., mitomycin, actinomycin). Mycin, duocarmycin (e.g., CC-1065), auromycin, polymycin, calicheamicin, endomycin, phenomycin, doxorubicin, daunorubicin, calicheamicin, endomycin, phenomycin, doxorubicin, daunorubicin, cisplatin, ethidium bromide, bleomycin, mitomycin, scintillan, praldidine, podophyllotoxin, etoposide, mitoxantrone, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nerabine, carmustine, lomustine, methotrexate, phenylalanine mustard, tenoposide, glucocorticoids, etc.

Radioactive isotopes can be selected from, for example, ²¹² Bi, ²¹³ Bi, ¹³¹ I, ¹²⁵ I, ¹¹¹ In, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, and ⁹⁰ Y. Antibodies labeled with radioactive isotopes are also called radioimmunoconjugates.

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

In one embodiment, the therapeutic agent is a molecule with antitumor biological activity. Molecules with antitumor biological activity include, but are not limited to, cytotoxic agents, chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, antibodies targeting tumor-specific antigens, and other antitumor drugs. In a preferred embodiment, the therapeutic agent is a cytotoxic agent. In yet another preferred embodiment, the therapeutic agent is a radioisotope.

The therapeutic agent can be conjugated to the anti-CD47-CLDN18.2 bispecific antibody of the present invention via a linker using any technique known in the art. The linker may contain an active group for covalent conjugation, such as an amine, hydroxylamine, maleimide, carboxyl, phenyl, thiol, mercapto, or hydroxyl group. The linker may be cleavable or non-cleavable. Cleavable linkers include, 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).

In one embodiment, the linker comprises an active group selected from amines, hydroxylamines, maleimides, carboxyl groups, phenyl groups, thiols, thiols, and hydroxyl groups. In one embodiment, the linker is a chemical bond. In one embodiment, the linker comprises an amino acid or a peptide consisting of 2-10 amino acids. The amino acid can be natural or non-natural.

In some embodiments, the therapeutic agent and the linker are first conjugated to form an intermediate before conjugation 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 thiol group of the anti-CD47-CLDN18.2 bispecific antibody of the present invention. The structure and preparation methods of such intermediates, as well as methods for preparing antibody conjugates using them, are described, for example, in International Patent Application Publication No. WO2019114666, the entire contents of which are incorporated herein by reference.

Pharmaceutical Composition

The present invention also provides pharmaceutical compositions comprising the anti-CD47-CLDN18.2 bispecific antibody or antibody conjugate of the present invention, and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers may include, but are not limited to: diluents, binders and adhesives, lubricants, disintegrants, preservatives, mediators, dispersants, glidants, sweeteners, coatings, excipients, preservatives, antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, etc. Suitable carriers include sugar alcohols, tartaric acid, phosphoric acid, solubilizers, gelling agents, softeners, solvents (e.g., water, alcohol, acetic acid, and syrup), buffers (e.g., phosphate buffers, histidine buffers, and acetate buffers), surfactants (e.g., nonionic surfactants such as polysorbate 80, polysorbate 20, poloxamer, or polyethylene glycol), antibacterial agents, antifungal agents, isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption delay agents, chelating agents, and emulsifiers. For compositions containing antibodies or antibody conjugates, suitable carriers may be selected from buffers (e.g., citrate buffers, acetate buffers, phosphate buffers, histidine buffers, histidine buffers), isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer), or combinations thereof.

The pharmaceutical compositions provided herein can 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 epidermal administration (e.g., by injection or infusion). For compositions containing antibodies or antibody conjugates, preferred dosage forms are typically, for example, injections and lyophilized powders.

The pharmaceutical compositions provided herein may be administered to a subject by any method known in the art, such as systemic or local administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavitary), local (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or local). Those skilled in the art will understand that the exact dosage will depend on various factors, such as the pharmacokinetic 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. Administration may be by, for example, injection or infusion.

As a general guideline, the dosage range of the anti-CD47-CLDN18.2 bispecific antibody of the present invention can be from about 0.0001 to 100 mg/kg, more typically from 0.01 to 20 mg/kg of subject body weight. For example, the dosage can 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 in the range of 1-20 mg/kg. Exemplary treatment regimens require administration once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every three months, once every three to six months, or with a slightly shorter initial dosing interval followed by longer intervals. Administration may be by intravenous infusion.

treat

In another aspect, 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 preparation of a medicament for treating a disease in a subject.

This invention also relates to the anti-CD47-CLDN18.2 bispecific antibody, antibody conjugate, or pharmaceutical composition of the present invention for the treatment of diseases.

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

In one embodiment, the disease described above is cancer. As used herein, "cancer" includes, but is not limited to, hematologic malignancies and solid tumors. Cancer can also be metastatic. "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 can be used to treat CLDN18.2-positive cancers. In a preferred embodiment, the anti-CD47-CLDN18.2 bispecific antibody is used to treat CD47 and CLDN18.2-positive cancers. In some embodiments, the cancer is gastric cancer.

Reagent test kit

The present invention also 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 also comprise a suitable container. In some embodiments, the kit further comprises a device for administration. Typically, the kit also includes a label indicating the intended use and/or method of use of the kit contents. The term "label" includes any written or recorded material provided on or with the kit or otherwise accompanied by the kit.

Beneficial effects

The anti-CD47-CLDN18.2 bispecific antibody of the present invention can achieve at least one of the following beneficial effects:

1) Blocking the binding of CD47 on the surface of cancer cells to SIRPα;

2) Induces macrophages to phagocytose cancer cells expressing CD47 and CLDN18.2;

3) Mediates ADCP targeting cancer cells expressing CLDN18.2; and

4) It binds to cancer cells expressing CD47 and CLDN18.2 but does not bind to or barely binds to red blood cells.

Furthermore, compared to monoclonal antibodies that target CD47 alone, the anti-CD47-CLDN18.2 bispecific antibody of the present invention has higher binding activity against CD47 and CLDN18.2 positive tumor cells and a stronger blocking effect on the interaction between CD47 and SIRPα.

Example

The following examples are intended to illustrate the invention only and should not be construed as limiting the invention in any way.

Materials and Methods

1. Construction of antibody expression plasmids

DNA fragments encoding the antibody heavy and light chains were prepared using recombinant DNA technology, and then cloned into the expression vector pcDNA3.4-TOPO (Invitrogen) to obtain antibody heavy and light chain expression plasmids. The plasmids were amplified in *E. coli* DH5α, and subsequently extracted and purified.

2. Antibody expression and purification

All antibodies were expressed using the ExpiCHO transient expression system (Thermo Fisher, A29133) (see WO2020238730A1) and affinity purified using a MabSelect SuRe LX (GE, 17547403).

3. SDS-PAGE for antibody purity assessment

Preparation of reducing solution: Add 2 μg of the test antibody or IPI (Ipilimlumab; reference) to 5×SDS loading buffer (containing DTT to a final concentration of 5 mM), heat in a dry bath at 100℃ for 10 min, cool to room temperature, centrifuge at 12000 rpm for 5 min, and collect the supernatant. Add the supernatant to a Bis-tris 4-15% gradient gel (GenScript) for protein gel electrophoresis. Subsequently, stain the protein bands with Coomassie Brilliant Blue to reveal the color, destain, and scan with an EPSON V550 color scanner. Calculate the purity of the protein bands using ImageJ according to the peak area normalization method.

4. SEC-HPLC was used to identify antibody purity.

An Agilent HPLC 1100 column (XBridge BEH SEC 3.5 μm, 7.8 mm I.D. × 30 cm, Waters) was used with a flow rate of 0.8 mL/min and an injection volume of 20 μL. The VWD detector wavelengths were 280 nm and 214 nm. The mobile phase was 150 mmol/L phosphate buffer, pH 7.4. All samples were diluted to 0.5 mg/mL using the mobile phase, and then blank and sample solutions were injected sequentially. The percentages of high molecular weight aggregates, antibody monomers, and low molecular weight aggregates in the samples were calculated using the area normalization method.

5. Differential scanning fluorescence method for identifying antibody stability

Differential scanning fluorimetry (DSF) can provide information about protein structural stability based on fluorescence changes in protein maps, detect conformational changes in proteins, and obtain the melting temperature (Tm) of proteins.

Prepare a 0.2 mg/mL antibody sample solution, using PBS and IPI (Ipilimlumab; 0.2 mg/mL) as references. Three aliquots of the test sample were added to each well of a 96-well plate (Nunc), with 1 μL of 100×SYPRO orange dye added to each well. The mixture was then pipetted and ready for PCR. The thermal stability of the samples was tested using an ABI 7500FAST RT-PCR instrument. The experiment type was selected as melting curve, continuous mode was used, the scanning temperature range was 25–95 °C, the heating rate was 1%, and equilibration was performed at 25 °C for 5 min. Data was collected during the heating process. The reporter group was selected as “ROX”, the quencher group as “None”, and the reaction volume was 20 μL. The melting temperature Tm of the antibody was determined by the temperature corresponding to the first peak-to-trough of the first derivative of the melting curve.

Example 1: Design of anti-CD47-CLDN18.2 bispecific antibody

This embodiment describes an exemplary anti-CD47-CLDN18.2 bispecific antibody: wherein the arm binding to CLDN18.2 uses an anti-CLDN18.2 single-domain antibody NA3S-H1 that specifically recognizes human CLDN18.2 but not CLDN18.1 (the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:13, 14, and 15, respectively; the amino acid sequence of VHH is shown in SEQ ID NO:12), and the arm binding to CD47 uses the antigen-binding domain of the anti-CD47 humanized antibody A7H3L3 (the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:4, 5, and 6, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:7, 8, and 9, respectively; 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 NO:11). The heavy chain variable region of antibody A7H3L3 was fused to the constant region of the human IgG1 heavy chain (SEQ ID NO:2) to form the heavy chain of antibody A7H3L3, and the light chain variable region was fused to the constant region of the human κ light chain (SEQ ID NO:3) to form the light chain of antibody A7H3L3. The anti-CLDN18.2 single-domain antibody NA3S-H1 has been published in WO2020238730A1, and its in vitro ADCC and CDC cytotoxic effects, as well as tumor suppression experiments in the human CLDN18.2-HEK29T-SCID tumor transplantation model, have shown excellent efficacy. The humanized antibody A7H3L3 binds weakly to CD47 on erythrocytes, making it a relatively ideal candidate antibody for bispecific antibody development. Furthermore, the design of the bispecific antibody, by binding to CLDN18.2, allows the binding arm of the anti-CD47 antibody A7H3L3 to better bind to tumor cells expressing both targets, while also better blocking the SIRPα inhibitory signal.

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

Table 1. Structure of anti-CD47-CLDN18.2 bispecific antibody

Table 2. Amino acid sequence of anti-CD47-CLDN18.2 bispecific antibody

Antibody name Heavy chain amino acid sequence Light chain amino acid sequence B8 SEQ ID NO:16 SEQ ID NO:17 B9 SEQ ID NO:18 SEQ ID NO:19 B10 SEQ ID NO:20 SEQ ID NO:17 B11 SEQ ID NO:16 SEQ ID NO:19 B12 SEQ ID NO:21 SEQ ID NO:17

Example 2: Binding activity of anti-CD47-CLDN18.2 bispecific antibody to CD47 on erythrocytes

The binding of the bispecific antibody to erythrocytes was determined by flow cytometry. Another anti-CD47 antibody developed by the applicant, F4AM4-IgG1 (abbreviated as F4AM4 in the attached figure; the amino acid sequence of the heavy chain is SEQ ID NO:24, and the amino acid sequence of the light chain is SEQ ID NO:25), was used as a positive control antibody. F4AM4-IgG1 showed strong binding to CD47 on erythrocytes in a series of functional verification experiments (conducted prior to the experiments in this embodiment), and was therefore selected as a positive control in this embodiment.

The specific method is as follows: Red blood cells were separated from 1 mL of anticoagulated human blood, centrifuged, and the supernatant was removed. After washing twice with PBS, 1 mL of PBS was added and the cells were resuspended. The red blood cells were diluted to 1 × ^10⁷ /mL with PBS, and 50 μL of red blood cells were added to each well of a 96-well round-bottom cell culture plate. Then, serially diluted test antibodies were added in equal volumes, mixed thoroughly, and incubated at 4°C for 1 h. Next, the cells were washed three times with FACS buffer, and 0.5 μg of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596) was added. The cells were incubated at 4°C for 1 h. Afterward, the cells were washed three times with FACS buffer, and 200 μL of FACS buffer was added to resuspend the cells. Finally, the amount of antibody bound to the red blood cells was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102) (expressed as mean fluorescence intensity (MFI)).

The binding activity of the antibodies to erythrocytes 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 showed almost no binding to erythrocytes or extremely low binding activity compared to the strong binding of F4AM4-IgG1 to erythrocytes; at high concentrations (15 μg/mL), the binding of B9, B10, and B11 to erythrocytes was not significantly different from that of the negative control IgG1, and the specific values are shown in Table 3. Therefore, the bispecific antibodies of this invention exhibit low binding activity to CD47 on erythrocytes and are unlikely to induce erythrocyte agglutination.

Table 3. Mean fluorescence intensity (MFI) of bispecific antibody binding on erythrocytes.

B8 B9 B10 B11 B12 A7H3L3 IgG1 F4AM4-IgG1 15 μg/mL MFI 988 239 534 238 1615 463 303 63000 150 μg/mL MFI 2145 1220 2493 847 2526 608 901 >63000

Example 3: Binding activity of anti-CD47-CLDN18.2 bispecific antibody on tumor cells expressing single and dual targets.

The binding activity of the anti-CD47-CLDN18.2 bispecific antibody B10 to NUGC-4 cells (expressing endogenous CD47, purchased from the BNCC bacterial bank, catalog number BNCC341962) and hCLDN18.2-NUGC-4 cells (a gastric cancer cell line NUGC-4 overexpressing exogenous human CLDN18.2 (amino acid sequence SEQ ID NO: 1) and simultaneously expressing endogenous CD47, constructed via lentiviral transfection) was determined. For comparison, the binding activities of antibody 1F8 (antibody 1F8 from WO2018075857A1) and F4AM4-IgG1 to these two cell lines were also determined. Human IgG1 was used as an isotype negative control.

The specific method is as follows: Take 1× ^10⁵ NUGC-4 cells or hCLDN18.2-NUGC-4 cells, centrifuge at low speed (300g) and remove the supernatant; rinse the cells at the bottom of the centrifuge tube once with prepared FACS buffer (1×PBS buffer containing 2% FBS by volume), then add serially diluted test antibody to the rinsed cells and incubate at 4℃ for 1h; then rinse three more times with the above FACS buffer, add 0.5μg of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596), and incubate at 4℃ for 1h; then rinse the cells three times with FACS buffer and resuspend them in 200μL of FACS buffer, and finally detect the amount of antibody bound to red blood cells by flow cytometry (Beckman, CytoFLEX AOO-1-1102) (expressed as mean fluorescence intensity (MFI)).

The binding activities of the antibody on NUGC-4 cells and hCLDN18.2-NUGC-4 cells are shown in Figures 3a and 3b, respectively. As shown in Figure 3a, the binding activity of bispecific antibody B10 on NUGC-4 tumor cells is weak, and slightly weaker than that of antibody 1F8. As shown in Figure 3b, bispecific antibody B10 exhibits significantly superior binding activity to antibody 1F8 on hCLDN18.2-NUGC-4 cells that express both CD47 and CLDN18.2. Based on this, although bispecific antibody B10 binds weakly to 1F8 on cells expressing only CD47, it binds better than 1F8 on cells expressing both CD47 and CLDN18.2. This result demonstrates that bispecific antibody B10 can simultaneously bind to CLDN18.2 and CD47 in tumor cells, increasing its ability to bind to tumor cells.

Example 4: The ability of anti-CD47-CLDN18.2 bispecific antibody to block CD47 binding to SIRPα on tumor cells expressing single and dual targets.

The ability of the anti-CD47-CLDN18.2 bispecific antibody to block the binding of CD47 to SIRPα on NUGC-4 and hCLDN18.2-NUGC-4 cells was determined by flow cytometry. For comparison, the ability of antibodies 1F8, F4AM4-IgG1, and A7H3L3 to block the binding of CD47 to SIRPα on NUGC-4 and hCLDN18.2-NUGC-4 tumor cells was also determined.

The specific method is as follows: Take 1× ^10⁵ NUGC-4 cells or hCLDN18.2-NUGC-4 cells, centrifuge at low speed (300g) and remove the supernatant. Cells at the bottom of the centrifuge tube were rinsed once with prepared FACS buffer (1×PBS buffer containing 2% FBS); then, serially diluted test antibody was added to the rinsed cells and incubated for 1 h; after rinsing the cells twice with FACS buffer, 100 μL of 1 μg/mL SIRPα-mFc (ACRO, SIA-H52A8) was added and incubated at 4 °C for 1 h; after rinsing three times with FACS buffer, 100 μL of PE-labeled goat anti-mouse Fc secondary antibody (Abcam, ab98742) diluted 1:200 was added and incubated at 4 °C for 1 h, then centrifuged to remove the supernatant, and the cells were resuspended in 200 μL of FACS buffer. Finally, the amount of SIRPα-mFc bound to the cells was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102) (expressed as mean fluorescence intensity (MFI)).

The results on NUGC-4 cells are shown in Figure 4a: Antibody F4AM4-IgG1 effectively blocked the binding of CD47 to SIRPα, with an _IC50 of 0.033 μg/mL (0.226 nM); Antibody 1F8 had a weak blocking effect, with an _IC50 of 15.36 μg/mL (105.6 nM); while the bispecific antibody B10 had almost no blocking ability.

The results on hCLDN18.2-NUGC-4 cells are shown in Figure 4b: Antibody F4AM4-IgG1 still showed strong blocking ability on these tumor cells, with an _IC50 of 0.056 μg/mL (0.383 nM); compared with its blocking ability on NUGC-4 cells, the bispecific antibody B10 showed significantly improved blocking ability on hCLDN18.2-NUGC-4 cells, and was superior to antibody 1F8; the _IC50 values of B10 and 1F8 for blocking CD47 binding to SIRPα on hCLDN18.2-NUGC-4 cells were 0.765 μg/mL (4.476 nM) and 11.98 μg/mL (82.34 nM), respectively; in addition, the anti-CLDN18.2 single-domain antibody NA3S-H1 did not have any blocking effect because it only bound to CLDN18.2. Based on this, although the blocking activity of bispecific antibody B10 is weaker than that of antibody 1F8 on cells expressing CD47 as a single target, its blocking activity is significantly better than that of antibody 1F8 on cells expressing both CD47 and CLDN18.2 as dual targets, and it will exert a stronger ability to block the binding of CD47 to receptor SIRPα.

The blocking ability of other bispecific antibodies on hCLDN18.2-NUGC-4 cells was also determined, 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 suppression experiment of anti-CD47-CLDN18.2 bispecific antibody

5.1 In vivo tumor suppression experiment 1

Six- to seven-week-old female nude mice (16-18g) were housed in individually ventilated enclosures with constant temperature and humidity (21-24℃, 30-53%). Three × ^10⁶ hCLDN18.2-NUGC-4 cells were subcutaneously injected into the left axilla of each mouse (day 0). When the subcutaneous tumor volume reached approximately 300-400 ^mm³ (day 20), mice with significantly different tumor volumes were excluded. The mice were then randomly assigned to four groups (n=8 per group) based on tumor volume: PBS treatment group, NA3S-H1 monoclonal antibody group, A7H3L3 monoclonal antibody group, NA3S-H1 + A7H3L3 combined treatment group, and bispecific antibody B10 treatment group. Using NA3S-H1 monoclonal antibody 5 mg/kg as the standard, all other drugs were administered at equimolar doses: A7H3L3 monoclonal antibody 9.4 mg/kg, NA3S-H1 + A7H3L3 combination 5 mg/kg + 9.4 mg/kg, and bispecific antibody B10 10.6 mg/kg. Administered twice weekly, alternating between intraperitoneal injection (ip) and intravenous injection (iv). Tumor length (mm) and width (mm) were continuously observed and recorded, and tumor volume (V) was calculated as: V = (length × ^width² ) / 2. Tumor inhibition rate (TGI) (%) = (1 - mean tumor volume in the treated group / mean tumor volume in the PBS-treated group) × 100%.

The results of antibody tumor suppression are shown in Figure 5a and Table 4. It can be seen that at this equimolar dose, the NA3S-H1 monoclonal antibody group showed almost no tumor suppression effect, while the other groups showed some tumor suppression effect. Among them, the bispecific antibody B10 had the best effect, reaching a tumor inhibition rate of nearly 54.54% before day 39. The tumor size was close to the initial tumor volume on day 20, and it was better than the results of combination therapy (A7H3L3+NA3S-H1 (9.4+5mpk)). This indicates that the binding of bispecific antibody B10 to CLDN18.2 can enhance its blocking effect on the binding of CD47 and SIRPα.

Table 4. Tumor inhibition rate of bispecific antibodies in mice.

Days NA3S-H1 A7H3L3 B10 A7H3L3+NA3S-H1 25 2.96% -1.31% 3.41% 12.73% 28 8.76% 2.17% 27.69% 18.40% 32 -5.33% 3.73% 40.26% 19.30% 35 -6.94% 2.10% 44.31% 19.49% 39 2.01% 15.00% 54.54% 33.71% 42 -7.26% 14.65% 51.45% 30.84% 46 -8.69% 24.65% 48.33% 32.59% 49 -5.94% 25.18% 45.12% 25.62%

5.2 In vivo tumor suppression experiment 2

Mice were randomly assigned to three groups (n=8 per group): a PBS treatment group, a monoclonal antibody NA3S-H1 administration group, and a bispecific antibody B10 administration group. The first administration was given on day 0 (vaccination day), followed by administration twice weekly. The NA3S-H1 monoclonal antibody was administered at a standard dose of 2.5 mg/kg, and the bispecific antibody B10 was administered at an equimolar dose of 5.3 mg/kg. The rest of the procedure was the same as in Example 5.1.

As shown in Figure 5b, at this equimolar dose, the NA3S-H1 monoclonal antibody group exhibited a certain tumor-suppressive effect, with a tumor inhibition rate of approximately 54.99% (day 34); in the bispecific antibody B10 group, tumors in all mice were completely inhibited, with a tumor inhibition rate approaching 100% (day 34). These results demonstrate that the tumor-suppressive effect of the bispecific antibody B10 is significantly superior to that of the anti-CLDN18.2 monoclonal antibody NA3S-H1.

Example 6: Physicochemical Properties Determination of Anti-CD47-CLDN18.2 Bispecific Antibody

6.1 SDS-PAGE Purity Identification

The purity of the bispecific antibody B10 was determined by reducing SDS-PAGE. The apparent relative molecular weights of the heavy and light chains of the bispecific antibody B10 were approximately 65 kDa and 25 kDa, respectively, which were consistent with the expected sizes, and the purity was approximately 90%.

6.2 SEC-HPLC purity identification

The monomeric purity of the bispecific antibody B10 was determined using SEC-HPLC. The monomeric purity of the bispecific antibody B10, as determined by SEC-HPLC, was greater than 94%.

6.3 Detection of DSF thermal stability of bispecific antibody B10

The thermal stability of bispecific antibody B10 was assessed by detecting the Tm value using the DSF method. The results showed that bispecific antibody B10 exhibited two melting peaks: the first peak had a Tm value of 69.85 ± 0.06 °C, and the second peak had a Tm value of 80.60 ± 0.16 °C, indicating that bispecific antibody B10 possesses good thermal stability.

Example 7 ADCP activity of anti-CD47-CLDN18.2 bispecific antibody

Antibody-dependent cell-mediated phagocytosis (ADCP) is an important mechanism by which therapeutic antibodies combat viral infections or tumor cells. This study evaluated the ADCP activity of the bispecific antibody of this invention using a bioluminescent reporter gene assay. The method used genetically engineered Jurkat cells (BPS Bioscience Inc., 71273) as effector cells, which stably express the FcγRIIa receptor and luciferase expression driven by the NFAT response element (Int Immunopharmacol. 2021 Nov; 100:108-112.). After recognizing target cells, the antibody binds to FcγRIIa on the effector cell surface, stimulating the intracellular NFAT response element, which in turn drives luciferase expression. Luciferase activity can be quantified using a bioluminescent method.

The test samples (B10, A7H3L3, NA3S-H1) were diluted to an initial reaction concentration of 200 nM, and ten 2-fold serial dilutions were performed. 50 μL of the diluted samples were added to each well of a 96-well white substrate plate, with three replicates for each concentration gradient. 150 μL/well of PBS solution was added to the edge wells of the 96-well plate. Then, 50 μL/well of hCLDN18.2-NUGC-4 cells (target cells) at 5 × ^10⁵ cells/mL were added to the 96-well plate and incubated at room temperature for 30 min. Next, 50 μL/well of effector cells at 1.5 × ^{10⁶ cells} /mL as described above were added. The 96-well plate was incubated at 37°C in a 5% _CO₂ incubator for 6 h. Detection: The 96-well plate was removed and allowed to stand at room temperature for 30 min. Add 50 μL of luciferase substrate (Bio-Glo ^™ Luciferase Assay System Promega G7940) to each well and incubate at room temperature in the dark for 5–10 min. Detect the luminescence intensity (expressed as relative light units (RLU)) using a microplate reader. Plot the logarithm of the antibody concentration against the corresponding RLU values, analyze the data using Graphpad Prism software, and calculate the _EC50 value.

Experimental results: As can be seen from Figure 6 and Table 5, when the target cells are hCLDN18.2-NUGC-4 cells, the bispecific antibody B10 of this invention exhibits higher ADCP activity compared with anti-CD47 monoclonal antibody A7H3L3 and anti-CLDN18.2 monoclonal antibody NA3S-H1.

Table 5 ADCP activity of bispecific antibodies

NA3S-H1 B10 A7H3L3 <![CDATA[EC₅₀]]> 7.169 11.69 25.64 <![CDATA[R²]]> 0.9948 0.9895 0.9962

Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings disclosed herein, and all such changes are within the scope of protection of the invention. The scope of protection of the invention is given by the appended claims and any equivalents thereof.

sequence list

SEQ ID NO:1 person CLDN18.2

SEQ ID NO:2 Human IgG1 Heavy Chain Constant Region

SEQ ID NO: 3 people Kapp light chain constant region

SEQ ID NO:4 A7H3L3-HCDR1

SEQ ID NO:5 A7H3L3-HCDR2

SEQ ID NO: 6 A7H3L3-HCDR3

SEQ ID NO:7 A7H3L3-LCDR1

SEQ ID NO:8 A7H3L3-LCDR2

SEQ ID NO:9 A7H3L3-LCDR3

SEQ ID NO:10 A7H3L3-VH

SEQ ID NO:11 A7H3L3-VL

SEQ ID NO:12 NA3S-H1

SEQ ID NO:13 NA3S-H1 CDR1

SEQ ID NO:14 NA3S-H1 CDR2

SEQ ID NO:15 NA3S-H1 CDR3

SEQ ID NO:16 B8/B11 heavy chain

SEQ ID NO:17 B8/B10/B12 Light Chain

SEQ ID NO:18 B9 heavy chain

SEQ ID NO:19 B9/B11 Light Chain

SEQ ID NO: 20 B10 heavy chain

SEQ ID NO:21 B12 heavy chain

SEQ ID NO:22 Linker1

SEQ ID NO:23 Linker2

SEQ ID NO:24 F4AM4-IgG1 heavy chain

SEQ ID NO:25 F4AM4-IgG1 light chain

sequence list

<110> Baochuan Biomedical Technology (Shanghai) Co., Ltd.

Sanyou Biopharmaceutical (Shanghai) Co., Ltd.

<120> Anti-CD47-CLDN18.2 Bispecific Antibody and Its Uses

<130> P2022TC6860CS

<150> CN202111214360.8

<151> 2021-10-19

<160> 25

<170> PatentIn version 3.5

<210> 1

<211> 261

<212> PRT

<213> Homo sapiens

<400> 1

Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile

1 5 10 15

Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

65 70 75 80

Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

85 90 95

Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

100 105 110

Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser

115 120 125

Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val

130 135 140

Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly

145 150 155 160

Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe

165 170 175

Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met

180 185 190

Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala

195 200 205

Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly

210 215 220

Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile

225 230 235 240

Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser

245 250 255

Lys His Asp Tyr Val

260

<210> 2

<211> 330

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IgG1 heavy chain constant region

<400> 2

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 3

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Human Kappa light chain constant region

<400> 3

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 4

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> A7H3L3-HCDR1

<400> 4

Gly Phe Asn Ile Lys Asp Ile Tyr Ile Tyr

1 5 10

<210> 5

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> A7H3L3-HCDR2

<400> 5

Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys

1 5 10

<210> 6

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> A7H3L3-HCDR3

<400> 6

Gly Tyr Gly Ser Gly Phe Ala Tyr

1 5

<210> 7

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> A7H3L3-LCDR1

<400> 7

Arg Ala Ser Gln Asp Ile Ser Asn His Leu Asn

1 5 10

<210> 8

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> A7H3L3-LCDR2

<400> 8

Tyr Thr Ser Arg Ile His Ser

1 5

<210> 9

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> A7H3L3-LCDR3

<400> 9

Gln Gln Gly Tyr Thr Leu Pro Phe Thr

1 5

<210> 10

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> A7H3L3-VH

<400> 10

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ile

20 25 30

Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 11

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> A7H3L3-VL

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Ile His Ser Gly Val Pro Ser Ser Phe Arg Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> NA3S-H1

<400> 12

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro

20 25 30

Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 13

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> NA3S-H1 CDR1

<400> 13

Gly Ser Ile Phe Asn Ile Pro Val

1 5

<210> 14

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NA3S-H1 CDR2

<400> 14

Ile Ser Thr Gly Gly Thr Thr

1 5

<210> 15

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> NA3S-H1 CDR3

<400> 15

Asn Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val

1 5 10

<210> 16

<211> 577

<212> PRT

<213> Artificial Sequence

<220>

<223> B8/B11 heavy chain

<400> 16

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro

20 25 30

Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys

145 150 155 160

Asp Ile Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

165 170 175

Trp Ile Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Gln

180 185 190

Lys Phe Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr

195 200 205

Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

210 215 220

Tyr Cys Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

245 250 255

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

260 265 270

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

275 280 285

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

290 295 300

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

305 310 315 320

Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys Pro

325 330 335

Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys

340 345 350

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

355 360 365

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

370 375 380

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

385 390 395 400

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

405 410 415

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

420 425 430

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

435 440 445

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

450 455 460

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

465 470 475 480

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

485 490 495

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

500 505 510

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

515 520 525

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

530 535 540

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

545 550 555 560

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

565 570 575

Lys

<210> 17

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> B8/B10/B12 Light Chain

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Ile His Ser Gly Val Pro Ser Ser Phe Arg Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 18

<211> 447

<212> PRT

<213> Artificial Sequence

<220>

<223> B9 heavy chain

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ile

20 25 30

Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 19

<211> 344

<212> PRT

<213> Artificial Sequence

<220>

<223> B9/B11 Light Chain

<400> 19

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro

20 25 30

Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

130 135 140

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser

145 150 155 160

Asn His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

165 170 175

Leu Ile Tyr Tyr Thr Ser Arg Ile His Ser Gly Val Pro Ser Ser Phe

180 185 190

Arg Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu

195 200 205

Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu

210 215 220

Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val

225 230 235 240

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

245 250 255

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

260 265 270

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

275 280 285

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

290 295 300

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

305 310 315 320

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

325 330 335

Lys Ser Phe Asn Arg Gly Glu Cys

340

<210> 20

<211> 577

<212> PRT

<213> Artificial Sequence

<220>

<223> B10 heavy chain

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ile

20 25 30

Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser

450 455 460

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

465 470 475 480

Ala Ser Gly Ser Ile Phe Asn Ile Pro Val Met Gly Trp Tyr Arg Gln

485 490 495

Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Gly Ile Ser Thr Gly Gly

500 505 510

Thr Thr Asn Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

515 520 525

Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro

530 535 540

Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Leu Val Val Ser Gly Ile

545 550 555 560

Gly Ser Thr Leu Glu Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser

<210> 21

<211> 572

<212> PRT

<213> Artificial Sequence

<220>

<223> B12 heavy chain

<400> 21

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro

20 25 30

Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Val Gln

115 120 125

Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys

130 135 140

Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ile Tyr Ile Tyr

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Lys Ile

165 170 175

Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Gln Lys Phe Gln Gly Arg

180 185 190

Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr Leu Glu Leu

195 200 205

Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly

210 215 220

Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

245 250 255

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

260 265 270

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

275 280 285

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

290 295 300

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

305 310 315 320

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

325 330 335

Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

340 345 350

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

355 360 365

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

370 375 380

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

385 390 395 400

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

405 410 415

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

420 425 430

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

435 440 445

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

450 455 460

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

465 470 475 480

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

485 490 495

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

500 505 510

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

515 520 525

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

530 535 540

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

545 550 555 560

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

565 570

<210> 22

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker1

<400> 22

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 23

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker2

<400> 23

Gly Gly Gly Gly Ser

1 5

<210> 24

<211> 452

<212> PRT

<213> Artificial Sequence

<220>

<223> F4AM4-IgG1 Heavy Chain

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Thr Asp Gly Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Phe Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Arg Pro Tyr Tyr Gly Thr Arg Tyr Gly Ser Trp Phe Ala Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 25

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> F4AM4-IgG1 light chain

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Asn Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Lys Asn Tyr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims (30)

1. A bispecific antibody or an antigen-binding fragment thereof, comprising a first antigen-binding moiety binding to CD47 and a second antigen-binding moiety binding 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 includes: 1) HCDR1, whose amino acid sequence is shown in SEQ ID NO:4; 2) HCDR2, whose amino acid sequence is shown in SEQ ID NO:5; and 3) HCDR3, whose amino acid sequence is shown in SEQ ID NO:6; and The light chain variable region includes: 1) LCDR1, whose amino acid sequence is shown in SEQ ID NO:7; 2) LCDR2, whose amino acid sequence is shown in SEQ ID NO:8; and 3) LCDR3, whose amino acid sequence is shown in SEQ ID NO:9; The second antigen-binding portion includes an immunoglobulin single variable domain (VHH) that binds to CLDN18.2, said immunoglobulin single variable domain comprising: CDR1, whose amino acid sequence is shown in SEQ ID NO:13; CDR2, whose amino acid sequence is shown in SEQ ID NO:14; and CDR3, whose amino acid sequence is shown in SEQ ID NO:15. 2. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10 and/or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11. 3. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 10. 4. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 10. 5. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10. 6. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 10. 7. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the heavy chain variable region comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 10. 8. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the light chain variable region comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11. 9. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the light chain variable region comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 11. 10. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the light chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11. 11. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the light chain variable region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 11. 12. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the light chain variable region comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 11. 13. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO:12. 14. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the immunoglobulin single variable domain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 12. 15. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the immunoglobulin single variable domain comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 12. 16. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the immunoglobulin single variable domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 12. 17. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the immunoglobulin single variable domain comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12. 18. The bispecific antibody of claim 1 or its antigen-binding fragment, wherein the immunoglobulin single variable domain comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 12. 19. A bispecific antibody or an antigen-binding fragment thereof of any one of claims 1-18, wherein the first antigen-binding portion and the second antigen-binding portion are connected by a connector. 20. The bispecific antibody or antigen-binding fragment thereof of any one of claims 1-18, further comprising the heavy chain constant region (CH) and light chain constant region (CL) of an immunoglobulin. 21. The bispecific antibody of claim 20 or its antigen-binding fragment, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, and/or the light chain constant region is the light chain constant region of human κ. 22. The bispecific antibody of claim 20 or its antigen-binding fragment, 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. 23. The bispecific antibody of claim 1 or its antigen-binding fragment, comprising a first polypeptide and a second polypeptide. in The first polypeptide has the following structure from the N-terminus to the C-terminus: VH-CH-Linker-VHH, The second polypeptide has the following structure from the N-terminus to the C-terminus: VL-CL, in VH and VL are the heavy chain variable region and the light chain variable region as defined in claim 1 or 2, respectively; VHH is an immunoglobulin single variable domain as defined in claim 1 or 13; CH and CL are the heavy chain constant region and light chain constant region of immunoglobulin, respectively; Linker is a connector. 24. The bispecific antibody of claim 23 or an antigen-binding fragment thereof, 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. 25. An isolated polynucleotide encoding a bispecific antibody or an antigen-binding fragment thereof of any one of claims 1-24. 26. An expression vector comprising the polynucleotide of claim 25. 27. A host cell comprising the polynucleotide of claim 25 or the expression vector of claim 26. 28. An antibody conjugate comprising a bispecific antibody or an antigen-binding fragment thereof of any one of claims 1-24 conjugated to at least one therapeutic agent. 29. A pharmaceutical composition comprising a bispecific antibody of any one of claims 1-24 or an antigen-binding fragment thereof, or an antibody conjugate of claim 28, and a pharmaceutically acceptable carrier. 30. Use of the bispecific antibody or antigen-binding fragment thereof of any one of claims 1-24, the antibody conjugate of claim 28, or the pharmaceutical composition of claim 29 in the preparation of a medicament for treating cancer, wherein the cancer is gastric cancer.