WO2023134742A1

WO2023134742A1 - Three-target anti-tumor drug, and preparation method therefor and use thereof

Info

Publication number: WO2023134742A1
Application number: PCT/CN2023/072066
Authority: WO
Inventors: 姚高峰; 黄岩山
Original assignee: 浙江道尔生物科技有限公司
Priority date: 2022-01-14
Filing date: 2023-01-13
Publication date: 2023-07-20

Abstract

A three-target anti-tumor drug, and a preparation method therefor and the use thereof. A fusion protein can be efficiently expressed, has a long half-life in vivo, a high stability and a good tumor inhibitory effect.

Description

A kind of antitumor drug with three targets, its preparation method and application

technical field

The invention relates to the field of biology, in particular to a three-target antitumor drug specifically targeting CLD18A2, human serum albumin and CD3, its preparation method and application.

Background technique

Claudin 18 (CLD18) is a transmembrane protein with a molecular weight of about 28kD, located in the tight junction of epithelium and endothelium, and is tightly connected between adjacent cells. In normal epithelial tissue, claudin on the cell surface is difficult to access due to the tight intercellular space, while the intercellular space in tumor cells is relatively loose. Therefore, claudin on tumor cells has become a potential target for extracellular antibodies and immunotherapy. CLD18 has four hydrophobic regions, which form two extracellular domains as transmembrane regions, in which hydrophobic region 1 and hydrophobic region 2 surround to form extracellular domain 1, and hydrophobic region 3 and hydrophobic region 4 surround to form extracellular domain 2. The human CLD18 gene has two different exon 1, which can be alternatively spliced after transcription to generate two protein isoforms with different sequences only at the N-terminus: CLD18A1 and CLD18A2. CLD18A1 is expressed in normal lung tissue, whereas CLD18A2 is expressed only in gastric tissue; more importantly, CLD18A2 is restricted to differentiated short-lived gastric epithelial cells but absent from gastric stem cells (Niimi T, et al. Biol .2001;21(21):7380-7390).

In addition, CLD18A2 is highly expressed in primary gastric cancer and its metastatic cancer types, and its activated expression is often observed in pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer. These properties of CLD18A2 make it a potential target for clinical treatment of gastric cancer and other related tumors.

However, although several drugs targeting CLD18A2 are currently in the clinical stage, there are still no drugs approved for marketing. In the existing drug research on the CLD18A2 target, there are still aspects to be improved in terms of drug expression system, drug half-life in vivo, drug efficacy, and targeting.

Contents of the invention

The object of the present invention is to provide a three-target anti-tumor drug specifically targeting CLD18A2, human serum albumin and CD3, its preparation method and application.

In a first aspect of the present invention, a fusion protein is provided, comprising: an anti-CLD18A2 domain, an anti-human serum albumin (HSA) domain and an anti-CD3 domain; the anti-CLD18A2 domain is a single-domain antibody with SEQ ID NO: CDR1 shown in No. 26-33 in 1, CDR2 shown in No. 51-57 in SEQ ID NO: 1, CDR3 shown in No. 96-109 in SEQ ID NO: 1; Said anti The human serum albumin domain is a single domain antibody with CDR1 shown in positions 155-162 in SEQ ID NO:1, CDR2 shown in positions 180-187 in SEQ ID NO:1, and in SEQ ID NO:1 CDR3 indicated at positions 226-233.

In one or more embodiments, the anti-CLD18A2 domain is a single domain antibody, and its framework region (FR) has a P→A mutation in the amino acid sequence; preferably, its FR1 region has a P→A mutation; more Preferably, the P→A mutation corresponds to the 14th position of SEQ ID NO:1.

In one or more embodiments, the anti-human serum albumin domain is a single domain antibody, and its framework region (FR) has a P→A mutation in its amino acid sequence; preferably, its FR1 region has a P→A mutation Mutation; more preferably, the amino acid of its P→A mutation is the 143rd amino acid corresponding to the sequence of SEQ ID NO:1.

In one or more embodiments, the anti-CD3 domain is a single chain antibody.

In one or more embodiments, the anti-CD3 domain has HCDR1 shown in positions 279-286 in SEQ ID NO: 1, HCDR2 shown in positions 304-313 in SEQ ID NO: 1, SEQ ID NO: 1 HCDR3 shown in No. 352-367 in ID NO:1, LCDR1 shown in No. 419-427 in SEQ ID NO:1, LCDR2 shown in No. 445-447 in SEQ ID NO:1, SEQ ID NO : LCDR3 shown in bits 484-492 in 1.

In one or more embodiments, the anti-CD3 domain is located at the N-terminal or C-terminal of the anti-human serum albumin domain.

In one or more embodiments, the amino acid sequence of the anti-CLD18A2 domain is shown in positions 1-120 of SEQ ID NO:1.

In one or more embodiments, the amino acid sequence of the anti-human serum albumin domain is shown in positions 130-244 of SEQ ID NO:1.

In one or more embodiments, the amino acid sequence of the anti-CD3 domain is shown in positions 254-502 of SEQ ID NO:1.

In one or more embodiments, the amino acid sequences of the anti-CLD18A2 domain, the anti-human serum albumin domain and the anti-CD3 domain are directly connected, or connected through a linker (connecting peptide); preferably, the The amino acid sequence of the fusion protein is shown in SEQ ID NO:1.

In one or more embodiments, the linker includes, but is not limited to: a flexible polypeptide containing glycine (G), serine (S) and/or alanine (A).

In one or more embodiments, when linked by two linkers, the two linkers are the same; alternatively, the two linkers are different.

In one or more embodiments, the linker is, for example, 2A, such as P2A, E2A, T2A.

In one or more embodiments, the anti-CLD18A2 VHH, anti-HSA VHH and anti-CD3 scFv, from N-terminus to C-terminus, are: CLD18A2VHH, anti-HSA VHH and anti-CD3 scFv.

In one or more embodiments, the anti-CLD18A2 VHH, anti-HSA VHH and anti-CD3 scFv, from N-terminus to C-terminus, are: CLD18A2VHH, anti-CD3 scFv and anti-HSA VHH.

In another aspect of the present invention, there is provided a polynucleotide or an expression construct comprising the polynucleotide (such as expression vector), the polynucleotide encodes the fusion protein.

In another aspect of the present invention, a fusion protein expression system is provided. The expression system contains the expression construct or the exogenous polynucleotide integrated in its genome.

In one or more embodiments, the expression system is a host cell.

In one or more embodiments, the expression system is a eukaryotic expression system.

In one or more embodiments, the eukaryotic expression system is an animal cell expression system or a yeast expression system.

In one or more embodiments, the animal cell expression system includes (but not limited to): CHO, NSO, BHK, HEK-293 or PER-C6.

In one or more embodiments, the animal cell expression system is CHO.

In one or more embodiments, the expression system is a prokaryotic expression system, such as a prokaryotic host cell.

In another aspect of the present invention, there is provided a method for preparing the fusion protein, comprising: using the expression system to express the fusion protein; preferably, the method further includes: isolating the fusion protein .

In another aspect of the present invention, a method for modifying a CLD18A2 binding molecule to improve its therapeutic effect is provided, comprising: using an anti-CLD18A2 domain as a CLD18A2 binding molecule, and linking it to an anti-human serum albumin domain and an anti-CD3 domain ; The anti-CLD18A2 structural domain is a single domain antibody, with CDR1 shown in the 26th-33rd position in SEQ ID NO: 1, CDR2 shown in the 51-57th position in SEQ ID NO: 1, SEQ ID NO: 1 The CDR3 shown in the 96th-109th position; the anti-human serum albumin domain is a single domain antibody, with the CDR1 shown in the 155th-162th position in SEQ ID NO:1, and the 180th position in SEQ ID NO:1 - CDR2 shown in position 187, CDR3 shown in positions 226-233 in SEQ ID NO:1.

In another aspect of the present invention, there is provided the use of any of the aforementioned fusion proteins, the expression system or its culture in the preparation of drugs for alleviating or treating diseases related to CLD18A2 positive cells; preferably, the CLD18A2 Positive cell-related diseases include tumors; preferably, the tumors include (but not limited to): pancreatic cancer, gastric cancer, esophageal cancer, lung cancer, ovarian cancer, breast cancer, colorectal cancer, liver cancer, gallbladder cancer or head and neck cancer.

In another aspect of the present invention, a pharmaceutical composition is provided, which includes: said fusion protein.

In another aspect of the present invention, there is provided a pharmaceutical composition, wherein said fusion protein expression system, or its culture.

In another aspect of the present invention, a kit is provided, which includes: a container or package, and the fusion protein therein.

In another aspect of the present invention, a kit is provided, which includes: a container or package, and the expression system of the fusion protein or its culture located therein.

In another aspect of the present invention, a kit is provided, comprising: a container or package, and a The pharmaceutical composition described in.

In another aspect of the present invention, a method for alleviating or treating CLD18A2-positive cell-related diseases is provided, the method comprising: administering an effective amount of the fusion protein to a subject in need of alleviating or treating the disease, or a protein expressing the fusion protein An expression system or its culture, or a pharmaceutical composition containing the fusion protein.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1 C57BL/6 mice were single-administered 1mg/kg DR303X2 and 1mg/kg DR303X8 by tail vein injection, and the relationship between blood drug concentration and administration time.

Figure 2. Intravenous injection of DR303X8, when the administration concentration (3mg/kg, 0.6mg/kg, 0.03mg/kg) ratio of 100:20:1 in the high, middle and low dose groups, the C _max ratio is 80:17:1, AUC _{The 0-t} ratio was 116:20:1, and the systemic exposure was linearly related to the administered dose.

Figure 3. Anti-tumor efficacy of anti-CLD18A2/HSA/CD3 trispecific fusion protein in PBMC immune system humanized BxPC3/hCLDN18.2 pancreatic cancer subcutaneous xenograft model. biwX5 means administration twice a week for a total of 5 weeks; qd X17 means administration once a day for 17 days; and so on in the following figure.

Figure 4. The anti-tumor efficacy of anti-CLD18A2/HSA/CD3 trispecific fusion protein in PBMC immune system humanized NUGC4/hCLDN18.2 subcutaneous xenograft model of gastric cancer.

Figure 5. Antitumor efficacy of fusion proteins DR303X8 and X910 in PBMC immune system humanized NUGC4/hCLDN18.2 subcutaneous xenograft model of gastric cancer.

Detailed ways

After in-depth research, the inventors provided a fusion protein with three-target targeting, which included: (1) a single-domain antibody (VHH) specifically binding to the CLD18A2 antigen, (2) a CD3-specific Bound single chain antibody (scFv) and (3) single domain antibody (VHH) that specifically binds human serum albumin (HSA). The fusion protein can be efficiently expressed, has a long half-life in vivo, high stability, and has good tumor suppressing effect.

fusion protein

In the previous studies of the inventors, it was found that the anti-CLD18A2 single-domain antibody had poor stability in vivo, and the application of IgG Fc, which is commonly used in the field to improve expression and stability, to be fused to it still cannot achieve substantial improvement. After extensive research and screening, the inventors fused the anti-CLD18A2 domain with the anti-CD3 domain and the anti-HSA domain to obtain an anti-CLD18A2/HSA/CD3 trispecific fusion protein.

As used herein, the "anti-CLD18A2/HSA/CD3 trispecific fusion protein", "trispecific fusion protein", and "triple target-targeting fusion protein" can be used interchangeably.

The term "specificity" in the present invention means that the trispecific fusion protein of the present invention does not or substantially does not or less cross-reacts with other proteins other than the target antigen. The degree of its specificity can be judged by immunological techniques, including but not limited to western blotting, immunoaffinity chromatography, flow cytometry and the like. For example, the specific recognition is preferably determined by flow cytometry, and the criteria for specific recognition in specific cases can be judged by those of ordinary skill in the art based on their common knowledge in the field.

As a preferred mode of the present invention, the anti-CLD18A2 domain is a single domain antibody, referred to herein as "anti-CLD18A2VHH". The anti-HSA domain is a single domain antibody, which is referred to as "anti-HSA VHH" for short herein. The overall molecular weight of the single-domain antibody provided by the present invention can be about one-half that of the ScFv single-chain antibody, so the molecular weight of the overall structure can be effectively reduced, thereby enhancing its tissue penetration and reaching target tissues and organs more effectively , improve the therapeutic effect, and this structure is easier to prepare than the structure with two ScFvs in series. The anti-CD3 structural domain is a single-chain antibody, which is referred to as "anti-CD3 scFv" for short in the present invention.

As a preferred mode of the present invention, the inventors have optimized and transformed the anti-CLD18A2 domain and the anti-HSA domain, the FR1 region of the anti-CLD18A2 domain undergoes a P→A mutation; the FR1 region of the anti-HSA domain undergoes a mutation P→A mutation. The mutations very significantly increased the stability and half-life of the fusion protein.

As a preferred mode of the present invention, the anti-CD3 domain is a single-chain antibody, and preferably its amino acid sequence is shown in positions 254-502 of SEQ ID NO:1.

In the present invention, the anti-CLD18A2 domain can specifically bind to the CLD18A2 antigen in tumor tissue; the anti-HSA domain can reversibly and non-covalently bind to albumin in serum; the anti-CD3 scFv can bind to T The CD3 subunit in the cellular TCR complex binds, activates T cells, and releases cytokines to kill tumor target cells. The sequence (N-terminal-C-terminal) of each domain in the anti-CLD18A2/HSA/CD3 trispecific fusion protein can be: anti-CLD18A2VHH-anti-HSA VHH-anti-CD3scFv or anti-CLD18A2VHH-anti-CD3scFv-anti-HSA VHH.

In the absence of tumor cells and T cells, the anti-CLD18A2/HSA/CD3 trispecific fusion protein non-covalently binds to HSA in peripheral blood, prolonging its in vivo half-life through the circulating mechanism of HSA binding to FcRn. In the tumor microenvironment, the anti-CLD18A2/HSA/CD3 trispecific fusion protein cross-links with tumor cells and T cells expressing CLD18A2 antigen to produce TDCC effect (T-cell dependent cellular cytotoxicity) to kill tumor cells.

In the fusion protein, a connecting peptide may also be included. For example, anti-CLD18A2VHH and anti-HSA Linking peptides may be provided between VHHs, and between anti-HSA VHHs and anti-CD3 scFv. The connecting peptide fragment can generally be a flexible polypeptide rich in G, S and/or A (mainly composed of glycine (G), serine (S) and/or alanine (A)) with a suitable length, so that Adjacent protein domains are free to move relative to each other. For example, the amino acid sequence of the connecting peptide fragment may include (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGS)nG, (GGGGA)nA, (GGGGG )nA and other sequences, wherein n is selected from an integer between 1-10. The length of the amino acid sequence of the connecting peptide fragment can be 3-30, 3-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-22, 22-24, 24-26, 26-28 or 28-30.

The fusion protein may also contain a tag protein, and the tag protein is 6×His, Fc, Myc, GST, Flag or HA. In some specific embodiments, the tag protein is 6×His, and the nucleotide sequence of the tag protein is shown in SEQ ID NO:9.

On the basis of the preferred domain sequences listed in the present invention, the present invention also includes their homofunctional variants. The difference between these variants with the same function and the preferred structural domains listed in the present invention may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. It should be understood that the change is more likely or usually occurs in the non-CDR region of the domain, but when considering the preferred mutation scheme of the present invention, the P→A mutation in the FR1 region corresponding to the anti-CLD18A2 domain remains conservative, corresponding The P→A mutation in the FR1 region of the anti-HSA domain remains conservative.

On the basis of the preferred structural domains listed in the present invention, the present invention includes sequences with high homology with the described structural domains (such as 70% or higher homology with the specific protein sequences listed; preferably 80% or higher homology; more preferably 90% or higher homology, such as 95%, 98% or 99% homology), and have the same function. Alternatively, the present invention also includes one or more (such as 1-20, 1-15, 1-10, 1-8, 1-5, 1-3 or 1-2) amino acid residues A domain formed by substitution, deletion or addition of a group and having the same function. Likewise, the change is more likely or usually occurs in the non-CDR regions of the domain, but Ala at position 14, Ala at position 143 corresponding to the sequence shown in SEQ ID NO: 1 is conserved.

polynucleotide

The present invention also provides an isolated polynucleotide encoding the anti-CLD18A2/HSA/CD3 trispecific fusion protein of the present invention, the polynucleotide may be RNA, DNA or cDNA, etc. Methods for providing the isolated polynucleotide should be known to those skilled in the art, for example, it can be prepared by automatic DNA synthesis and/or recombinant DNA technology, etc., or it can be isolated from a suitable natural source. In a specific embodiment of the present invention, the nucleic acid sequence of the isolated polynucleotide may include the sequence shown in SEQ ID NO:4.

Expression vector

The present invention also provides an expression vector containing the isolated polynucleotide of the present invention. The method for constructing the expression vector should be known to those skilled in the art. For example, the expression vector can be constructed by methods such as in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. More specifically, it can be It is constructed by inserting the isolated polynucleotide into the multiple cloning site of the expression vector. The expression vector in the present invention generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors. The vector may also include one or more regulatory sequences operably linked to the polynucleotide sequence, which may include a suitable promoter sequence. The promoter sequence is usually operably linked to the coding sequence for the amino acid sequence to be expressed. The promoter can be any nucleotide sequence that shows transcriptional activity in the host cell of choice, including mutated, truncated, and hybrid promoters, and can be derived from an extracellular sequence that encodes either homologous or heterologous to the host cell. Or intracellular polypeptide gene acquisition. The regulatory sequences may also include suitable transcription terminator sequences, sequences recognized by a host cell to terminate transcription. A terminator sequence is attached to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the host cell of choice may be used in the present invention.

In general, suitable vectors will contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites and one or more selectable markers. For example, these promoters may be lac or trp promoters including but not limited to E. coli; bacteriophage lambda PL promoter; eukaryotic promoters including CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters , the methanol oxidase promoter of Pichia pastoris and some other known promoters that can control the expression of genes in prokaryotic or eukaryotic cells or their viruses. Marker genes may be used to provide phenotypic traits for selection of transformed host cells, for example, may include but are not limited to dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), Or tetracycline or ampicillin resistance for E. coli etc. When the polynucleotide is expressed, an enhancer sequence can also be included in the expression vector. If the enhancer sequence is inserted into the vector, the transcription will be enhanced. The enhancer is a cis-acting factor of DNA, usually about There are 10 to 300 base pairs and act on the promoter to enhance the transcription of the gene.

expression system

The present invention also provides an expression system of an anti-CLD18A2/HSA/CD3 trispecific fusion protein, the expression system contains the expression vector of the present invention or the exogenous polynucleotide of the present invention is integrated in the genome. Any cell suitable for expressing the expression vector can be used as a host cell, for example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher Eukaryotic cells, such as mammalian cells, specifically include but are not limited to Escherichia coli; Streptomyces sp; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9 ; A combination of one or more of CHO, COS, HEK293 cells, or animal cells such as Bowes melanoma cells. The method for constructing the expression system should be known to those skilled in the art, for example, it may include but not limited to microinjection method, particle gun method, electroporation method, virus-mediated transformation method, electron bombardment method , calcium phosphate precipitation method, etc. in one or more combinations. As a preferred mode of the present invention, the expression system is an animal cell expression system, such as CHO, NSO, BHK, HEK-293 or PER-C6. In a more specific embodiment, said cell is CHO.

Preparation method of fusion protein

The invention also provides a preparation method of the anti-CLD18A2/HSA/CD3 trispecific fusion protein. Those skilled in the art can choose an appropriate method to prepare the fusion protein. For example, the preparation method may include: cultivating the expression system of the fusion protein of the present invention under conditions suitable for expressing the fusion protein, so as to express The fusion protein is obtained, and the fusion protein is purified and isolated.

Pharmaceutical composition/kit

The present invention also provides a pharmaceutical composition, including the anti-CLD18A2/HSA/CD3 trispecific fusion protein of the present invention, the expression system of the present invention or its culture.

Various pharmaceutically acceptable carriers in the art may also be included in the pharmaceutical composition. The pharmaceutically acceptable carrier may include various excipients and diluents, these carriers themselves are not essential active ingredients, and there is no excessive toxicity after administration. Suitable carriers will be well known to those skilled in the art, for example, a full discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J., 1991).

In the pharmaceutical composition, the content of the fusion protein is usually an effective amount, and the content of the active ingredient corresponding to the effective amount can be determined according to the object to be treated and the specific administration method.

The present invention also provides a kit containing the fusion protein, the expression system or its expression product (culture) or the pharmaceutical composition. Instructions for use can also be included in the kit to guide the application of those skilled in the art or clinicians.

use

The present invention also provides the use of the anti-CLD18A2/HSA/CD3 trispecific fusion protein, the expression system or its culture in the preparation of medicines for alleviating or treating diseases related to CLD18A2 positive cells.

As used herein, the "CLD18A2-positive cell (related) disease" includes "CLD18A2 abnormal expression (related) disease" or "CLD18A2 high expression (related) disease", and these terms may be Used interchangeably.

CLD18A2 is expressed in many tumor cells. In view of this characteristic of CLD18A2, CLD18A2 can be used as a therapeutic target for related tumors. In the present invention, the CLD18A2-positive cells may include all CLD18A2-expressing tumors, and these tumors may usually express CLD18A2-positively. The positive expression of CLD18A2 usually refers to the expression of CLD18A2, or the expression level of CLD18A2 is higher than a certain standard. The expression of CLD18A2 protein can be detected. For another example, CLD18A2 positive can be that the mRNA expression level of CLD18A2 in the tumor tissue is higher than that of its surrounding healthy tissue. For another example, CLD18A2 positive can be that the expression level of CLD18A2 protein in the tumor tissue is higher than its surrounding healthy tissue. These diseases may specifically include but are not limited to gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, breast cancer, colorectal cancer, liver cancer, gallbladder cancer, head and neck cancer, etc. These cancers may be early, middle or late, such as metastasis cancer etc.

treatment method

The present invention also provides a treatment method comprising: administering to an individual an effective amount of the anti-CLD18A2/HSA/CD3 trispecific fusion protein, the expression system or its culture, or the pharmaceutical composition.

The "therapeutically effective amount" of the fusion protein and pharmaceutical composition provided by the present invention preferably leads to a reduction in the severity of disease symptoms, an increase in the frequency and duration of the asymptomatic period of the disease, or prevention of damage or disability caused by the suffering of the disease . For example, for the treatment of CLD18A2-related tumors (including, for example, gastric cancer), a "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least About 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%. The ability to inhibit tumor growth can be evaluated in animal model systems predictive of efficacy against human tumors. Alternatively, it can also be assessed by examining the ability to inhibit cell growth, which can be determined in vitro by assays well known to those skilled in the art. A therapeutically effective amount of a fusion protein, pharmaceutical composition, generally reduces tumor size, or otherwise alleviates symptoms in a subject. Those skilled in the art can select an appropriate therapeutically effective amount according to the actual situation, for example, the size of the subject, the severity of the subject's symptoms and the selected specific composition or administration route. Prescriptions for treatment (e.g., dosage decisions, etc.) can be determined by a physician, generally taking into account factors including, but not limited to, the disease being treated, individual patient conditions, site of delivery, method of administration, and other factors. Prophylactically effective dose refers to the amount that is effective in achieving the desired preventive effect at the necessary dose and time. quantity.

The fusion protein and pharmaceutical composition of the present invention can be administered as a single active ingredient, or in combination therapy, that is, in combination with other agents. For example, the combination therapy may be the fusion protein or the pharmaceutical composition combined with at least one other anti-tumor drug (for example, paclitaxel, etc.). For another example, the combination therapy may be the combination of the fusion protein, the pharmaceutical composition and an immune checkpoint inhibitor, and the immune checkpoint inhibitor includes but is not limited to a PD-1 inhibitor, a PD-L1 inhibitor, or A combination of one or more of CTLA-4 inhibitors, etc., said inhibitors may be monoclonal antibodies.

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the examples, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the examples of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field conventional technology. These techniques have been fully described in the existing literature. For details, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons , New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Ch romatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999 et al.

Embodiment 1, the fusion expression of trispecific fusion protein

1. Design of trispecific fusion protein

DR303X8 sequence (SEQ ID NO: 1):

In the above sequence, the "A" at the 14th and 143rd positions is the P/A mutation site introduced by the inventors. Positions 1-120 are anti-CLDN18.2 VHH, positions 130-244 are anti-HSA VHH, positions 254-502 are anti-CD3 scFv.

Amino acids underlined in italics are linker sequences.

Amino acid fragments marked in bold underline are CDRs. Among them, for the anti-CLDN18.2VHH, the 26th to 33rd positions are CDR1, the 51st to 57th positions are CDR2, and the 96th to 109th positions are CDR3. For anti-HSA VHH, positions 155-162 are CDR1, positions 178-187 are CDR2, and positions 226-233 are CDR3. For anti-CD3 scFv, the 279th-286th position is HCDR1, the 304th-313rd position is HCDR2, the 352th-367th position is HCDR3; the 419th-428th position is LCDR1, the 445th-447th position is LCDR2, and the 484th-492nd position is LCDR3. The CDR region was marked using the IMGT numbering scheme (antibody numbering scheme).

DR303X6 sequence (SEQ ID NO:2):

In the above sequence, positions 1-120 are anti-CLDN18.2 VHH; positions 130-378 are anti-CD3 scFv (excluding anti-HSA VHH).

Amino acid fragments marked in bold underline are CDRs. Among them, for the anti-CLDN18.2VHH, the 26th to 33rd positions are CDR1, the 51st to 57th positions are CDR2, and the 96th to 109th positions are CDR3. For anti-CD3 scFv, 155th to 162th is HCDR1, 180th to 189th is HCDR2, 228th to 243rd is HCDR3; Bits 295 to 303 are LCDR1, bits 321 to 323 are LCDR2, and bits 360 to 368 are LCDR3. The CDR region was marked using the IMGT numbering scheme (antibody numbering scheme).

DR303X2 sequence (SEQ ID NO: 3)

In the above sequence, positions 1-120 are anti-CLDN18.2 VHH, positions 130-244 are anti-HSA VHH, positions 254-502 are anti-CD3 scFv.

Amino acids underlined in italics are linker sequences.

2. Recombinant expression

The anti-CLD18A2 domain, anti-CD3 domain and anti-human serum albumin (HSA) domain were combined in tandem through connecting peptides to form an anti-CLD18A2/HSA/CD3 trispecific fusion protein (Table 1), and entrusted to General Biosystems ( Anhui) Co., Ltd. optimized the synthesis of the base sequence. After adding a signal peptide (METDTLLLWVLLLWVPGSTG) at the 5' end, it was constructed into an animal cell expression vector for secreted expression. Use the endotoxin-free plasmid extraction kit (Biomiga) to extract the recombinant expression plasmid, measure the plasmid and transfection reagent at a mass ratio of 1:3, and dilute them with medium respectively, mix the dilutions and let stand at room temperature for 30 minutes After that, it was added to CHO cells, cultured in a shaker incubator at 37° C. with 5% CO ₂ for 7 days, and centrifuged to obtain cell expression supernatant for purification.

Table 1

X910: According to the sequence of SEQ ID NO: 132 of US_2020_0055932_A1, it was expressed and prepared in CHO cells.

3. Purification and preparation

The cell expression supernatant was centrifuged at high speed, then filtered through a 1.2 μm membrane, and loaded onto an AT Protein A Diamond affinity chromatography column (Borgeron (Shanghai) Biotechnology Co., Ltd.). Equilibrium solution is 20mmol/L Tris-HCl, 150mmol/L NaCl, pH7.4 solution; eluent is 20mmol/L HAc-NaAc pH5.0 solution; eluent is 100mmol/L Gly-HCl, pH3.0 solution ; The cleaning solution is 0.1mmol/L NaOH solution; the neutralizing solution is 1mol/L Tris-HCl, pH8.5. Equilibrate the chromatographic column with the balance liquid, load the sample with a retention time of 5 minutes according to the load of no more than 20 mg target protein/ml filler, equilibrate the chromatographic column with the balance liquid after loading the sample, and then rinse with the eluent for 3-5 times the volume of the column bed, and finally elute the target peak with the eluent, adjust the pH of the target peak to 7.0 with neutralizing solution, centrifuge to remove the precipitate, and take the supernatant. Adjust the conductance of the centrifuged supernatant to less than 5mS/cm, pass through a 1.2μm filter membrane, and then load the sample onto the Q Bestarose FF anion exchange chromatography column (Borgeron (Shanghai) Biotechnology Co., Ltd.), the flow-through mode removes impurities, and balances The solution is 20mmol/L PB, pH7.0 solution. The anion-exchange flow-through sample was loaded onto a Diamond SP cation-exchange chromatography column (Borgeron (Shanghai) Biotechnology Co., Ltd.), 0-100% B linear gradient elution (0-500mmol/L NaCl, 20mmol/L PB , pH7.0 solution, 20CV), obtain SDS-PAGE electrophoresis purity≥95% sample.

The expression results showed that even with IgG Fc with improved expression and stability, the expression of X910 was lower than that of DR303X8, which were 0.67g/L and 2.27g/L, respectively.

Embodiment 2, in vitro activity detection

Construction of CHO-C18.2-gfpx stably transfected cell line (target cell highly expressing CLDN18A2): CLD18A2 full-length gene (sequence shown in SEQ ID NO: 7) and egfp gene (sequence shown in SEQ ID NO: 8 Shown) double expression cassette plasmid pDR05-C18.2-gfp, the plasmid was extracted with an endotoxin-free plasmid kit, electroporated into CHO-S cells after sterile filtration, and cultured in 96-well plates with MSX under pressure. Pick the stably transfected cells growing in separate clusters on the 96-well plate, and gradually amplify them under the culture condition of adding MSX, and use the prepared positive control antibody zolbetuximab analog (the heavy chain and sequence number of sequence number 118 in the patent US9751934B2 No. 125 light chain) for immunofluorescence detection and Anti-Claudin18 antibody [34H14L15] (Abcam) for spotting The CLD18A2 positive expression cell line CHO-C18.2-gfpx. stably transfected cell line was obtained by Dot blotting detection.

Construction of Jurkat-NFAT-Luc2p (effector cell) stably transfected cell line: Jurkat cells were transformed into the NFAT-responsive luciferase system to obtain the TDCC activity detection effector cell line Jurkat-NFAT-Luc2p.

In vitro activity detection adopts T cell activation fluorescence signaling pathway method. Take an appropriate amount of CHO-C18.2-gfpx and Jurkat-NFAT-Luc2p cells, centrifuge at 1000rpm for 5/10min, discard the supernatant, add RPMI1640 medium (containing 1% FBS) to resuspend, count and dilute to the specified concentration, press a certain The ratio (E:T=1:4, T=15000) of the two kinds of cells was mixed and added to a 96-well cell culture plate (50 μl/well) for later use. The protein was diluted to the specified concentration with RPMI1640 medium (containing 1% FBS), and then an appropriate volume (25 μl/well) was added to a 96-well plate, and cultured overnight for 18 hours. Add 10 μl/well of detection solution (luciferase detection kit, Promega, E2620), shake for 1.5 min, transfer 60 μl of the solution to a white plate, and detect in a microplate reader (Molecular Devices I3).

The results showed that the EC ₅₀ values of DR303X6, DR303X8 and X910 were about 69, 61 and 75pM, respectively. The presence of anti-HSA VHH did not affect the in vitro activity of the fusion protein. In another independent activity assay, the EC ₅₀ value of DR303X2 was about 75pM.

Embodiment 3, serum stability

The reporter gene method in Example 2 was used to compare the stability of DR303X2 and DR303X8 incubated in human serum for 7 days at 37°C.

Take an appropriate amount of CHO-C18.2-gfpx and Jurkat-NFAT-Luc2p-10F2 cells, centrifuge at 1000rpm for 5/10min, discard the supernatant, add 1640 medium (containing 1% FBS) to resuspend, count, and use the analysis medium to adjust Effector cell Jurkat-NFAT-luc2p-10F2 cell density to 2.4×10 ⁶ cells/ml, adjust target cell CHO-C18.2-gfpx cell density to 6×10 ⁵ cells/ml, mix 1:1 and spread in 96-well plate , 50 μl per well, spare. Dilute the protein with 1640 medium (containing 1% FBS) to the specified concentration (initial concentration is 2μg/ml, prepared concentration × 2, 5-fold gradient dilution), then take 50μl and add it to a 96-well plate, and incubate for 18 hours . Add 20 μl/well of detection solution (luciferase detection kit, Promega, E2620), shake for 1 min 30 s, and detect the photometric value in a microplate reader. The TDCC activity of the two samples was detected in parallel.

The TDCC activity of DR303X2 and DR303X8 samples in human serum showed a downward trend with increasing storage time at 37°C.

Among them, the EC ₅₀ of the DR303X2 sample in human serum (HS) was 0.04012 on day 0, and the EC ₅₀ on day 7 was 0.07349, and the residual activity rate was 55%.

The EC ₅₀ of the DR303X8 sample in human serum (HS) was 0.01426 at day 0, and the EC ₅₀ at day 7 was 0.02265, and the residual activity rate was 63%.

Activity residual rate=EC _{50 (0 day)} /EC _{50 (7 day)} *100%.

Therefore, DR303X8 has better stability in human serum than DR303X2.

Embodiment 4, half-life in vivo of C57BL/6 mice

C57BL/6 mice were given 1 mg/kg DR303X2 and 1 mg/kg DR303X8 by tail vein injection, and blood samples were collected 3 days before the drug, 1 h, 6 h, 24 h, 30 h, 48 h, 72 h, 96 h, and 120 h after the drug to analyze the drug concentration. As shown in Figure 1, the pharmacokinetic parameters were calculated using the non-compartmental model using PK solver 2.0 software. When the dose of DR303X2 and DR303X8 mice was administered through the tail vein of 1mg/kg, the half-lives were 26.8h and 28.8h respectively.

Embodiment 5, half-life in cynomolgus monkeys

Cynomolgus monkeys were given 0.03mg/kg, 0.6mg/kg, 3mg/kg DR303X8 or DR303X6 intravenously in a single dose, and the blood was sampled and analyzed before administration, immediately after administration, 4h, 8h, 24h, 48h, 72h, 120h and 168h Drug concentrations were calculated, and pharmacokinetic parameters were calculated using non-compartmental models.

DR303X8 or DR303X6 was intravenously injected into cynomolgus monkeys: when the dose was 0.03mg/kg, the C _max was 1.02±0.02μg/ml, the AUC _0-t was 33.12±0.12μg/ml*h, and the t _1/2 was 46.03 ±16.32h; when the dose was 0.6mg/kg, C _max was 17.33±0.65μg/ml, AUC _0-t was 641.74±102.66μg/ml*h, t _1/2 was 37.37±1.11h; At 3mg/kg, C _max was 81.01±2.44μg/ml, AUC _0-t was 3846.04±497.80μg/ml*h, t _1/2 was 41.8±5.76h.

Intravenous injection of DR303X8, when the administration concentration (3mg/kg, 0.6mg/kg, 0.03mg/kg) ratio of 100:20:1 in the high, middle and low dose groups, the C _max ratio was 80:17:1, and the AUC _0-t The ratio was 116:20:1, and the systemic exposure was linearly related to the administered dose. There was no significant difference in the half-life, retention time and clearance rate of the high, middle and low dose groups, as shown in Figure 2.

Since the blood concentration of DR303X6 has been lower than the lower limit of detection after 24 hours, it is not shown in Figure 2.

According to the above, DR303X8 has a long half-life in vivo and ideal stability in vivo.

Example 6. Anti-tumor efficacy of anti-CLD18A2/HSA/CD3 trispecific fusion protein in PBMC immune system humanized BxPC3/hCLDN18.2 pancreatic cancer subcutaneous xenograft tumor model

To establish a PBMC immune system humanized BxPC3/hCLDN18.2 pancreatic cancer subcutaneous xenograft model, select male NCG mice, about 7 weeks old. BxPC3/hCLDN18.2 tumor cells were inoculated subcutaneously in the right flank of mice, 5x10 ⁶ /mouse, PBMCs were derived from normal human peripheral blood, and injected into tumor-bearing mice by tail vein, 2x10 ⁶ /mouse. When the average tumor volume reached about ^60mm3 , the drugs were divided into groups, divided into 5 groups, 6 animals in each group, and blank preparation (vehicle, 2 times a week, continuous administration for 5 weeks), 0.03mg/kg DR303X8 (twice a week, continuous administration for 5 weeks), 0.03mg/kg DR303X8 (once a day, continuous administration for 17 days), 0.1mg/kg DR303X8 (twice a week, continuous administration for 5 weeks), 0.1mg /kg DR303X8 (once a day, continuous administration of 17 Days), drug withdrawal and observation. The tumor volume was measured twice a week: the maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V=L×W ² /2, when the tumor in the blank group grew to 2000 mm ³ Or the animal state is not enough to support the test to end the test.

The experimental results are shown in Figure 3, DR303X8 in the low-concentration administration group and high-concentration administration group had significant tumor inhibitory effects on the PBMC immune system humanized BxPC3/hCLDN18.2 pancreatic cancer subcutaneous xenograft tumor model.

Example 7. Anti-tumor efficacy of anti-CLD18A2/HSA/CD3 trispecific fusion protein in PBMC immune system humanized NUGC4/hCLDN18.2 gastric cancer subcutaneous xenograft tumor model

The PBMC immune system humanized NUGC4/hCLDN18.2 gastric cancer subcutaneous xenograft model was established, and male NCG mice, about 7 weeks old, were selected. NUGC4/hCLDN18.2 tumor cells were inoculated subcutaneously in the right flank of mice, 5x10 ⁶ /mouse, PBMCs were derived from normal human peripheral blood, and injected into tumor-bearing mice by tail vein, 2x10 ⁶ /mouse. When the average tumor volume reached about ^60mm3 , the drugs were divided into groups, divided into 5 groups, 6 animals in each group, and blank preparation (vehicle, 2 times a week, continuous administration for 5 weeks), 0.03mg/kg DR303X8 (twice a week, continuous administration for 5 weeks), 0.03mg/kg DR303X8 (once a day, continuous administration for 17 days), 0.1mg/kg DR303X8 (twice a week, continuous administration for 5 weeks), 0.1mg /kg DR303X8 (once a day, continuous administration for 17 days). The tumor volume was measured twice a week: the maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V=L×W ² /2, and the average tumor volume of the control group reached 2000mm ³ The test was terminated when the time or the state of the animal was not enough to support the test.

The experimental results are shown in Figure 4, DR303X8 in the low-concentration administration group and high-concentration administration group had significant tumor inhibitory effects on the PBMC immune system humanized NUGC4/hCLDN18.2 gastric cancer subcutaneous xenograft tumor model.

Example 8. Antitumor efficacy of fusion proteins DR303X8 and X910 in PBMC immune system humanized NUGC4/hCLDN18.2 gastric cancer subcutaneous xenograft tumor model

The PBMC immune system humanized NUGC4/hCLDN18.2 gastric cancer subcutaneous xenograft model was selected, and male NCG mice were selected, about 7 weeks old. NUGC4/hCLDN18.2 tumor cells were inoculated subcutaneously in the right flank of mice, 5x10 ⁶ /mouse; PBMCs were derived from normal human peripheral blood, and injected into tumor-bearing mice by tail vein, 2x10 ⁶ /mouse. When the average tumor volume reached about ^60mm3 , the drugs were divided into groups, divided into 5 groups, with 6 animals in each group, and the blank preparation (vehicle, once a day, continuously administered for 29 days), 0.01 mg/kg DR303X8 ( Once a day, continuous administration for 29 days), 0.03mg/kg DR303X8 (twice a week, continuous administration for 5 times), 0.03mg/kg DR303X8 (once a day, continuous administration for 17 days), 0.06mg/kg X910 (once a day, continuous administration for 32 days). The tumor volume was measured twice a week: the maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V=L×W ² /2, and the average tumor volume of the control group reached 2000mm ³ The test was terminated when the time or the state of the animal was not enough to support the test.

The experimental results are shown in Figure 5, DR303X8 has a significant tumor inhibitory effect on the PBMC immune system humanized NUGC4/hCLDN18.2 gastric cancer subcutaneous xenograft tumor model.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims. Also, all documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference.

Claims

A fusion protein comprising: an anti-CLD18A2 domain, an anti-human serum albumin domain and an anti-CD3 domain;

The anti-CLD18A2 structural domain is a single domain antibody with CDR1 shown in positions 26-33 of SEQ ID NO:1, CDR2 shown in positions 51-57 of SEQ ID NO:1, and CDR2 shown in positions 51-57 of SEQ ID NO:1. CDR3 indicated at positions 96-109;

The anti-human serum albumin domain is a single-domain antibody, having CDR1 shown in positions 155-162 in SEQ ID NO: 1, CDR2 shown in positions 180-187 in SEQ ID NO: 1, and SEQ ID NO : CDR3 shown in positions 226-233 in 1.
The fusion protein according to claim 1, wherein the anti-CLD18A2 domain is a single domain antibody, and its framework region has a P→A mutation in its amino acid sequence; preferably, its FR1 region has a P→A mutation; More preferably, its P→A mutation corresponds to the 14th position of SEQ ID NO:1 sequence; and/or

The anti-human serum albumin domain is a single-domain antibody, and its framework region has a P→A mutation in its amino acid sequence; preferably, its FR1 region has a P→A mutation; more preferably, its P→A mutated amino acid It is the 143rd amino acid corresponding to the sequence of SEQ ID NO:1.
The fusion protein according to claim 1, wherein the anti-CD3 domain is a single-chain antibody; preferably, it has HCDR1 shown in positions 279-286 in SEQ ID NO:1, SEQ ID NO HCDR2 shown in No. 304-313 positions in :1, HCDR3 shown in No. 352-367 positions in SEQ ID NO: 1, LCDR1 shown in No. 419-427 positions in SEQ ID NO: 1, SEQ ID NO: 1 and/or

The anti-CD3 structural domain is located at the N-terminal or C-terminal of the anti-human serum albumin structural domain.
The fusion protein according to any one of claims 1 to 3, wherein the amino acid sequence of the anti-CLD18A2 domain is shown in positions 1 to 120 in SEQ ID NO:1;

The amino acid sequence of the anti-human serum albumin domain is shown in the 130th to 244th positions in SEQ ID NO: 1; and/or

The amino acid sequence of the anti-CD3 domain is shown in the 254th to 502nd positions in SEQ ID NO:1.
The fusion protein according to claim 1, wherein the amino acid sequences of the anti-CLD18A2 domain, the anti-human serum albumin domain and the anti-CD3 domain are directly connected, or connected by a linker, and the linker The same or different; preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO:1.
A polynucleotide or an expression construct containing the polynucleotide, characterized in that the polynucleotide encodes the fusion protein according to any one of claims 1-5.
A fusion protein expression system, the expression system contains the expression construct of claim 6 or the exogenous polynucleotide of claim 6 integrated in its genome.
The method for preparing the fusion protein according to any one of claims 1 to 5, characterized in that the method comprises: expressing the fusion protein using the expression system according to claim 7; preferably, the method further comprises : the fusion protein is isolated.
A method for modifying a CLD18A2 binding molecule to improve its curative effect, characterized in that the method comprises: using an anti-CLD18A2 domain as a CLD18A2 binding molecule, linking it to an anti-human serum albumin domain and an anti-CD3 domain;

The anti-CLD18A2 structural domain is a single domain antibody with CDR1 shown in positions 26-33 of SEQ ID NO:1, CDR2 shown in positions 51-57 of SEQ ID NO:1, and CDR2 shown in positions 51-57 of SEQ ID NO:1. CDR3 indicated at positions 96-109;

The anti-human serum albumin domain is a single-domain antibody, having CDR1 shown in positions 155-162 in SEQ ID NO: 1, CDR2 shown in positions 180-187 in SEQ ID NO: 1, and SEQ ID NO : CDR3 shown in positions 226-233 in 1;

The anti-CD3 structural domain is located at the N-terminal or C-terminal of the anti-human serum albumin structural domain.
The method according to claim 9, wherein the anti-CLD18A2 domain is a single domain antibody, and the framework region thereof is subjected to a P→A mutation of the amino acid sequence; preferably, a P→A mutation is performed on the FR1 region ; More preferably, its P → A mutation corresponds to the 14th position of SEQ ID NO: 1 sequence; and/or

The anti-human serum albumin domain is a single-domain antibody, and its framework region is subjected to a P→A mutation in its amino acid sequence; preferably, its FR1 region is subjected to a P→A mutation; more preferably, its P→A mutation The amino acid is the 143rd amino acid corresponding to the sequence of SEQ ID NO:1; and/or

The anti-CD3 structural domain is a single-chain antibody; preferably, it has HCDR1 shown in positions 279-286 in SEQ ID NO: 1, HCDR2 shown in positions 304-313 in SEQ ID NO: 1, SEQ ID NO: 1 HCDR3 shown in No. 352-367 in ID NO:1, LCDR1 shown in No. 419-427 in SEQ ID NO:1, LCDR2 shown in No. 445-447 in SEQ ID NO:1, SEQ ID NO : LCDR3 shown in bits 484-492 in 1.
The fusion protein according to any one of claims 1 to 5, the expression system according to claim 7 or its culture The use of nutrients in the preparation of drugs for alleviating or treating CLD18A2 positive cell-related diseases; preferably, the CLD18A2 positive cell-related diseases include tumors; preferably, the tumors include: pancreatic cancer, gastric cancer, esophageal cancer, lung cancer , ovarian, breast, colorectal, liver, gallbladder, or head and neck cancer.
A kind of pharmaceutical composition, is characterized in that, described pharmaceutical composition comprises:

The fusion protein of any one of claims 1-5; or

The expression system of the fusion protein according to claim 7, or its culture.
A kind of medicine box, it is characterized in that, described medicine box comprises: container or packing, and be positioned at wherein:

The fusion protein according to any one of claims 1-5; or

The expression system of the fusion protein according to claim 7, or its culture; or

The pharmaceutical composition of claim 12.