阅读说明：本技术 一种用于治疗晚期胃癌的抗cldn18全人源化抗体 (anti-CLDN 18 fully humanized antibody for treating advanced gastric cancer ) 是由 许铮 李响 刘影 熊国裕 李峰 史继峰 上官丽娟 于 2020-07-13 设计创作，主要内容包括：本发明涉及一种用于治疗晚期胃癌的抗Claudin18.2全人源单克隆抗体,相对于Ganymed现有Claudin18.2抗体IMAB362,人源化程度更高,具有相对更低的免疫原性,选择性结合CLDN18.2,相对具有更高亲和力。(The invention relates to an anti-Claudin 18.2 fully human monoclonal antibody for treating advanced gastric cancer, which has higher humanization degree, relatively lower immunogenicity, selective binding to CLDN18.2 and relatively higher affinity compared with the existing Claudin18.2 antibody IMAB362 of Ganyed.)

1. An anti-Claudin 18.2 fully humanized antibody for the treatment of advanced gastric cancer, wherein the antibody has heavy chain variable region (VH) and light chain variable region (VL) sequences as described below,

the amino acid sequence is SEQ ID NO: 1. VH shown in 3 or 5, and

the amino acid sequence is SEQ ID NO: 2.4 or 6.

2. The anti-claudin18.2 fully humanized antibody of claim 1, wherein said antibody has an amino acid sequence of SEQ ID NO: 3 and the amino acid sequence is SEQ ID NO: 4, VL shown in fig. 4.

3. The anti-claudin18.2 fully humanized antibody according to claim 1 or 2, wherein the constant region of the heavy chain is selected from the human IgG series, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG 1; the constant region of the light chain is selected from a kappa or lambda chain.

4. An isolated nucleic acid molecule encoding the anti-claudin18.2 fully humanized antibody of any of claims 1-3.

5. An expression vector comprising the coding gene of claim 2.

6. An engineered bacterium comprising the expression vector of claim 5.

7. A pharmaceutical composition comprising a humanized antibody according to any of claims 1-3, and a pharmaceutically acceptable excipient.

8. Use of a humanized antibody according to any of claims 1 to 3 for the manufacture of a medicament for the treatment and/or prevention of cancer; wherein the cancer may be, for example, gastrointestinal cancer, pancreatic cancer, esophageal cancer, or non-small cell lung cancer; the gastrointestinal cancer is preferably advanced gastric cancer.

9. An article of manufacture or kit comprising a container comprising the humanized antibody of any one of claims 1-3, or the pharmaceutical composition of claim 7, and a package insert carrying instructions for use of the medicament.

Technical Field

The invention relates to an anti-CLDN 18.2(Claudin18.2) fully humanized antibody for treating advanced gastric cancer, belonging to the technical field of biology.

Background

Stomach cancer is one of the most prevalent cancers worldwide and is the second most common tumor in china. According to the disclosure of the 12 th international stomach cancer conference called Beijing in 2017 in 4 months, the number of newly discovered cases of Chinese stomach cancer is about 68 ten thousand every year, which accounts for about half of the total number of global diseases, and compared with 44.65 ten thousand cases published in 2012, the annual average growth rate exceeds 13%. The death rate of stomach cancer in China is 4-8 times that of developed countries in Europe and America, and about 1 Chinese can die of stomach cancer every 2-3 minutes. Compared with other countries, the situation of the stomach cancer in China is more severe.

When most of the gastric cancer patients in China are diagnosed, the condition of the stomach cancer patients already enters the middle and late stage, the postoperative effect is extremely unsatisfactory, the prognosis is extremely poor, and the stomach cancer patients are extremely intractable malignant tumors clinically.

Intercellular Tight Junctions (TJs) are a transmembrane protein complex, the stability of which requires the coordinated activities of several different proteins to be maintained, while Claudin protein is the major protein that ensures the specificity of tight junction permeability. To date 27 Claudin family members have been found in mammals. The molecular weight of the Claudin protein family is 20-27 KD, the structure comprises 4 transmembrane regions, two extracellular loops and one intracellular loop, and the N end and the C end of the Claudin protein family are in cytoplasm. The two extracellular loops make it an ideal antibody target. Claudin protein is a skeleton protein forming a tight connection structure, is positioned on the top side of adjacent cell gaps, has tissue and organ specificity in distribution, and has the functions of mainly intercellular adhesion, cell polarity maintenance, cell bypass permeability regulation, and participation in cell proliferation and differentiation regulation.

The molecular weight of the Claudin18 protein is about 26KD, and the Claudin protein can be changed into Claudin subtypes with different properties by selective shearing: claudin18.1 and Claudin18.2. While there are only eight amino acid differences between the first extracellular domains of claudin18.1 and claudin18.2, the expression profiles are different, with claudin18.1 being selectively expressed in normal lung and stomach epithelium, claudin18.2 being expressed only on transiently differentiated gastric epithelial cells, and completely undetectable in any other normal human organ, while claudin18.2 is significantly upregulated in a number of malignancies, including 80% gastrointestinal adenomas, 60% pancreatic tumors, 30% esophageal cancer, and 25% non-small cell lung cancer. In tumors, the tight junctions between cells are disrupted and Claudin18.2 fails to perform its normal function. Thus, claudin18.2 is a suitable target for tumor therapy.

The Claudin18 gene has 2 different first exons, so two subunits, Claudin18.1 and Claudin18.2, can be produced. The two molecules differ in structure by 69 amino acids at the N-terminus, which is located within the loop structure of the first extracellular region. The two subtypes of Claudin18 were transcriptionally amplified in different tissues, respectively, wherein Claudin18.1 was mainly expressed in lung tissue and Claudin18.2 was specifically expressed in stomach tissue.

In 2006, Sanada et al found that the expression of Claudin18 gene was down-regulated in 57% of gastric cancers, and through immunohistochemical analysis, Claudin18 was expressed on the cell membrane of normal gastric mucosa and duodenal Pan cells, but in some intestinal and 90% microadenomas, Claudin18 expression was reduced, and at the same time, the reduction in intestinal type gastric cancer was more common than other types of gastric cancer, presumably involved in the development of early stage gastric cancer. Survival analysis showed that the reduction of Claudin18 expression in gastric cancer was associated with poor prognosis in advanced patients, and it was considered that the reduction of Claudin18 expression was a factor in poor prognosis in gastric cancer patients.

In 2008, the study of Sahin et al proves that the expression of claudin18.2 exists in 77% of primary gastric adenocarcinoma tissues, and most importantly, the expression level of 56% of the primary gastric adenocarcinoma tissues reaches more than 60%. The expression of Claudin18.2 protein in intestinal gastric cancer is Xiaotiao, and the expression of Claudin18.2 protein in diffuse gastric cancer is higher. However, tissues such as pancreas, esophagus and ovary have no Claudin18.2 protein expression under normal state, and the corresponding tumor tissues can express Claudin18.2 protein in a large amount. Therefore, the Claudin18.2 protein can be used as a target for clinical tumor diagnosis and treatment.

Claudin18 splice variant 2, claudin18.2 (CLDN18.2), is a member of the claudin family of tight junction proteins. A transmembrane protein having CLDN18.2 of 27.8kDa comprising 4 transmembrane domains with two small extracellular loops.

In normal tissues (except stomach), expression of CLDN18.2 was not detectable by RT-PCR. Immunohistochemistry for CLDN 18.2-specific antibodies showed the stomach to be the only positive tissue.

CLDN18.2 is a highly selective gastric lineage antigen expressed only on transiently differentiated gastric epithelial cells. CLDN18.2 remains in the process of malignant transformation and is therefore frequently displayed on the surface of human gastric cancer cells, and in addition, in esophageal, pancreatic and lung adenocarcinoma, the pan-tumor antigen is ectopically activated at significant levels. The CLDN18.2 protein is also localized in lymph node metastases of gastric adenocarcinoma and in particular distant metastases in the ovary (krukenberg tumors).

A chimeric antibody IMAB362 of IgG1 directed against CLDN18.2 has been developed by Ganymed Pharmaceuticals AG. IMAB362 recognizes the first extracellular domain of CLDN18.2 (ECD1) with high affinity and specificity. IMAB362 does not bind to any other claudin family members, including the closely related claudin18 splice variant 1(CLDN 18.1). IMAB362 exhibits precise tumor cell specificity and combines four independent highly effective mechanisms of action. Upon target binding, IMAB362 mediates cell killing by ADCC, CDC, induction of apoptosis induced by target cross-linking on the surface of tumor cells, and direct constant proliferation. Thus, IMAB362 efficiently lyses CLDN18.2 positive cells, including human gastric cancer cell lines in vitro and in vivo. Mice with CLDN18.2 positive cancer cell lines had survival benefits, and up to 40% of mice exhibited regression of their tumors when treated with IMAB 362.

Disclosure of Invention

The present invention aims to provide an anti-CLDN 18.2 fully humanized antibody having a relatively higher affinity for CLDN18.2 while ensuring a higher degree of humanization.

The anti-CLDN 18.2 fully humanized antibody of the invention has heavy chain variable region (VH) and light chain variable region (VL) sequences as described below,

the amino acid sequence is SEQ ID NO: 1. VH shown in 3 or 5, and

the amino acid sequence is SEQ ID NO: 2.4 or 6.

Specifically, the antibodies VH and VL of the invention are: the amino acid sequence is SEQ ID NO:1, and the amino acid sequence is SEQ ID NO: 2 VL (KW-1); or, the amino acid sequence is SEQ ID NO: 3, and the amino acid sequence is SEQ ID NO: 4 VL (KW-2); or, the amino acid sequence is SEQ ID NO: 5, and the amino acid sequence is SEQ ID NO: VL (KW-3) shown as 6.

In a preferred embodiment, the antibody has an amino acid sequence of SEQ ID NO: 3 and the amino acid sequence is SEQ ID NO: 4, VL shown in fig. 4.

The anti-CLDN 18.2 fully humanized antibody of the present invention, wherein the constant region of the heavy chain is selected from the human IgG series, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG 1; the constant region of the light chain is selected from a kappa or lambda chain.

The invention also relates to an isolated nucleic acid molecule encoding an anti-CLDN 18.2 fully humanized antibody of the invention; and an expression vector comprising the nucleic acid molecule of the invention, and a host cell, preferably a eukaryotic cell, comprising the expression vector of the invention.

The present invention also provides a method of preparing an anti-CLDN 18.2 fully humanized antibody of the present invention, the method comprising expressing a nucleic acid molecule of the present invention under conditions conducive to expression of the antibody of the present invention and recovering the expressed antibody or antigen binding fragment thereof.

The medium used to culture the cells can be any conventional medium used to culture the host cells, such as minimal medium or complex medium containing suitable additives. Suitable media can be obtained commercially or prepared according to published procedures. The polypeptide produced by the host cell can then be recovered from the culture medium by conventional methods, for example, by precipitating the protein component of the supernatant or filtrate with a salt such as ammonium sulfate, and further purified by various chromatographic methods such as, for example, exchange chromatography, gel filtration chromatography, affinity chromatography, etc., depending on the kind of the desired peptide.

The coding DNA sequence described above may be inserted into any suitable vector. In general, the choice of vector will often depend on the host cell into which the vector is to be introduced, and thus, the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector may be of a type which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequence encoding the peptide is operably linked to other segments of the DNA required for transcription, such as a promoter. Examples of promoters suitable for directing transcription of DNA encoding a peptide of the invention in a variety of host cells are well known in the art, see for example Sambrook, J, Fritsch, EF and maniotis, T, molecular cloning: a guide to the experimental work, Cold Spring Harbor Laboratory Press, New York, 1989.

The vector may also contain a selectable marker, e.g., a gene the gene product of which complements a defect in the host cell or which confers resistance to a drug, e.g., ampicillin, doxorubicin, tetracycline, chloramphenicol, neomycin, streptomycin, or methotrexate.

To introduce the expressed peptides of the invention into the secretory pathway of a host cell, a secretory signal sequence (also referred to as a leader sequence) may be provided in the recombinant vector. The secretory signal sequence is linked in the correct reading frame to the DNA sequence encoding the peptide. The secretion signal sequence is usually located 5' to the DNA sequence encoding the peptide. The secretory signal sequence may be one normally linked to the peptide, or may be derived from a gene encoding another secretory protein.

Methods for ligating the DNA sequence encoding the peptide of the present invention, the promoter and optionally the terminator and/or secretion signal peptide sequence, respectively, and inserting them into a suitable vector containing information necessary for replication are known to those skilled in the art.

Expression vectors for use in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for termination of transcription and stabilization of mRNA, such sequences typically being obtained from the 5 'and (occasionally) 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions comprise nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the humanized antibody that binds claudin 18.2.

The host cell into which the DNA sequence or recombinant vector is to be introduced may be any cell capable of producing the peptide of the invention, including bacterial, viral, yeast, fungal and higher eukaryotic cells. Examples of suitable hosts known and used by those skilled in the art include, but are not limited to, viruses.

The antibody or antigen-binding fragment of the invention may be recovered in various forms from the culture medium or host cell lysate, and if membrane-bound, may be released from the membrane using a suitable detergent solution (e.g., Triton-X100) or by enzymatic cleavage, and the cells used to express the antibody of the invention that binds to claudin18.2 may be disrupted by various physical or chemical methods such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysis reagents.

It may be desirable to purify the antibodies or antigen-binding fragments of the invention from recombinant cellular proteins, and the following methods are exemplary of suitable purification methods: separation by fractionation on an ion exchange column; ethanol precipitation; reversed phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; carrying out chromatographic focusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein a Sepharose column to remove contaminants such as IgG.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.

Suitable pharmaceutically acceptable carriers include, but are not limited to: antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methyl paraben, ethyl or n-propyl paraben), emulsifiers, suspending agents, dispersants, solvents, fillers, bulking agents, buffers, carriers, diluents, and/or adjuvants. For example, a suitable carrier may be a physiological saline solution or citrate buffered saline, possibly supplemented with other materials common in parenterally administered pharmaceutical compositions. Medium buffered saline or saline mixed with serum albumin are other exemplary carriers. Those skilled in the art will readily recognize the wide variety of buffers that may be employed in the pharmaceutical compositions and dosage forms used in the present invention. Typical buffering agents include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. The buffer component also comprises water-soluble materials, such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid and their salts.

The pharmaceutical compositions and powders may be stored in sterile vials as solutions, suspensions, gels, emulsions, solids, or dehydrated or lyophilized powders. These compositions may be stored as a ready to use form, a freeze-dried form which requires reconstitution prior to use, a liquid form which requires dilution prior to use, or other useful forms.

In another aspect, the invention relates to the use of an antibody or antigen-binding fragment thereof for the manufacture of a medicament for the prevention or treatment of a cancer-related disease, in particular gastric cancer.

In a fifth aspect of the invention, there is provided a method of preventing or treating cancer comprising administering an antibody that binds to Claudin18.2, according to the invention; wherein the cancer may be, for example, gastrointestinal cancer, pancreatic cancer, esophageal cancer, or non-small cell lung cancer; the gastrointestinal cancer is preferably advanced gastric cancer.

In a sixth aspect of the invention, there is provided an article of manufacture or a kit comprising a container holding an antibody or antigen-binding fragment thereof according to the invention, or a pharmaceutical composition according to the invention, and a package insert carrying instructions for use of the medicament. In a preferred embodiment, the article of manufacture or kit further comprises one or more containers comprising one or more additional agents for preventing or treating cancer.

Suitable containers include, for example, ampoules, vials, syringes and the like. The container may be formed of a variety of substances (e.g., glass or plastic) that hold or contain a small composition for treatment and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody or antigen-binding fragment of the invention. The label or package insert indicates that the composition is to be used to treat a metabolic-related disease, disorder, or condition in an individual suffering from the metabolic-related disease, disorder, or condition under specific guidance regarding the time interval between doses of the antibody and any other drugs provided. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable dilution buffer, bacteriostatic water for injection, phosphate buffer, Ringer's solution, and dextrose solution. The article may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes. Instructions for use of the "package insert" typically include instructions in commercial packaging of the therapeutic product that contain information regarding instructions, usage, dosages, administrations, contraindications, other therapeutic products in combination with the packaged product, and/or warnings regarding use of such therapeutic products, and the like.

The article may also include other components, each component of the article may be packaged in a single container and all of the plurality of containers may be placed in a single package.

The fully humanized antibody of the present invention has more excellent affinity for CLDN18.2 than the chimeric anti-CLDN 18.2 antibody IMAB362 of Ganymed Pharmaceuticals AG.

Drawings

Fig. 1 is a graph of anti-CLDN 18.2 antibody screening-cell ELISA binding activity sigmoidal. .

Fig. 2 is a bar graph comparing the relative binding activity of each anti-CLDN 18.2 antibody.

Detailed Description