CN113929780A - Humanized antibody for treating cancer by combining claudin - Google Patents

Humanized antibody for treating cancer by combining claudin Download PDF

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CN113929780A
CN113929780A CN202110516771.6A CN202110516771A CN113929780A CN 113929780 A CN113929780 A CN 113929780A CN 202110516771 A CN202110516771 A CN 202110516771A CN 113929780 A CN113929780 A CN 113929780A
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许铮
李响
刘影
熊国裕
李峰
史继峰
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Beijing Kawin Technology Share Holding Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

The invention relates to a humanized antibody which is combined with claudin18.2 and is used for treating cancers, wherein the antibody is not combined with claudin18.1, but is selectively combined with claudin18.2 to destroy the tight connection among cells, so that claudin18.2 cannot play the normal function of the antibody, and the antibody can be used for preventing or treating tumors, particularly late gastric cancer.

Description

Humanized antibody for treating cancer by combining claudin
Technical Field
The invention relates to a humanized antibody for treating cancer, which is combined with claudin18.2 and belongs to the technical field of antibody medicines.
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.
Disclosure of Invention
In a first aspect of the invention, there is provided a humanized antibody that binds to claudin18.2, said antibody comprising a heavy chain hypervariable region (HCDR) and a light chain hypervariable region (LCDR) comprising:
the amino acid sequence is GYX1HCDR1 for FTNYG, HCDR2 for the amino acid sequence INTNTGEP, and HCDR3 for the amino acid sequence ARLGFGNAMDY;
the light chain hypervariable region comprises:
the amino acid sequence is QX2X3LNX4X5NX6LCDR1 of KNY and the amino acid sequence is WAX7LCDR2 and amino acid sequence QX8DYX9LCDR3 of YPLT (Localx),
wherein amino acid X in the HCDR11Selected from S or T;
amino acid X in the LCDR12Selected from T or S, X3Is selected from L or V, X4 is selected from T or S, X5 is selected from G or S, X6Selected from Q or N;
amino acid X in the LCDR27Selected from T or S;
amino acid X in the LCDR38Selected from N or Q, X9Selected from T or S.
In a preferred embodiment, the antibody comprises HCDR and LCDR as described below,
CDR1(HCDR1) having an amino acid sequence of GYSFTNYG (SEQ ID NO: 1) or GYTFTNYG (SEQ ID NO: 2);
CDR2(HCDR2) having the amino acid sequence INTNTGEP (SEQ ID NO: 3);
CDR3(HCDR3) having amino acid sequence ARLGFGNAMDY (SEQ ID NO: 4); and
CDR1(LCDR1) having an amino acid sequence of QTLLNTGNQKNY (SEQ ID NO:5), QSVLNSGNQKNY (SEQ ID NO:6), QSLLNSSNNKNY (SEQ ID NO:7), QSVLNSSNNKNY (SEQ ID NO:8), QTLLNSGNQKNY (SEQ ID NO:9), QTLLNSGNNKNY (SEQ ID NO:10), QTLLNSSNQKNY (SEQ ID NO:11), QTLLNSSNNKNY (SEQ ID NO:12), QSLLNSSNQKNY (SEQ ID NO:13), QSLLNSGNNKNY (SEQ ID NO:14), QSLLNSGNQKNY (SEQ ID NO:15), QSVLNSSNQKNY (SEQ ID NO:16), QSVLNSGNNKNY (SEQ ID NO:17) or QTVLNSGNNKNY (SEQ ID NO: 18);
CDR2(LCDR2) having the amino acid sequence WAT (SEQ ID NO: 19) or WAS (SEQ ID NO: 20);
CDR3(LCDR3) having an amino acid sequence of QNDYTYPLT (SEQ ID NO:21), QQDYTYPLT (SEQ ID NO:22), QQDYSYPLT (SEQ ID NO:23) or QNDYSYPLT (SEQ ID NO: 24).
In a more preferred embodiment of the invention, the humanized antibody comprises a heavy chain hypervariable region and a light chain hypervariable region in combination:
(1) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 5, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 21; or
(2) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 22; or
(3) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 7, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 21; or
(4) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 8, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 23; or
(5) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 9, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24; or
(6) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 5, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24; or
(7) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 9, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 21; or
(8) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 10, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24; or
(9) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 11, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24; or
(10) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 12, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24; or
(11) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24; or
(12) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 13, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24; or
(13) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 14, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24; or
(14) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 15, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 23; or
(15) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 7, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24; or
(16) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 13, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 23; or
(17) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 16, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24; or
(18) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 14, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 23; or
(19) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 17, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24; or
(20) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 8, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24; or
(21) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 7, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 23; or
(22) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 18, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24; or
(23) HCDR1 of SEQ ID NO. 17, HCDR2 of SEQ ID NO. 32, HCDR3 of SEQ ID NO. 54, and LCDR1 of SEQ ID NO. 61, LCDR2 of SEQ ID NO. 63, LCDR3 of SEQ ID NO. 66.
In a second aspect of the invention, there is provided an antibody having the amino acid sequences set forth below, which antibody has the heavy chain variable region (VH) and light chain variable region (VL) sequences set forth below:
the amino acid sequence is QVQLVQSGXa1ELKKPGASVKISCKASZH1MNWVRQAPGQGLKWMGZH2TYAEEFKGRFVFS LDTSVSTAYLQISSLKAEDTAVYXa2CZH3VH shown by WGQGTLVTVSS; and
the amino acid sequence is DIVMTQSPDSLAVSLGERATMCKSSZL1LTWYQQKPGQPPKLLIYZL2T RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCZL3FGAGTKLEIK is shown in the form of a VL,
wherein Xa1Is P or S, Xa2Is F or Y, ZH1、ZH2And ZH3Three CDR regions of the heavy chain, ZL1、ZL2And ZL3Three CDR regions of the light chain,
wherein ZH1The amino acid sequence of (A) is GYX1FTNYG，ZH2The amino acid sequence of (A) is INTNTGEP, ZH3ARLGFGNAMDY; zL1Has the amino acid sequence of QX2X3LNX4X5NX6KNY，ZL2Has the amino acid sequence of WAX7，ZL3Has the amino acid sequence of QX8DYX9YPLT；
X1Selected from S or T;
X2selected from T or S; x3Is selected from L or V; x4Selected from T or S; x5Selected from G or S; x6Selected from Q or N; x7Selected from T or S; x8Is selected from N or Q; x9Selected from T or S.
In a more specific embodiment of the invention, the humanized antibody has VH and VL sequences,
the amino acid sequence is SEQ ID NO: 25. 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 51, and
the amino acid sequence is SEQ ID NO: 26. 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52.
Exemplary humanized antibodies have VH and VL sequences as set forth in any one of the following groups:
SEQ ID NO: 25, and the amino acid sequence of SEQ ID NO: 26 (BY 5-1); SEQ ID NO: 27, and the VH of the amino acid sequence shown in SEQ ID NO: 28 (BY 5-5); SEQ ID NO: 29, and the VH of the amino acid sequence shown in SEQ ID NO: 30 (BY 6-1); SEQ ID NO: 31, and the VH of the amino acid sequence shown in SEQ ID NO:32 (BY 6-2); SEQ ID NO: 33, and the VH of the amino acid sequence shown in SEQ ID NO: 34 (BY 6-3); SEQ ID NO: 35, and the VH of the amino acid sequence shown in SEQ ID NO: 36 (BY 6-4); SEQ ID NO: 37, and the amino acid sequence of SEQ ID NO: 38 (BY 6-6); SEQ ID NO: 39, and the VH of the amino acid sequence shown in SEQ ID NO: 40 (BY 6-7); SEQ ID NO: 41, and the amino acid sequence of SEQ ID NO: 42 (BY 6-8); SEQ ID NO: 43, and the VH of the amino acid sequence shown in SEQ ID NO: 44 (BY 6-9); SEQ ID NO: 45, and the amino acid sequence of SEQ ID NO: 46 (BY 6-13); SEQ ID NO: 47, and the amino acid sequence of SEQ ID NO: 48 (BY 6-15); SEQ ID NO: 49, and the VH of the amino acid sequence shown in SEQ ID NO: 50 (BY 6-19).
In each of the candidate antibodies, the heavy constant region 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, preferably a kappa chain.
In a specific embodiment, the names and corresponding sequences of the antibodies of the present invention are as follows:
VH sequence:
QVQLVQSGXa1ELKKPGASVKISCKASGYX1FTNYGMNWVRQAPGQGLKWMGINTNTGEPTYAEEFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYXa2CARLGFGNAMDYWGQGTLVTVSS；
a VL sequence:
DIVMTQSPDSLAVSLGERATMNCKSSQX2X3LNX4X5NX6KNYLTWYQQKPGQPPKLLIYWAX7TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQX8DYX9YPLTFGAGTKLEIK is shown in the form of a VL,
TABLE 1 humanized antibody names binding to Claudin18.2 and corresponding VH, VL sequences
Figure BDA0003061803480000041
Figure BDA0003061803480000051
BY0-0 VH and VL amino acid sequences (SEQ ID NOS: 51 and 52)
VH complementarity determining region: CDR1 GYTFTNYG, CDR2 INTNTGEP; CDR3: ARLGFGNAMDY
VH framework region: FR 1: QIQLVQSGPELKKPGETVKISCKAS, respectively; FR 2: MNWVKQAPGKGLKWMGW, respectively; FR 3: TYAEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFC, respectively; FR 4: WGQGTSVTVSS
VL hypervariable region/complementarity determining region: CDR1: QSLLNSGNQKNY, respectively; CDR2: WAS; CDR3: QNDYSYPLT are provided.
VL framework region: FR 1: DIVMTQSPSSLTVTAGEKVTMSCKSS, respectively; FR 2: LTWYQQKPGQPPKLLIY, respectively; FR 3: TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC, respectively; FR 4: FGAGTKLELK are provided.
The antibody or antigen-binding fragment of the invention, wherein the constant region of the HVR is selected from the human IgG series, such as IgG1, IgG2, IgG3, or IgG4, preferably IgG 1; the constant region of the LVR is selected from a kappa or lambda chain, preferably a kappa chain.
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.
The invention also relates to an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to the invention; 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.
In a fourth aspect of the invention, there is provided a method of producing an antibody or antigen-binding fragment thereof that specifically binds to claudin18.2, comprising expressing a nucleic acid molecule of the invention under conditions conducive to expression of the antibody or antigen-binding fragment thereof, 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 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.
Detailed Description
1. Defining:
an "antibody" as described herein is an immunoglobulin molecule consisting of four peptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, each heavy chain comprising a heavy chain variable region (HVR or VH) comprising three domains CH1, CH2 and CH3 and a heavy chain constant region comprising a light chain variable region (LVR or VL) and a light chain constant region comprising one domain (CL1), the VH and VL regions being further divided into hypervariable regions known as Complementarity Determining Regions (CDRs) interspersed with more conserved regions known as Framework Regions (FRs), each VH and VL consisting of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. Herein, the heavy chain constant region may be selected from IgG1, IgG2, IgG3 or IgG4, preferably IgG4, and the light chain constant region is selected from κ or λ.
The term "variable region" is used herein to describe regions where certain portions of an antibody differ between antibody sequences and relate to the binding and specificity of a particular antibody for its particular antigen. However, variability is typically not evenly distributed throughout the variable region of an antibody. It is typically concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The relatively conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FRs, which mostly adopt a β -sheet conformation, connected by three CDRs which form loops connecting, and in some cases forming part of, the β -sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen binding site of the antibody. The constant regions are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.
The terms "complementarity determining regions", "hypervariable regions" and "CDRs" refer to one or more of the hypervariable regions or Complementarity Determining Regions (CDRs) present in the variable regions of an antibody light or heavy chain (see Kabat, e.a. et al, sequences of proteins of immunologicalcalemtest, national institutes of health, Bethesda, Md., (1987)). These terms include the hypervariable regions defined by Kabat et al ("Sequences of proteins of immunologicalcalemtest," Kabat E., et al, USDept. of health and Humanservices, 1983) or the hypervariable loops in the three-dimensional structure of antibodies (Chothia and Lesk, J mol. biol.196901-917 (1987)). The CDRs in each chain are held in close proximity by the framework regions and, together with the CDRs from the other chains, facilitate the formation of an antigen binding site.
The terms "framework regions" and "FR" refer to one or more of the framework regions within the variable regions of antibody light and heavy chains (see Kabat, e.a. et al, sequences of proteins of immunologicalcalest, national institutes of health, Bethesda, Md., (1987)). These terms include those amino acid sequences in the antibody light and heavy chains that are located between the amino terminus and the first CDR, those that are intermediate between the CDRs and those that are between the third CDR and the start of the constant region.
CDR and FR residues can be determined according to standard sequence definitions (Kabat et al, sequence of proteins of immunologicals Interes, national institutes of health, BethesdaMd. (1987)) and structural definitions (Chothia and Lesk, J.Mot.biol.196: 901-217 (1987)).
When referred to herein, reference is made to the numbering scheme of Kabat, e.a., et al, sequences of proteins of immunologicalcalest (national institutes of health, Bethesda, Md. (1987) and (1991).
The "affinity" of an antibody for an antigen or epitope is a term well understood in the art and refers to the degree or strength of binding of an antibody to an epitope. Affinity can be measured and/or expressed in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (KD or KD, which can be defined as the ratio of the dissociation rate to the association rate of an antibody, i.e., Koff/Kon) Apparent equilibrium dissociation constant (KD 'or KD'), EC50 (the amount required to achieve 50% binding to antigen), and IC50 (the amount required to achieve 50% inhibition in a competition assay); the relative affinity of the humanized antibody can also be determined by comparison with, for example, a related murine or chimeric antibody. For the purposes of the present invention, affinity is the average affinity of a particular population of antibodies that bind an antigen or epitope. The affinity of the antibody can be measured using an enzyme-linked immunosorbent assay (ELISA) or a Fluorescence Activated Cell Sorting (FACS) assay, and the affinity of the antibody of the invention for binding to claudin18.2 is determined using an ELISA method in the examples herein, see examples.
2. Method for producing antibody of the present invention
The antibodies of the invention can be produced by any useful method, such as recombinant expression techniques. Nucleic acids encoding the light and heavy chain variable regions operably linked to the constant regions can be inserted into an expression vector. The light and heavy chains may be cloned in the same or different expression vectors. The DNA segment encoding the immunoglobulin chain may be operably linked to the control sequences of an expression vector that ensure expression of the immunoglobulin polypeptide. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. In one embodiment, the expression control sequence is a prokaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is introduced into an appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of the antibody.
The expression vector may be replicable in any host organism, either as an episome or as an integral part of the host chromosomal DNA. In one embodiment, the expression vector contains a selectable marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) to allow for detection of those cells transformed with the desired DNA sequence.
Expression vectors can be used to express the antibodies of the invention from any host cell, including prokaryotic host cells (e.g., E.coli), yeast host cells, mammalian host cells, plant host cells, and insect host cells.
In one embodiment, the antibodies of the invention are produced using E.coli. Other prokaryotic hosts suitable for such applications include bacilli, such as bacilli and other enterobacteriaceae, such as salmonella, saxiella and various pseudomonas species. In these prokaryotic hosts, expression vectors can also be prepared, which typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell. In addition, there will be any number of a variety of well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or a promoter system from bacteriophage lambda. Promoters generally control expression, optionally together with operator sequences, and have ribosome binding site sequences and the like for initiating and completing transcription and translation.
Other microorganisms, such as yeast, may also be used to express the antibodies of the invention. For example, Saccharomyces can be used as a yeast host, with a suitable promoter containing expression control sequences (e.g., a promoter), an origin of replication, termination sequences, and other sequences as desired. Promoters useful in yeast expression techniques include 3-phosphoglycerate kinase and other glycolytic enzyme promoters. Inducible yeast promoters include, but are not limited to, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
In another embodiment, mammalian tissue cell cultures can be used for expression and production of the antibodies of the invention. Any mammalian tissue cell can be used for such methods, and many suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art, including mammalian BHK or CHO cell lines, various Cos cell lines, HeLa cell lines, preferably myeloma cell lines or transformed B cells or hybridomas. In one embodiment, the cell is non-human. Mammalian cell expression vectors can include expression control sequences such as origins of replication, promoters, and enhancers, as well as necessary processing signal sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. In one embodiment, the expression control sequence is a promoter derived from an immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like.
Vectors containing the polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred to a host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, bioballistics or viral transfection may be used for other cellular hosts. Other methods for transforming mammalian cells include the use of polybrene (polybrene), protoplast fusion, liposomes, electroporation, and microinjection. To produce transgenic animals, the transgene may be microinjected into a fertilized egg, or may be introduced into the genome of an embryonic stem cell, and the nucleus of such cell transferred into an enucleated oocyte.
When the nucleic acid molecules encoding the heavy and light chains are cloned into separate expression vectors, the vectors may be co-transfected to obtain expression and assemble the intact immunoglobulin. Once expressed, the whole antibody, its dimer, individual light and heavy chains, or other immunoglobulin forms of the antibody may be purified according to standard methods in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis, and the like. Substantially pure immunoglobulins of at least about 90 to 95% homogeneity may be prepared for pharmaceutical use. In another embodiment, substantially pure humanized antibodies of at least about 98 to 99% or greater homogeneity can be produced for use in pharmaceutical formulations and methods.
Accordingly, the invention provides a method of expressing an antibody of the invention comprising: (a) transforming a host cell with a nucleic acid molecule encoding an antibody described herein, and (b) culturing the transformed host cell under conditions that allow expression of the antibody. Known techniques for including a selectable marker on a vector can be used so that host cells expressing the marker of the antibody can be readily selected.
An "antigen-binding fragment" of the antibody, or "antigen-binding portion" of an antibody, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., Claudin18.2). It has been demonstrated that the antigen binding function of an antibody can be performed by certain fragments of a full-length antibody. Examples of "antigen-binding fragments" of antibodies include, but are not limited to: (i) fab fragments, monovalent fragments consisting of the VL, VH, CL1 and CH1 domains; (ii) a F (ab ') 2 fragment, i.e., a bivalent fragment consisting of two F (ab') fragments linked by disulfide bonds of the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the single-arm VL and VH domains of an antibody; (v) a dAb fragment consisting of a VH domain; and (vi) a CDR. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be joined together by recombinant means by a synthetic linker into separate linked chains in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv, scFv). Such single chain antibodies are also encompassed within the scope of "antigen-binding fragments" of antibodies.
The antibody of the present invention can be produced by a method comprising culturing a host cell containing a DNA sequence encoding the antibody and capable of expressing the antibody under conditions permitting expression of the antibody, and recovering the produced antibody from the culture.
The medium used to culture the cells can be any conventional medium used to culture the host cells, such as minimal medium or a minimal medium containing suitable additives. Suitable media can be obtained commercially or prepared according to published procedures. The polypeptide produced by the cells can then be recovered from the culture medium by conventional methods including separation of the host cells from the culture medium by centrifugation or filtration, precipitation of the protein component in the supernatant or filtrate with a salt such as ammonium sulfate, and purification by various chromatographic methods such as, for example, exchange chromatography, gel filtration chromatography, affinity chromatography, etc., depending on the kind of the desired peptide.
3. Therapeutic methods and agents
The present invention provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof of the invention that binds to claudin 18.2. The pharmaceutical compositions of the present invention will be administered with suitable carriers, excipients and other formulations. These formulations are included in formulations to improve delivery and tolerability, among other things. Pharmacopoeias well known to all pharmaceutical chemists: a number of suitable formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
The administration dose is adjusted depending on the age and body size of the subject, the target disease, symptoms, administration route, and the like. When the antibodies of the invention are used to treat various cancer-related conditions and diseases in adults, the antibodies of the invention may be administered intravenously, typically in a single dose of about 0.01 to about 20mg/kg body weight, more preferably about 0.1 to about 15, about 1 to about 10, or about 3 to about 10mg/kg body weight. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted.
Various drug delivery systems are known for administration of the pharmaceutical compositions of the invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing variant viruses, receptor-mediated endocytosis (see, e.g., Wu et al (1987), J.biol.chem.262: 4429-4432). Methods of administering drugs include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical compositions may be administered by any convenient route, such as by infusion or intravenous bolus injection, absorption through epithelial and mucosal layers (e.g., oral mucosa, rectal and small intestinal mucosa), and may be administered with other biologically active agents. The mode of administration may be systemic or local.
The pharmaceutical composition may also be delivered by a vesicle, in particular a liposome vesicle (see Langer (1990) Science 249: 1527-.
In some cases, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. biomed. Eng.14: 201). In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974.) for a discussion of other Controlled Release systems, see Langer (1990) Science 249: 1527-.
The injectable preparation may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, instillation and the like. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared by dissolving, suspending or emulsifying the antibody or salt thereof in a sterile aqueous or oily medium conventionally used for injection. Examples of the aqueous medium for injection include physiological saline, an isotonic solution containing glucose and other auxiliary agents, and the like. They may be used in combination with a suitable solubilizer such as alcohol (e.g., ethanol), polyol (e.g., propylene glycol, polyethylene glycol), nonionic surfactant [ e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mole) adduct of hydrogenated castor oil), etc. The oil medium can be sesame oil, soybean oil, etc. They may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, and the like. The injection thus prepared is preferably enclosed in a suitable ampoule.
Monotherapy and combination therapy the antibodies and antibody fragments of the present invention can be improved on therapy by disrupting the tight junctions between cells, rendering claudin18.2 unable to perform its normal function.
The present invention encompasses combination therapies in which an antibody or antibody fragment that binds to claudin18.2 is administered in combination with one or more therapeutic agents (or second therapeutic agents). Co-administration and combination therapy are not limited to simultaneous administration, but also include treatment regimens in which the anti-claudin 18.2 antibody or antibody fragment is administered at least once during a course of treatment involving administration of at least one other therapeutic agent to the patient. The second therapeutic agent may be another therapeutic agent for gastric cancer (e.g., gastric cancer).
The invention also includes the use of any of the anti-claudin 18.2 antibodies or antigen-binding fragments described herein for the preparation of a medicament for the treatment of a disease or condition by disrupting intercellular tight junctions such that claudin18.2 does not perform its normal function.
In one aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention that binds to claudin18.2, and a pharmaceutically acceptable carrier.
In this context, drugs, pharmaceutical compositions and pharmaceutical preparations (medicaments) may be used interchangeably unless contradicted or otherwise specifically indicated. Pharmaceutically acceptable excipients in this context refer to nontoxic fillers, stabilizers, diluents, carriers, solvents or other formulation excipients. For example, diluents, excipients, such as microcrystalline cellulose, mannitol, and the like; fillers, such as starch, sucrose, and the like; binders, such as starch, cellulose derivatives, alginates, gelatin and/or polyvinylpyrrolidone; disintegrants, such as calcium carbonate and/or sodium bicarbonate; absorption promoters, such as quaternary ammonium compounds; surfactants such as cetyl alcohol; carriers, solvents, such as water, physiological saline, kaolin, bentonite, etc.; lubricants, such as talc, calcium/magnesium stearate, polyethylene glycol, and the like. In addition, the pharmaceutical composition of the present invention is preferably an injection.
In some embodiments of the invention, the antibody or antigen-binding fragment thereof in the pharmaceutical composition of the invention is present at a concentration of 1mg/ml to 1000mg/ml, preferably 10mg/ml to 1000mg/ml, more preferably 50mg/ml to 500mg/ml, more preferably 100mg/ml to 300 mg/ml.
The pharmaceutical composition of the present invention preferably has a pH of 3.0 to 9.0. Wherein, a buffer system, a preservative, a surface tension agent, a chelating agent, a stabilizer and a surfactant can be further included. In one embodiment of the invention, the pharmaceutical composition of the invention is an aqueous formulation. Such preparations are usually solutions or suspensions. In a particular embodiment of the invention, the pharmaceutical composition is a stable aqueous solution. In another embodiment of the invention, the pharmaceutical composition is a lyophilized formulation which is dissolved by the addition of a solvent and/or diluent by the physician or patient prior to use.
Drawings
FIG. 1 is a graph showing the binding activity of anti-Claudin18.2 antibody screening-cell ELISA.
FIG. 2 is a graph showing the anti-Claudin18.2 antibody screening-proliferation inhibitory activity.
FIG. 3 is a graph of anti-Claudin18.2 antibody screening-antibody dependent cytotoxicity.
FIG. 4 is a graph of anti-Claudin18.2 antibody screening-complement dependent cytotoxicity.
FIG. 5(5-1,5-2) is a graph of the effect of candidate antibodies on KATO cell nude mouse graft tumor model versus tumor volume (RTV).
FIG. 6(6-1,6-2) is a graph of the effect of candidate antibodies on the nude mouse transplanted tumor model Relative Tumor Volume (RTV) for NUGC cells.
Detailed Description
Example 1 construction of HEK293 cells expressing Claudin18.1 and Claudin18.2 antigens
HEK293 cells (purchased from ATCC) were transfected with pcDNA3.1 vectors encoding human Claudin18.1 and Claudin18.2 antigens (purchased from Invitrogen), and HEK293 cells stably expressing Claudin18.1 and Claudin18.2 antigens were obtained using 200. mu.g/mL geneticin as the selection pressure. The antibody IMAB362 (self-made, CHO-S cell transient expression, further chromatography purification, reference examples 2 and 3, BY0-0 in the present invention) of Claudin18.2 from Ganymed corporation was used as a positive antibody, and HEK293 cells stably expressing human Claudin18.1 and Claudin18.2 antigens were selected BY FACS method.
EXAMPLE 2 construction of expression vector for candidate antibody BY6-4
The HindIII cleavage site (b) (ii)AAGCTT) KoZAK sequence (A)GCCGCCACC) ATG, signal peptide gene GAGAGAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT and heavy chain encoding genes of the antibody (including heavy chain variable region encoding gene SEQ ID NO: 59 and constant region IgG1 encoding the gene SEQ ID NO: 63) the terminator TAA and the EcoRI coding gene GAATTC are sequentially fused in series, and a gene fragment is obtained by using a chemical synthesis mode. The above fragment was inserted into eukaryotic expression plasmid pCDNA3.4(+) ((purchased from Invitrogen) via EcoRI and HindIII sites and verified BY sequencing to give expression plasmid pCDNA3.4(+) -BY6-4 for antibody heavy chain.
The HindIII cleavage site (b) (ii)AAGCTT) KoZAK sequence (A)GCCGCCACC) ATG, signal peptide gene GAGAGAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT and the light chain encoding genes of the antibody (including the light chain variable region encoding gene SEQ ID NO: 60 and a constant region kappa coding gene SEQ ID NO:64), a terminator TAA and an EcoRI coding gene GAATTC are sequentially fused in series, and a gene fragment is obtained by using a chemical synthesis mode. The above fragment was inserted into eukaryotic expression plasmid pCDNA3.4(+) through EcoRI and HindIII sites and verified BY sequencing to obtain expression plasmid PCDNA3.4(+) -BY6-4 of antibody light chain.
The heavy chain coding gene (SEQ ID NO: 53) and the light chain coding gene (SEQ ID NO: 54) of BY6-1, the heavy chain coding gene (SEQ ID NO: 55) and the light chain coding gene (SEQ ID NO: 56) of BY6-2, the heavy chain coding gene (SEQ ID NO: 57) and the light chain coding gene (SEQ ID NO: 58) of BY6-3, the heavy chain coding gene (SEQ ID NO: 61) and the light chain coding gene (SEQ ID NO: 62) of BY0-0 are substituted for the heavy and light chain coding gene of BY6-4, and corresponding heavy and light chain expression plasmids can be obtained.
Given the amino acid sequence of such antibodies, obtaining the genes encoding such antibodies is well known in the art. According to the same method, a series of expression plasmids of the candidate antibody can be obtained according to the amino acid sequences of different candidate antibodies: BY5-1, BY5-2, BY5-3, BY5-4, BY5-5, BY5-6, BY5-7, BY5-8, BY5-9, BY6-5, BY6-6, BY6-7, BY6-8, BY6-9, BY6-10, BY6-11, BY6-12, BY6-13, BY6-14, BY6-15, BY6-16, BY6-17, BY6-18, BY6-19 heavy and light chain expression plasmids
The heavy chain constant region of each candidate antibody was IgG1 and the light chain constant region was kappa.
Example 3 expression and purification of anti-Claudin18.2 antibody
Using the DNA constructs described in example 2, they were transiently transferred to CHO-S cells (purchased from Invitrogen) to express the desired antibody, according to the CHO-S cell manipulation Manual (Freedom)TM CHO-STMKituer GUIDE) cell density was adjusted to 1x10 one day before plasmid transfection6One/ml. On the day of plasmid transfection, the plasmid was mixed with transfection reagent and added to EXPICHO EXPRESSION MEDIUM cell culture MEDIUM (purchased from Invitrogen corporation), the cells were continuously cultured at 37 ℃ and 8% CO2 until the 8 th day, the cell fluid was collected, the cells were removed by centrifugation, filtered at 0.2 μm, purified by protein A affinity chromatography, the pH of the collected sample was adjusted to 5.5, and the sample was stored at 2-8 ℃. The purity of the purified antibody is over 95 percent by SDS PAGE and SEC detection.
Example 4 specificity and affinity identification
Using synthetic peptide part of Claudin18.2N-terminal extracellular domain as antigen, performing ELISA screening on antibody by using HEK293 cells stably transformed with Claudin18.2 and Claudin18.1 coated by ELISA, detecting by using double antibody sandwich method (coating 96-well cell culture plate with 0.1mg/mL polylysine, removing coating solution after 5min, adding 5 × 104HEK293-Claudin18.2 or HEK293-Claudin18.1 cells in wells at 37 ℃ with 5% CO2After culturing in an incubator for 24h for adherence, removing supernatant, adding PBST to each hole to wash the cells for 3 times, adding a gradient diluted antibody solution into each hole at 100 mu l/hole, washing the plate for 3 times by using a washing solution at 37 ℃ for 2 hours; a dilution (2% bovine serum albumin solution prepared using a washing solution) 1/5000 diluted HRP-labeled secondary antibody (accession No. ab6858, product from Abcam corporation), added to the microplate at 100. mu.l/well, and incubated at 37 ℃ for 1 hour; washing the plate with washing solution for 3 times; adding TMB color development solution into 100 mu 1/hole, placing at room temperature for reaction in a dark place for 5-10 minutes, and adding human stop solution into 50 mu l/hole to stopReacted and absorbance was measured at a wavelength of 450 nm), the EC50 value of each candidate antibody with claudin18.2 was measured, and the relative binding activity of each candidate antibody was calculated based on the EC50 value of BY0-0 (see table 2 for structure). The calculation method is as follows, relative binding activity ═ EC50 for reference BY 0-0/EC 50 value for each candidate antibody.
TABLE 2 relative binding Activity of each candidate antibody
Figure BDA0003061803480000131
Figure BDA0003061803480000141
The results of cell ELISA binding activity show that the candidate antibody has better specificity, does not bind to Claudin18.1, but has significant difference with the Claudin18.2 binding activity, wherein the binding activity of BY6-1, BY6-2, BY6-3, BY6-4, BY6-6, BY6-7, BY6-8, BY6-9, BY6-13 and BY6-19 is better than that of Claudin18.2, while the binding activity of BY5-2, BY5-3, BY5-4, BY5-6, BY5-7, BY5-8, BY6-10, BY6-12, BY6-14, BY6-17, BY6-18 and Claudin18.2 is poorer.
Example 5 in vitro functional Activity of cells
The anti-claudin18.2 antibody mediates four independent highly effective mechanisms of action to induce killing and apoptosis of tumor cells: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), induction of apoptosis induced by target cross-linking on the surface of tumor cells, and direct inhibition of proliferation. The second round of antibody screening is to carry out further functional (ADCC, CDC and proliferation inhibition) identification on cell models of 7 antibodies (BY6-1, BY6-2, BY6-3, BY6-4, BY6-6, BY6-7 and BY6-9) with higher affinity screened from the first round of fully human antibody library, and provides basis for determination of clinical candidate drugs.
(1) Proliferation inhibitory Activity
The CCK8 method was used to study the proliferation inhibitory effect of anti-Claudin18.2 antibodies on KATOIII cells (purchased from ATCC): taking KATO III cells growing in logarithmic phase,cell concentration was adjusted to 2X 10 using IMDM (purchased from Invitrogen) + 10% FBS medium5In a 5% CO2 incubator at 37 deg.C/ml for use. The samples and reference (Ch163, or BY0-0) were pre-diluted to a final concentration of 400. mu.g/ml in IMDM + 5% FBS medium, followed BY 2-fold dilutions for a total of 14 gradients: 400ug/ml, 200. mu.g/ml, 100. mu.g/ml, 50. mu.g/ml, 25. mu.g/ml, 12.5. mu.g/ml, 6.25. mu.g/ml, 3.125. mu.g/ml, 1.56. mu.g/ml, 0.78. mu.g/ml, 0.39. mu.g/ml, 0.19. mu.g/ml, 0.097. mu.g/ml, 0. mu.g/ml. 60ul of cell suspension was added to each well of a flat-bottom 96-well plate, followed by 60ul of serially diluted anti-CLDN 18.2 antibody. Shaking gently, and mixing. Cell culture plate containing 5% CO2The incubator at 37 ℃ was incubated for 72 hours. After the incubation is finished, adding CCK-8 color development solution into the mixture, placing the mixture into a hole with the concentration of 12 mu l/hole and placing the mixture containing 5 percent CO2The incubator at 37 ℃ is incubated for 2-4 h. The absorbance values of each well were read at a wavelength of 450 nm.
Data processing IC50 values were calculated for samples and references using graphpad prism v5.01 software. The test results are shown in FIG. 2.
The proliferation inhibition activity result shows that the in vitro cell proliferation inhibition effect of the antibodies of the candidate antibodies BY6-2, BY6-3 and BY6-4 is obvious.
(2) Antibody Dependent Cellular Cytotoxicity (ADCC)
The ADCC activity of the antibody to be tested was examined using the transfected HEK18.2 cells as target cells and PBMC cells isolated from fresh human peripheral blood as effector cells. 7 antibodies were detected using trypsinized NUGC cells (purchased from JCRB) and HEK18.2 cells, 5000 cells per well, respectively, as target cells.
PBMCs from 30ml blood of 2 healthy volunteers were extracted one day prior to the experiment and cultured overnight in RPMI-1640 (from Invitrogen) + 10% FBS using 2.5X 10 cells per well5And (4) respectively. The initial concentration of the test agent was 10. mu.g/ml (diluted to 40. mu.g/ml before use), and the test agent was diluted to 8 samples by a 5-fold dilution method. RPMI-1640+ 2% FBS (inactivated serum) was used as the test medium for ADCC.
Result calculation method
ER ═ ER pore absorbance-CMB pore absorbance
ESR-pore Absorbance-CMB pore Absorbance
TSR ═ TSR well absorbance-CMB well absorbance
TMR ═ TMR pore absorbance-VCC pore absorbance
Cytotoxicity (%) ═ ER-ESR-TSR)/(TMR-TSR) × 100%)
The amount of dead and damaged cells was measured by the cytoxicity LDH Assay Kit-WST Kit (purchased from Homalvere Chemicals, Japan). The kit is used for measuring cell damage by measuring the activity of Lactate Dehydrogenase (LDH) released by cells into a culture medium. The test results are shown in FIG. 3.
ADCC activity results show that the antibodies of the candidate antibodies BY6-1, BY6-2, BY6-3 and BY6-4 have obvious in-vitro ADCC activity inhibition effect.
(3) Complement dependent cytotoxicity
The CDC effect of the anti-Claudin18.2 antibody, human serum and HEK8.2 cell after the action is detected by a CCK8 method. Serum for complement lysis test was obtained from healthy volunteers, centrifuged at 600g for 20min, harvested and stored at-20 ℃. HEK18.2 cells were cultured in RPMI-1640 complete medium at 37 ℃ with 5% CO2Culturing under the condition to logarithmic growth phase. HEK18.2 cells were digested and washed twice using serum-free RPMI-1640 medium. Counting cells, and preparing into 5 × 10 medium with serum-free RPMI-1640 medium5Cell suspension in ml. Screening antibodies were routinely diluted 1000. mu.g/mL (in RPMI-1640 medium) and further diluted in a 5-fold gradient (1000, 300, 100, 30, 10. mu.g/mL); the positive control antibody Rituxan (purchased from Roche) was routinely diluted 500. mu.g/mL (in RPMI-1640 medium) and 5-fold gradient diluted (500, 100, 20, 4. mu.g/mL). 100. mu.l per well, wherein; HEK18.2 cells suspended in RPMI-1640 medium in a volume of 85. mu.L, and the number of cells was 5.0X 104Well, 5 μ L of human serum (complement) (1:30 dilution); antibody 10 μ L at different concentrations (positive control plus RPMI-1640 medium for dilution of antibody, with cell-free medium control wells set up); the final concentration of the screening antibody was (100, 30, 10, 3, 1. mu.g/mL) and the final concentration of Rituxan mab was (50, 10, 2, 0.4. mu.g/mL). The 96-well cell culture plate is placed in 5% CO2After incubation at 37 ℃ for 4 hours, 10. mu.l of CCK8 were added. After 4 hours, absorbance was measured: placing into a microplate readerAnd (3) measurement and reading: the wavelength is 450 nm;
calculate the actual absorbance reading for the treated sample: sample well absorbance readings-cell free media control well absorbance readings.
Cell death rate (%) calculation:
[ (maximum actual absorbance for wells without antibody drug-actual absorbance for treated samples) ÷ maximum actual absorbance for wells without antibody drug ]. times.100%
The test results are shown in fig. 4.
The CDC activity results show that the in vitro cell CDC activity effect of the candidate antibodies BY6-1, BY6-3 and BY6-4 is obvious.
Example 6 in vivo antitumor Activity
A nude mouse (from Kyoto laboratory animal technology Co., Ltd., Beijing Wintolite) subcutaneous transplantation tumor model was constructed using human gastric cancer KATO (from ATCC) expressing Claudin18.2 and NUGC cell line (from JCRB), and tumor mass inoculation was used.
Collecting KATO and NUGC cells under sterile conditions, adjusting cell density to 1 × 10 with sterilized normal saline7vc/ml, 0.2ml is inoculated to the axilla of the nude mouse subcutaneously, and when the tumor grows to 1000mm in diameter3Size, taking out under aseptic condition, cutting into tumor blocks of 1mm × 1mm, and uniformly inoculating to axillary subcutaneous of nude mouse. The volume of the tumor to be treated is 100-300 mm3When the tumor size is large, screening is carried out according to the tumor size, people with excessive tumor size and non-tumor formation are not selected, 10 mice are randomly grouped into each group, 5 groups of KATO cells and NUGC cells are respectively subjected to intraperitoneal injection, the dosage of administration is 10 mg/kg/mouse, PBS is administered to a negative control group, the administration is carried out twice a week and a week for 7 times in a continuous manner, the dosage on the day of the first administration is D0, the tumor growth of the mice is detected twice a week (the weight of the mice is measured, and the tumor size calculation volume is measured BY vernier caliper (the tumor size of the control group exceeds 1000 mm)3The experiment can be ended)).
Tumor weight inhibition ratio (%) (tumor weight in control group-tumor weight in administration group)/tumor weight in control group × 100%
The test results are shown in tables 3-1 and 3-2, FIGS. 5-1,5-2 and 6-1/6-2.
TABLE 3-1 Effect of anti-Claudin18.2 antibodies on the growth of KaTO cell nude mouse transplantable tumors
Figure BDA0003061803480000161
TABLE 3-2 Effect of anti-Claudin18.2 antibodies on the growth of nude mouse transplanted tumors with NUGC cells
Figure BDA0003061803480000162
The results of two human gastric cancer cell line mouse xenograft model experiments show that the function of the candidate antibody for inhibiting the growth of the human gastric cancer KATO and NUGC cell nude mouse xenograft tumor is positively correlated with the dose. Has obvious inhibiting effect on the growth of human gastric cancer KATO and NUGC cell nude mouse xenograft tumor within certain dosage and administration time. Of the three candidate antibodies BY6-1, BY6-2 and BY6-4, BY6-4 performed the most effectively in two types of nude mouse xenografts with gastric cancer cells.
Example 7 stability experiment
And (3) inspecting the stability of the candidate antibody under the influence of factors such as high temperature, strong light, repeated freeze thawing, strong acid and strong alkali conditions and the like, and detecting according to inspection focus inspection items. The specific experimental design, detection items and sampling modes are shown in tables 4-1, 4-2 and 4-3; the stability results are shown in tables 4-4.
TABLE 4-1 design of candidate antibody pharmacy analysis experiments
Figure BDA0003061803480000171
Each investigation condition was placed with the same packaged buffer for blank control.
TABLE 4-2 analysis and detection method for candidate antibody druggability
Figure BDA0003061803480000172
TABLE 4-3 candidate antibody druggability analysis sampling Table
Figure BDA0003061803480000173
TABLE 4-4 results of candidate antibody stability experiments
Figure BDA0003061803480000174
Figure BDA0003061803480000181
The zero detection result shows that the purity of the antibody expressed BY CHO-S and the antibody with one-step affinity purity and SEC detection monomer is higher than 98%, the content of main peak detected BY IEX is about 70%, 8 antibodies have no obvious difference, the modification proportion of lysine is different after the enzyme digestion of carboxypeptidase, except BY6-15, the modification proportion of lysine of 7 antibodies is 3% -5%, the modification proportion of BY6-15 lysine is about 10%, the SDS-PAGE result shows that the molecular weight of the antibody is normal, the light and heavy chains are complete, and the 8 antibodies have no obvious difference, the relative binding activity difference with Claudin18.2 is larger, the activity of BY6-1, BY6-2, BY6-3 and BY6-4 is higher relative to BY0-0, the activity of BY6-6, BY6-15 and BY6-19 is lower relative to BY0-0, but the relative activity is consistent with that of an antibody screening stage.
The high-temperature stability experiment result shows that: 1) and (2) SEC detection: SEC detection of high-temperature stability of 8 antibodies has no significant difference, and SEC results of high-temperature detection at 50 ℃ for 20 days are equal to or better than that of an original research control antibody 163E 12; 2) IEX detection: except that BY6-15 has lower proportion of main peak at 50 ℃ in 20 days and higher proportion of acid peak, the other 7 antibodies have no significant difference in IEX detection of high temperature stability at 50 ℃ in 20 days; 3) SDS-PAGE: the purity of BY6-3 is slightly lower in the result of non-reduced SDS; 4) relative binding activity: the relative binding activity of 8 antibodies is in a descending trend along with the prolongation of the high-temperature incubation time at 50 ℃, wherein the relative binding activity of BY6-1 and BY6-4 is better than that of BY0-0, the relative binding activity of BY6-2 and BY6-3 is more consistent with that of BY0-0, and the relative binding activity of BY6-6, BY6-15 and BY7-1 is lower.
The strong acid stability experiment result shows that: 1) the main peak ratios of SEC, SDS and IEX of each antibody are all in a descending trend, wherein the main peak ratios of SEC and SDS of BY6-3/4 are more obvious compared with other antibodies. 2) The relative binding activity of the BY6-3/4 antibody declined during the stability study, but was still 80% of the relative activity of the control antibody; BY7-1 was relatively less active at the starting point (59.2%) but relatively more active in stability studies (130.4%); the relative binding activity of the remaining antibodies did not have a tendency to change significantly during the stability study. The relative binding activity start and end points of BY6-6 and BY6-15 were still low.
The illumination stability experiment result shows that: 1) and (2) SEC detection: during the stability study, the purity of the main peak of each antibody SEC is in a descending trend, but is not lower than that of a control antibody BY 0-0; 2) IEX detection: during the stability study period, the IEX main peak proportion of each antibody is reduced, the acid peak proportion is increased, and the main peak and the acid peak proportion of other antibodies have no obvious difference except that the main peak purity of BY6-3 is slightly low; 3) SDS-PAGE: except for the BY6-6, the purity of the main band of the reduced SDS-PAGE is low, and the rest antibodies have no obvious difference; 4) relative binding activity: there was no significant tendency for each antibody to change during the stability study.
IEX detection shows that the purity of the BY6-3 main peak is reduced remarkably BY about 20%, the purity of the BY6-1/2 main peak is reduced BY 10%, the relative binding activity of the BY6-6/16 and the BY6-19 is low, and other parts have no remarkable difference.
Repeated freeze-thaw stability experiment results show that: 1) SEC and IEX detection: during the stability study, each antibody had no significant tendency to change; 2) SDS-PAGE: the content of the BY6-6 light and heavy chains is low, and other antibodies have no obvious difference; 3) relative binding activity: the relative activity of BY6-1//2/3/4/6 is obviously reduced, only the relative activity of BY6-1 is 80% higher than that of the control, and the relative activity of BY6-15 and BY6-19 is not changed. BY6-1//2/3/4/6 was suggested to try to avoid repeated freezing and thawing.
Sequence listing
<110> Beijing Kaiyin science and technology Co., Ltd
<120> a humanized antibody for treating cancer which binds to claudin
<160> 64
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gly Tyr Ser Phe Thr Asn Tyr Gly
1 5
<210> 2
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gly Tyr Thr Phe Thr Asn Tyr Gly
1 5
<210> 3
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Ile Asn Thr Asn Thr Gly Glu Pro
1 5
<210> 4
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr
1 5 10
<210> 5
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Gln Thr Leu Leu Asn Thr Gly Asn Gln Lys Asn Tyr
1 5 10
<210> 6
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gln Ser Val Leu Asn Ser Gly Asn Gln Lys Asn Tyr
1 5 10
<210> 7
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gln Ser Leu Leu Asn Ser Ser Asn Asn Lys Asn Tyr
1 5 10
<210> 8
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Gln Ser Val Leu Asn Ser Ser Asn Asn Lys Asn Tyr
1 5 10
<210> 9
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Gln Thr Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr
1 5 10
<210> 10
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gln Thr Leu Leu Asn Ser Gly Asn Asn Lys Asn Tyr
1 5 10
<210> 11
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Gln Thr Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr
1 5 10
<210> 12
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Gln Thr Leu Leu Asn Ser Ser Asn Asn Lys Asn Tyr
1 5 10
<210> 13
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr
1 5 10
<210> 14
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Gln Ser Leu Leu Asn Ser Gly Asn Asn Lys Asn Tyr
1 5 10
<210> 15
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr
1 5 10
<210> 16
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Gln Ser Val Leu Asn Ser Ser Asn Gln Lys Asn Tyr
1 5 10
<210> 17
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Gln Ser Val Leu Asn Ser Gly Asn Asn Lys Asn Tyr
1 5 10
<210> 18
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Gln Thr Val Leu Asn Ser Gly Asn Asn Lys Asn Tyr
1 5 10
<210> 19
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Trp Ala Thr
1
<210> 20
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Trp Ala Ser
1
<210> 21
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Gln Asn Asp Tyr Thr Tyr Pro Leu Thr
1 5
<210> 22
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Gln Gln Asp Tyr Thr Tyr Pro Leu Thr
1 5
<210> 23
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Gln Gln Asp Tyr Ser Tyr Pro Leu Thr
1 5
<210> 24
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Gln Asn Asp Tyr Ser Tyr Pro Leu Thr
1 5
<210> 25
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 26
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Thr Leu Leu Asn Thr
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Thr Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Thr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 27
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 28
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Thr Leu Leu Asn Thr
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Thr Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Thr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 29
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 30
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Thr Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Thr Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 31
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 32
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Thr Leu Leu Asn Thr
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Thr Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 33
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 34
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Thr Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Thr Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Thr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 35
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 36
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Thr Leu Leu Asn Ser
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Thr Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 37
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 38
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Thr Leu Leu Asn Ser
20 25 30
Ser Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Thr Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 39
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 40
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 41
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 42
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 43
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 44
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 45
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 46
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 47
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 48
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Ser
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 49
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 50
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Met Asn Cys Lys Ser Ser Gln Thr Val Leu Asn Ser
20 25 30
Gly Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Thr Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 51
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210> 52
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 53
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
caggtgcagc tggtgcagag cggccccgag ctgaagaagc ccggcgccag cgtgaagatc 60
agctgcaagg ccagcggcta cagcttcacc aactacggca tgaactgggt gaggcaggcc 120
cccggccagg gcctgaagtg gatgggctgg atcaacacca acaccggcga gcccacctac 180
gccgaggagt tcaagggcag gttcgtgttc agcctggaca ccagcgtgag caccgcctac 240
ctgcagatca gcagcctgaa ggccgaggac accgccgtgt acttctgcgc caggctgggc 300
ttcggcaacg ccatggacta ctggggccag ggcaccctgg tgaccgtgag cagc 354
<210> 54
<211> 339
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gagggccacc 60
atgaactgca agagcagcca gaccctgctg aacagcggca accagaagaa ctacctgacc 120
tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc caccaccagg 180
gagagcggcg tgcccgacag gttcagcggc agcggcagcg gcaccgactt caccctgacc 240
atcagcagcc tgcaggccga ggacgtggcc gtgtactact gccagaacga ctacagctac 300
cccctgacct tcggcgccgg caccaagctg gagatcaag 339
<210> 55
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
caggtgcagc tggtgcagag cggccccgag ctgaagaagc ccggcgccag cgtgaagatc 60
agctgcaagg ccagcggcta cagcttcacc aactacggca tgaactgggt gaggcaggcc 120
cccggccagg gcctgaagtg gatgggctgg atcaacacca acaccggcga gcccacctac 180
gccgaggagt tcaagggcag gttcgtgttc agcctggaca ccagcgtgag caccgcctac 240
ctgcagatca gcagcctgaa ggccgaggac accgccgtgt acttctgcgc caggctgggc 300
ttcggcaacg ccatggacta ctggggccag ggcaccctgg tgaccgtgag cagc 354
<210> 56
<211> 339
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gagggccacc 60
atgaactgca agagcagcca gaccctgctg aacaccggca accagaagaa ctacctgacc 120
tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc caccaccagg 180
gagagcggcg tgcccgacag gttcagcggc agcggcagcg gcaccgactt caccctgacc 240
atcagcagcc tgcaggccga ggacgtggcc gtgtactact gccagaacga ctacagctac 300
cccctgacct tcggcgccgg caccaagctg gagatcaag 339
<210> 57
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
caggtgcagc tggtgcagag cggccccgag ctgaagaagc ccggcgccag cgtgaagatc 60
agctgcaagg ccagcggcta cagcttcacc aactacggca tgaactgggt gaggcaggcc 120
cccggccagg gcctgaagtg gatgggctgg atcaacacca acaccggcga gcccacctac 180
gccgaggagt tcaagggcag gttcgtgttc agcctggaca ccagcgtgag caccgcctac 240
ctgcagatca gcagcctgaa ggccgaggac accgccgtgt acttctgcgc caggctgggc 300
ttcggcaacg ccatggacta ctggggccag ggcaccctgg tgaccgtgag cagc 354
<210> 58
<211> 339
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gagggccacc 60
atgaactgca agagcagcca gaccctgctg aacagcggca accagaagaa ctacctgacc 120
tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc caccaccagg 180
gagagcggcg tgcccgacag gttcagcggc agcggcagcg gcaccgactt caccctgacc 240
atcagcagcc tgcaggccga ggacgtggcc gtgtactact gccagaacga ctacacctac 300
cccctgacct tcggcgccgg caccaagctg gagatcaag 339
<210> 59
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
caggtgcagc tggtgcagag cggccccgag ctgaagaagc ccggcgccag cgtgaagatc 60
agctgcaagg ccagcggcta cagcttcacc aactacggca tgaactgggt gaggcaggcc 120
cccggccagg gcctgaagtg gatgggctgg atcaacacca acaccggcga gcccacctac 180
gccgaggagt tcaagggcag gttcgtgttc agcctggaca ccagcgtgag caccgcctac 240
ctgcagatca gcagcctgaa ggccgaggac accgccgtgt acttctgcgc caggctgggc 300
ttcggcaacg ccatggacta ctggggccag ggcaccctgg tgaccgtgag cagc 354
<210> 60
<211> 339
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gagggccacc 60
atgaactgca agagcagcca gaccctgctg aacagcggca acaacaagaa ctacctgacc 120
tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc caccaccagg 180
gagagcggcg tgcccgacag gttcagcggc agcggcagcg gcaccgactt caccctgacc 240
atcagcagcc tgcaggccga ggacgtggcc gtgtactact gccagaacga ctacagctac 300
cccctgacct tcggcgccgg caccaagctg gagatcaag 339
<210> 61
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
cagatccagc tggtgcagag cggccccgag ctgaagaagc ccggcgagac cgtgaagatc 60
agctgcaagg ccagcggcta caccttcacc aactacggca tgaactgggt gaagcaggcc 120
cccggcaagg gcctgaagtg gatgggctgg atcaacacca acaccggcga gcccacctac 180
gccgaggagt tcaagggcag gttcgccttc agcctggaga ccagcgccag caccgcctac 240
ctgcagatca acaacctgaa gaacgaggac accgccacct acttctgcgc caggctgggc 300
ttcggcaacg ccatggacta ctggggccag ggcaccagcg tgaccgtgag cagc 354
<210> 62
<211> 339
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gagggccacc 60
atcaactgca agagcagcca gagcctgctg aacagcggca accagaagaa ctacctgacc 120
tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc cagcaccagg 180
gagagcggcg tgcccgacag gttcagcggc agcggcagcg gcaccgactt caccctgacc 240
atcagcagcc tgcaggccga ggacgtggcc gtgtactact gccagaacga ctacagctac 300
cccctgacct tcggcgccgg caccaagctg gagctgaag 339
<210> 63
<211> 990
<212> DNA
<213> Homo sapiens
<400> 63
gccagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc 60
ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc 120
tggaacagcg gcgccctgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 180
ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 240
tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggagccc 300
aagagctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct gctgggcggc 360
cccagcgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatcag caggaccccc 420
gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg 480
tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga gcagtacaac 540
agcacctaca gggtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag 600
gagtacaagt gcaaggtgag caacaaggcc ctgcccgccc ccatcgagaa gaccatcagc 660
aaggccaagg gccagcccag ggagccccag gtgtacaccc tgccccccag cagggacgag 720
ctgaccaaga accaggtgag cctgacctgc ctggtgaagg gcttctaccc cagcgacatc 780
gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccccgtg 840
ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gagcaggtgg 900
cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 960
cagaagagcc tgagcctgag ccccggcaag 990
<210> 64
<211> 321
<212> DNA
<213> Homo sapiens
<400> 64
aggaccgtgg ccgcccccag cgtgttcatc ttccccccca gcgacgagca gctgaagagc 60
ggcaccgcca gcgtggtgtg cctgctgaac aacttctacc ccagggaggc caaggtgcag 120
tggaaggtgg acaacgccct gcagagcggc aacagccagg agagcgtgac cgagcaggac 180
agcaaggaca gcacctacag cctgagcagc accctgaccc tgagcaaggc cgactacgag 240
aagcacaagg tgtacgcctg cgaggtgacc caccagggcc tgagcagccc cgtgaccaag 300
agcttcaaca ggggcgagtg c 321

Claims (11)

1. A humanized antibody that binds claudin18.2, said antibody comprising a heavy chain hypervariable region (HCDR) and a light chain hypervariable region (LCDR), said heavy chain hypervariable region comprising:
the amino acid sequence is GYX1HCDR1 for FTNYG, HCDR2 for the amino acid sequence INTNTGEP, and HCDR3 for the amino acid sequence ARLGFGNAMDY; the light chain hypervariable region comprises:
the amino acid sequence is QX2X3LNX4X5NX6LCDR1 of KNY and the amino acid sequence is WAX7LCDR2 and amino acid sequence QX8DYX9LCDR3 of YPLT (Localx),
wherein amino acid X in the HCDR11Selected from S or T;
amino acid X in the LCDR12Selected from T or S, X3Selected from L or V, X4Selected from T or S, said X5Selected from G or S, X6Selected from Q or N;
amino acid X in the LCDR27Selected from T or S;
amino acid X in the LCDR38Selected from N or Q, X9Selected from T or S.
2. The humanized antibody of claim 1, wherein the antibody comprises a heavy chain hypervariable region and a light chain hypervariable region wherein the heavy chain hypervariable region comprises CDR sequences,
the amino acid sequence is GYSFTNYG (SEQ ID NO: 1), the CDR1(HCDR1) of GYTFTNYG (SEQ ID NO: 2); CDR2(HCDR2) having the amino acid sequence INTNTGEP (SEQ ID NO: 3);
CDR3(HCDR3) having amino acid sequence ARLGFGNAMDY (SEQ ID NO: 4);
the light chain hypervariable region comprises the CDR sequences,
CDRs 1(LCDR1) having amino acid sequences of QTLLNTGNQKNY (SEQ ID NO:5), QSVLNSGNQKNY (SEQ ID NO:6), QSLLNSSNNKNY (SEQ ID NO:7), QSVLNSSNNKNY (SEQ ID NO:8), QTLLNSGNQKNY (SEQ ID NO:9), QTLLNSGNNKNY (SEQ ID NO:10), QTLLNSSNQKNY (SEQ ID NO:11), QTLLNSSNNKNY (SEQ ID NO:12), QSLLNSSNQKNY (SEQ ID NO:13), QSLLNSGNNKNY (SEQ ID NO:14), QSLLNSGNQKNY (SEQ ID NO:15), QSVLNSSNQKNY (SEQ ID NO:16), QSVLNSGNNKNY (SEQ ID NO:17), QTVLNSGNNKNY (SEQ ID NO: 18);
the amino acid sequence is WAT (SEQ ID NO: 19), CDR2(LCDR2) of WAS (SEQ ID NO: 20); CDR3(LCDR3) having an amino acid sequence of QNDYTYPLT (SEQ ID NO:21), QQDYTYPLT (SEQ ID NO:22), QQDYSYPLT (SEQ ID NO:23), QNDYSYPLT (SEQ ID NO: 24).
3. The humanized antibody of claim 2, comprising any set of heavy chain hypervariable regions and light chain hypervariable regions:
(1) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 5, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 21;
(2) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 22;
(3) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 7, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 21;
(4) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 8, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 23; (5) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 9, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24;
(6) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 5, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24;
(7) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 9, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 21;
(8) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 10, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24;
(9) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 11, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24;
(10) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 12, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24;
(11) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24;
(12) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 13, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24;
(13) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 14, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24;
(14) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 15, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 23;
(15) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 7, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24;
(16) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 13, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 23;
(17) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 16, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24;
(18) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 14, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 23;
(19) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 17, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24;
(20) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 8, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 24;
(21) HCDR1 of SEQ ID NO. 2, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 7, LCDR2 of SEQ ID NO. 20, LCDR3 of SEQ ID NO. 23;
(22) HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 3, HCDR3 of SEQ ID NO. 4, and LCDR1 of SEQ ID NO. 18, LCDR2 of SEQ ID NO. 19, LCDR3 of SEQ ID NO. 24; or
(23) HCDR1 of SEQ ID NO. 17, HCDR2 of SEQ ID NO. 32, HCDR3 of SEQ ID NO. 54, and LCDR1 of SEQ ID NO. 61, LCDR2 of SEQ ID NO. 63, LCDR3 of SEQ ID NO. 66.
4. The humanized antibody of any one of claims 1-3, wherein the antibody has heavy chain variable region (VH) and light chain variable region (VL) sequences as follows:
the amino acid sequence is QVQLVQSGXa1ELKKPGASVKISCKASZH1MNWVRQAPGQGLKWMGZH2TYAEEFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYXa2CZH3VH shown by WGQGTLVTVSS; and
the amino acid sequence is DIVMTQSPDSLAVSLGERATMCKSSZL1LTWYQQKPGQPPKLLIYZL2T RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCZL3FGAGTKLEIK is shown in the form of a VL,
wherein Xa1Is P or S, Xa2Is F or Y, ZH1、ZH2And ZH3Three CDR regions of the heavy chain, ZL1、ZL2And ZL3Three CDR regions of the light chain,
wherein ZH1The amino acid sequence of (A) is GYX1FTNYG，ZH2The amino acid sequence of (A) is INTNTGEP, ZH3ARLGFGNAMDY; the amino acid sequence of ZL1 is QX2X3LNX4X5NX6The amino acid sequence of KNY, ZL2 is WAX7, and the amino acid sequence of ZL3 is QX8DYX9YPLT；
X1Selected from S orT；
X2Selected from T or S; x3Is selected from L or V; x4Selected from T or S; x5Selected from G or S; x6Selected from Q or N; x7Selected from T or S; x8Is selected from N or Q; x9Selected from T or S.
5. The humanized antibody of claim 4, wherein the antibody has VH and VL sequences as described below,
the amino acid sequence is SEQ ID NO: 25. 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 51, and
the amino acid sequence is SEQ ID NO: 26. 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52.
6. The humanized antibody of claim 5, wherein the antibody has VH and VL sequences of any one of the following groups,
SEQ ID NO: 25, and the amino acid sequence of SEQ ID NO: 26 VL of an amino acid sequence set forth in seq id no; SEQ ID NO: 27, and the VH of the amino acid sequence shown in SEQ ID NO: 28, VL of the amino acid sequence set forth in seq id no; SEQ ID NO: 29, and the VH of the amino acid sequence shown in SEQ ID NO: 30 VL of an amino acid sequence set forth in seq id no; SEQ ID NO: 31, and the VH of the amino acid sequence shown in SEQ ID NO:32, VL of an amino acid sequence set forth in seq id no; SEQ ID NO: 33, and the VH of the amino acid sequence shown in SEQ ID NO: 34 VL of the amino acid sequence set forth in seq id no; SEQ ID NO: 35, and the VH of the amino acid sequence shown in SEQ ID NO: 36, VL of an amino acid sequence set forth in seq id no; SEQ ID NO: 37, and the amino acid sequence of SEQ ID NO: 38, VL of an amino acid sequence set forth in seq id no; SEQ ID NO: 39, and the VH of the amino acid sequence shown in SEQ ID NO: 40, VL of an amino acid sequence set forth in seq id no; SEQ ID NO: 41, and the amino acid sequence of SEQ ID NO: 42, VL of an amino acid sequence set forth in seq id no; SEQ ID NO: 43, and the VH of the amino acid sequence shown in SEQ ID NO: 44, VL of an amino acid sequence set forth in seq id no; SEQ ID NO: 45, and the amino acid sequence of SEQ ID NO: 46, VL of an amino acid sequence set forth in seq id no; SEQ ID NO: 47, and the amino acid sequence of SEQ ID NO: 48, VL of an amino acid sequence set forth in seq id no; SEQ ID NO: 49, and the VH of the amino acid sequence shown in SEQ ID NO: 50, VL of an amino acid sequence set forth in seq id no.
7. The humanized antibody of any of claims 1-6, 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.
8. An isolated nucleic acid molecule encoding the humanized antibody of any one of claims 1-7.
9. A pharmaceutical composition comprising a humanized antibody according to any of claims 1-7, and a pharmaceutically acceptable excipient.
10. Use of a humanized antibody according to any of claims 1 to 7 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.
11. An article of manufacture or kit comprising a container comprising the humanized antibody of any one of claims 1-7, or the pharmaceutical composition of claim 9, and a package insert carrying instructions for use of the medicament.
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