CN116253800A

CN116253800A - Heavy chain antibody against CLDN18.2, related products and uses

Info

Publication number: CN116253800A
Application number: CN202211525518.8A
Authority: CN
Inventors: 李嘉涛; 狄升蒙; 余学军; 张世伟; 李照润; 范艳秋; 马恒; 陈鑫; 冯冬歌
Original assignee: Huadao Shanghai Biopharma Co ltd
Current assignee: Huadao Shanghai Biopharma Co ltd
Priority date: 2022-11-30
Filing date: 2022-11-30
Publication date: 2023-06-13
Anticipated expiration: 2042-11-30
Also published as: CN116253800B; CN118005794A; CN118184780A; CN117924500A; CN117903310A; CN117924501A

Abstract

The application discloses a heavy chain antibody against CLDN18.2, related products and uses. The heavy chain antibody consists of a heavy chain variable region, and the amino acid sequence of CDR1 of the heavy chain variable region is shown as one of SEQ ID No.1-SEQ ID No. 6; the amino acid sequence of CDR2 of the heavy chain variable region is shown as one of SEQ ID No.7-SEQ ID No. 12; the amino acid sequence of CDR3 of the heavy chain variable region is shown as one of SEQ ID No.13-SEQ ID No. 18. The heavy chain antibody for resisting the CLDN18.2 only comprises a heavy chain variable region, has high affinity and specificity, can efficiently target the CLDN18.2 antigen without recognizing the CLDN18.1, has a simple structure, and is easy to prepare. In addition, the CAR-T cells prepared by the method can specifically identify CLDN18.2 positive tumor cells, and simultaneously can release various cytokines to kill the tumor cells with high efficiency.

Description

Heavy chain antibody against CLDN18.2, related products and uses

Technical Field

The application relates to the technical field of biological medicine, in particular to a heavy chain antibody for resisting CLDN18.2, a related product and application.

Background

Heavy chain antibodies (heavy chain antibody, hcAb), which are found in camels and shark animals, are antibodies that naturally lack light chains, consisting of only heavy chains. Cloning of the variable region results in a single domain antibody consisting of only the heavy chain variable region, known as VHH (Variable domain of heavy chain of heavy chain antibody), also known as nanobody (nanobody), which is the smallest functional antigen-binding fragment. Unlike common antibody, nanometer antibody is one peptide chain of about 110 amino acids and has molecular weight of about 1/10 that of common antibody, and has the advantages of small size, high stability, high solubility, easy expression, low production cost, etc. compared with common antibody and recombinant single chain antibody (single chain fragment variable, scFv), the nanometer antibody has wide application foreground in immunological experiment, diagnosis and treatment.

The Claudins (CLDNs) family has been found to be expressed in a variety of epithelial tissues and plays a central role in the close association between cells. Tens of members of the CLDNs family are transmembrane proteins, with a very high degree of identity within the transmembrane region. The CLDNs family plays an important role in maintaining the osmotic pressure of epithelial cells, protecting barrier effect and the like, and participates in the immune defense process; in addition, the CLDNs family was observed to express activities that are different from usual in the course of many tumor development and development, and was studied and discussed in various borders as tumor-specific marker proteins. CLDN18.2 is one member of CLDNs family, and has very low expression level in normal tissues, but studies find that it is abnormal to activate in the process of occurrence and development of primary malignant tumors of different organs and tissues of human body such as stomach, breast, bronchus, etc., and has over-expression level, especially high in digestive system malignant tumors. Therefore, CLDN18.2 is expected to be a new target for first-line treatment of gastric cancer and the like due to the characteristic of high selectivity and stable expression in malignant solid tumors such as gastric cancer and the like.

One of the difficulties in CLDN18.2 targeted therapies is the presence of a protein that is very similar in structure to CLDN18.1, another cleavage entity formed by the allele of the first exon of the CLDN18 gene. CLDN18.1 has a high degree of similarity to the structural sequence of CLDN18.2, differing only by 7 amino acid residues located on the ectodomain ECL1 sequence. CLDN18.1 is highly expressed only in normal alveolar tissue, with very high specificity. Most antibodies developed by researchers and practitioners that target CLDN18.2 are also often prone to off-target recognition of CLDN18.1, thus severely compromising the targeting and safety of antibody drugs. Therefore, development of antibody drugs specifically recognizing CLDN18.2 but not CLDN18.1 is a difficulty in the development process.

In view of the above, developing efficient CAR-T therapies against digestive system malignancies is of great importance in the field of digestive system malignancies.

Disclosure of Invention

In view of the above-described drawbacks of the prior art, it is an object of the present application to provide a heavy chain antibody against CLDN18.2, related products and uses for solving the problems of the prior art.

In order to achieve the above purpose, the present invention adopts the following technical scheme.

In a first aspect, the present application provides an anti-CLDN 18.2 heavy chain antibody consisting of a heavy chain variable region having the amino acid sequence of CDR1 as set forth in one of SEQ ID nos. 1-6; the amino acid sequence of CDR2 of the heavy chain variable region is shown as one of SEQ ID No.7-SEQ ID No. 12; the amino acid sequence of CDR3 of the heavy chain variable region is shown as one of SEQ ID No.13-SEQ ID No. 18.

In certain embodiments, the amino acid sequence of the heavy chain variable region comprises A1) or A2):

a1 An amino acid sequence as shown in one of SEQ ID No.38-SEQ ID No. 43;

a2 An amino acid sequence having at least 80% homology with the amino acid sequence shown in A1) and having the function of the amino acid sequence defined in A1).

In certain embodiments, the heavy chain variable region further comprises framework regions FR1-FR4: the amino acid sequence of the framework region FR1 comprises a sequence shown as one of SEQ ID No.19-SEQ ID No.23, the amino acid sequence of the framework region FR2 comprises a sequence shown as one of SEQ ID No.24-SEQ ID No.29, the amino acid sequence of the framework region FR3 comprises a sequence shown as one of SEQ ID No.30-SEQ ID No.35, and the amino acid sequence of the framework region FR4 comprises a sequence shown as one of SEQ ID No.36 or SEQ ID No. 37.

In a second aspect the present application provides an isolated polypeptide comprising an antigen binding domain comprising a heavy chain antibody as described above, a hinge region and a transmembrane domain.

In certain embodiments, the polypeptide is a chimeric antigen receptor.

In certain embodiments, the transmembrane domain comprises one or more of a CD8 a transmembrane region, a CD28 transmembrane region, or a DAP10 transmembrane region.

In certain embodiments, the hinge region comprises a CD8 a hinge region.

In certain embodiments, the polypeptide comprises, in order from the N-terminus to the C-terminus, the heavy chain antibody, a hinge region, and a transmembrane domain.

A third aspect of the present application provides a biological material associated with a heavy chain antibody as described above or a polypeptide as described above, the biological material comprising one or more of the following:

1) A nucleotide encoding a heavy chain antibody as described above or a nucleotide encoding a polypeptide as described above;

2) A recombinant expression vector comprising 1) said nucleotide;

3) The bioengineering bacteria containing 1) the nucleotide or the bioengineering bacteria containing 2) the recombinant expression vector.

In certain embodiments, in 1), the sequence of the nucleotide comprises a sequence as set forth in one of SEQ ID No.44-SEQ ID No. 49.

In certain embodiments, the recombinant expression vector is selected from a lentiviral vector, a retroviral vector, or an adenoviral vector.

Preferably, the recombinant expression vector is a lentiviral vector.

In a fourth aspect the present application provides a cell expressing a polypeptide as described above.

In certain embodiments, the cells are selected from one or more of T lymphocytes, B lymphocytes, NK cells, mast cells, and macrophages.

A fifth aspect of the present application provides the use of a heavy chain antibody as described above or a polypeptide as described above or a biological material as described above or a cell as described above in the manufacture of an in vitro assay product, in the manufacture of a medicament for the prevention or treatment of a tumor.

In certain embodiments, the tumor is a tumor associated with expression of CLND 18.2, preferably selected from one or more of bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, and thyroid cancer.

The sixth aspect of the present application provides a test product comprising an antibody as described above or a polypeptide as described above or a biological material as described above or a cell as described above.

In certain embodiments, the product is used to detect CLND 18.2.

A seventh aspect of the present application provides a pharmaceutical composition comprising an antibody as described above or a polypeptide as described above or a biological material as described above or a cell as described above, and a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition is for preventing or treating a neoplastic disease associated with expression of CLND 18.2.

Preferably, the tumor associated with expression of CLND 18.2 is selected from one or more of bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, and thyroid cancer.

Compared with the prior art, the beneficial effects of this application are:

(1) The heavy chain antibody for resisting the CLDN18.2 only comprises a heavy chain variable region, has high affinity and specificity, can efficiently target the CLDN18.2 antigen, and has a simple structure and easy preparation.

(2) The invention constructs chimeric antigen receptor by using heavy chain antibody of anti-CLDN 18.2, and the chimeric antigen receptor can efficiently target CLDN18.2 without recognizing CLDN18.1.

(3) The chimeric antigen receptor cell of the invention can specifically identify CLDN18.2 positive tumor cells, perform high-efficiency killing, and simultaneously release various cytokines including TNF-alpha, IFN-gamma factors and the like to play a role in cell killing.

Drawings

FIG. 1A shows a graph of the affinity of the anti-CLDN 18.2-A93 VHH antibody detected using Biacore in example 2 of the present invention.

FIG. 1B shows a graph of the affinity of the anti-CLDN 18.2-A100 VHH antibody detected using Biacore in example 2 of the present invention.

FIG. 1C shows a graph of the affinity of the anti-CLDN 18.2-A102 VHH antibody detected using Biacore in example 2 of the present invention.

FIG. 1D shows a graph of the affinity of the anti-CLDN 18.2-A127 VHH antibody detected using Biacore in example 2 of the present invention.

FIG. 1E shows a graph of the affinity of the anti-CLDN 18.2-B123 VHH antibody detected using Biacore in example 2 of the present invention.

FIG. 1F shows a graph of the affinity of the anti-CLDN 18.2-B142 VHH antibody detected using Biacore in example 2 of the present invention.

FIG. 2A is a graph showing the results of FACS detection of the 6 anti-CLDN 18.2 VHH antibodies recognizing the CLDN18.2 antigen in example 3 of the present invention.

FIG. 2B is a graph showing the results of FACS detection of the 6 anti-CLDN 18.2 VHH antibodies recognizing the CLDN18.1 antigen in example 3 of the present invention.

FIG. 3A shows a plasmid map of a lentiviral vector (HD SIN03A93 41 BBz) expressing a chimeric antigen receptor of CLDN18.2 in example 4 of the invention.

FIG. 3B shows a plasmid map of a lentiviral vector (HD SIN03A100 41 BBz) expressing a chimeric antigen receptor of CLDN18.2 in example 4 of the invention.

FIG. 3C shows a plasmid map of a lentiviral vector (HD SIN03A102 41 BBz) expressing a chimeric antigen receptor of CLDN18.2 in example 4 of the invention.

FIG. 3D shows a plasmid map of a lentiviral vector (HD SIN03A127 41 BBz) expressing a chimeric antigen receptor of CLDN18.2 in example 4 of the invention.

FIG. 3E shows a plasmid map of a lentiviral vector (HD SIN03B123 41 BBz) expressing a chimeric antigen receptor of CLDN18.2 in example 4 of the invention.

FIG. 3F shows a plasmid map of a lentiviral vector (HD SIN03B142 41 BBz) expressing a chimeric antigen receptor of CLDN18.2 according to example 4 of the invention.

FIG. 4 shows a schematic structural diagram of a chimeric antigen receptor expressing an anti-CLDN 18.2 antibody in example 5 of the present invention.

FIG. 5 is a graph showing the expression rate of chimeric antigen receptor of T lymphocytes in example 9 of the present invention.

Figure 6A shows a graph of the killing effect of 6 CAR-T cells on 293T cells in example 10 of the present invention.

FIG. 6B is a graph showing the killing effect of 6 CAR-T cells on HGC-27-18.2 cells in example 10 of the present invention.

FIG. 6C is a graph showing the killing effect of 6 CAR-T cells on N87-18.2 cells in example 10 of the present invention.

FIG. 7 is a bar graph showing the levels of TNF- α cytokine secretion by 6 CAR-T cells in example 11 of the present invention.

FIG. 8 shows a bar graph of IFN-gamma cytokine secretion levels by 6 CAR-T cells in example 11 of the present invention.

FIG. 9 shows a map of HD SIN03 CD19 CAR-Kanar plasmid in example 4 of the invention.

Detailed Description

The invention adopts CHO cells expressing CLDN18.2 protein to stimulate alpaca to produce high-titer antibody, and adopts phage display technology to construct the alpaca with the reservoir capacity larger than 10 ⁹ 6 candidate antibodies including CLDN18.2-a93, CLDN18.2-a100, CLDN18.2-a102, CLDN18.2-a127, CLDN18.2-B123 and CLDN18.2-B142 are obtained by panning amplification of phage display camel VHH immune library. The reduction molecular weight is found to be less than 40Kda, which is 1/10 of that of the common antibody; through Biacore detection, the affinities of the 6 candidate antibodies are found to reach the nM level; it was further found that the 6 candidate antibodies specifically recognize CLDN18.2 antigen on the cell surface, but not CLDN18.1 antigen. Meanwhile, CAR-T cells containing 6 candidate antibodies are constructed, and the CAR-T cells are found to have killing activity on CLDN18.2 positive tumor cells and can secrete cytokines TNF-alpha and IFN-gamma with high efficiency. The present invention has been completed on the basis of this finding.

In a first aspect, the present application provides an anti-CLDN 18.2 heavy chain antibody, which consists of a heavy chain variable region, wherein the amino acid sequence of CDR1 of the heavy chain variable region is as shown in one of SEQ ID nos. 1-6; the amino acid sequence of CDR2 of the heavy chain variable region is shown as one of SEQ ID No.7-SEQ ID No. 12; the amino acid sequence of CDR3 of the heavy chain variable region is shown as one of SEQ ID No.13-SEQ ID No. 18.

The heavy chain antibody is composed of a heavy chain variable region, and has the advantages of small molecular weight, high affinity, weak immunogenicity to human bodies, easier storage and transportation, easier expression, genetic engineering modification and the like.

SEQ ID No.1:GRTFSSYH

SEQ ID No.2:GSIAHIAGINV

SEQ ID No.3:RSILSFSV

SEQ ID No.4:GRIFRINN

SEQ ID No.5:GRTFGNYN

SEQ ID No.6:GFTLDYYS

SEQ ID No.7:ISWSGGIR

SEQ ID No.8:IFYSGTT

SEQ ID No.9:IFNGGHT

SEQ ID No.10:VSLGGTT

SEQ ID No.11:ISWSGSIT

SEQ ID No.12:ISGSGVST

SEQ ID No.13:AAAGVTLSRNEGDYAY

SEQ ID No.14:YANHNIANY

SEQ ID No.15:NANDLGFLAQNY

SEQ ID No.16:NADVYETPFSSKNY

SEQ ID No.17:AAASFDLTRDASRYAY

SEQ ID No.18:IARPIGNSWYCSIAYTDFGS

In the invention, the amino acid sequence of CDR1 of the heavy chain variable region comprises a sequence shown as SEQ ID No.1, the amino acid sequence of CDR2 comprises a sequence shown as SEQ ID No.7, and the amino acid sequence of CDR3 comprises a sequence shown as SEQ ID No. 13.

In the present invention, the amino acid sequence of CDR1 of the heavy chain variable region comprises the sequence shown in SEQ ID No.2, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.8, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 14.

In the invention, the amino acid sequence of CDR1 of the heavy chain variable region comprises a sequence shown as SEQ ID No.3, the amino acid sequence of CDR2 comprises a sequence shown as SEQ ID No.9, and the amino acid sequence of CDR3 comprises a sequence shown as SEQ ID No. 15.

In the present invention, the amino acid sequence of CDR1 of the heavy chain variable region comprises the sequence shown in SEQ ID No.4, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.10, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 16.

In the present invention, the amino acid sequence of CDR1 of the heavy chain variable region comprises the sequence shown in SEQ ID No.5, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.11, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 17.

In the present invention, the amino acid sequence of CDR1 of the heavy chain variable region comprises the sequence shown in SEQ ID No.6, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.12, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 18.

In the present invention, the heavy chain variable region comprises framework regions FR1-FR4. Preferably, the amino acid sequence of FR1 comprises the sequence shown as SEQ ID No.19 or SEQ ID No.20 or SEQ ID No.21 or SEQ ID No.22 or SEQ ID No. 23. Preferably, the amino acid sequence of FR2 comprises the sequence shown as SEQ ID No.24 or SEQ ID No.25 or SEQ ID No.26 or SEQ ID No.27 or SEQ ID No.28 or SEQ ID No. 29. Preferably, the amino acid sequence of FR3 comprises the sequence shown as SEQ ID No.30 or SEQ ID No.31 or SEQ ID No.32 or SEQ ID No.33 or SEQ ID No.34 or SEQ ID No. 35. Preferably, the amino acid sequence of FR4 comprises the sequence shown as SEQ ID No.36 or SEQ ID No. 37.

SEQ ID No.19:QVQLVESGGGLVQVGGSLRLSCAAS

SEQ ID No.20:EVQLVESGGGLVQPGGSLRLSCAAS

SEQ ID No.21:EVQVVESGGGTVQPGGSLRLSCATS

SEQ ID No.22:QVQLVESGGGSVQPGGSLRLSCAAS

SEQ ID No.23:QLQLVESGGGLVQPGGSLRLSCAAS

SEQ ID No.24:MGWFRQAPGKEREFVAA

SEQ ID No.25:MGWYRQTPGKQRDFVAH

SEQ ID No.26:MGWYRQAPGNQREWVAH

SEQ ID No.27:MYWYRQAAGKQREFVAG

SEQ ID No.28:MGWFRQAPGKEREFVAG

SEQ ID No.29:IAWFRQAPGKEREGVSC

SEQ ID No.30:DYTSSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC

SEQ ID No.31:TYADSVKGRFIISRDSATNTMSLQMNNLKPEDTATYYC

SEQ ID No.32:NYADSVKGRATISRDNAKNTVYLQMSSLKPEDTAVYYC

SEQ ID No.33:NAADSVKGRFTVSRDNAKNTMYLQMSAMKPDDTAVYYC

SEQ ID No.34:DYGDSVKGRFTISRDNAENTLYLQMNNLKPEDTAVYFC

SEQ ID No.35:KYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYC

SEQ ID No.36:WGQGTQVTSSS

SEQ ID No.37:WGQGTQVTVSS

In the invention, the amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.19, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.24, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.30, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 36.

In the invention, the amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.20, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.25, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.31, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 37.

In the invention, the amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.21, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.26, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.32, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 37.

In the invention, the amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.22, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.27, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.33, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 37.

In the invention, the amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.23, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.28, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.34, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 37.

In the invention, the amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.23, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.29, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.35, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 37.

In the present invention, the amino acid sequence of the heavy chain variable region comprises A1) or A2): a1 An amino acid sequence as shown in one of SEQ ID No.38-SEQ ID No. 43; a2 An amino acid sequence having at least 80% homology with the amino acid sequence shown in A1) and having the function of the amino acid sequence defined in A1). Specific sequences of SEQ ID No.38, SEQ ID No.39, SEQ ID No.40, SEQ ID No.41, SEQ ID No.42, SEQ ID No.43 are shown below corresponding to CLDN18.2-A93, CLDN18.2-A100, CLDN18.2-A102, CLDN18.2-A127, anti-CLDN 18.2-B123 and CLDN18.2-B142, respectively.

SEQ ID No.38 (italics in bold represents the framework region,underline lineCDR regions):

SEQ ID No.39 (italics in bold represents the framework region,underline lineCDR regions):

SEQ ID No.40 (italics in bold represents the framework region,underline lineCDR regions):

SEQ ID No.41 (italics in bold represents the framework region,underline lineCDR regions):

SEQ ID No.42 (italics in bold represents the framework region, Underline lineCDR regions):

SEQ ID No.43 (italics in bold represents the framework region,underline lineCDR regions):

in the invention, the preparation method of the heavy chain antibody comprises the following steps: inserting the coding gene of the heavy chain antibody into an expression vector to obtain a recombinant expression vector, introducing the recombinant expression vector into cells, culturing, and separating and purifying to obtain the heavy chain antibody.

Another aspect of the present application provides an isolated polypeptide comprising an antigen binding domain comprising a heavy chain antibody as described above, a hinge region, and a transmembrane domain.

In the invention, the heavy chain antibody is utilized to construct the polypeptide, and the polypeptide can efficiently target CLDN18.2.

In the present invention, the hinge region comprises a CD8 a hinge region.

In the present invention, the transmembrane domain comprises one or more of a CD8 a transmembrane region, a CD28 transmembrane region, or a DAP10 transmembrane region.

In the present invention, the polypeptide further comprises a signal peptide or a signal transduction domain. Preferably, the signal peptide comprises a CD8 a signal peptide. Preferably, the signal transduction domain comprises an immunoreceptor tyrosine activation motif. More preferably, the signal transduction domain further comprises a co-stimulatory molecule comprising any one or a combination of at least two of 4-1BB, the intracellular domain of CD28, OX40, ICOS or the intracellular domain of DAP 10. In certain embodiments, the amino acid sequence of the signal peptide comprises the sequence set forth in SEQ ID No. 50. In certain embodiments, the amino acid sequence of the immunoreceptor tyrosine activation motif comprises the sequence depicted in SEQ ID No. 52.

MALPVTALLLPLALLLHAARP(SEQ ID No.50)。

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQID No.52)。

In the present invention, the polypeptides include a CD8 a signal peptide, the heavy chain antibody, a CD8 a hinge region, a CD8 a transmembrane region, and an immunoreceptor tyrosine activation motif. The amino acid sequences of the CD8 alpha hinge region and the transmembrane region are

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID No.51)。

Another aspect of the present application relates to a heavy chain antibody as described above or a polypeptide as described above, said biological material comprising one or more of the following:

2) A recombinant expression vector comprising 1) said nucleotide;

In the present invention, 1), the sequence of the nucleotide encoding the heavy chain antibody as described above comprises a sequence as shown in one of SEQ ID No.44 to SEQ ID No. 49.

SEQ ID No.44:

caggtgcagctggtggagagcggggggggcctggtgcaggtgggcgggagcctgaggctgagctgcgccgccagcggcaggacattcagcagctaccacatggggtggttccggcaggcccccggcaaggagagggagttcgtggccgccatcagctggagcgggggcatcagggactacacaagcagcgtgaagggcaggttcaccatcagccgggacaacgccaaaaacaccgtgtacctgcaaatgaacagcctcaagccagaggacaccgccgtgtactactgtgccgccgctggcgtcactctgtccaggaacgagggggattacgcttattgggggcagggcacacaggtgacaagcagcagc

SEQ ID No.45:

gaggtgcagctggtggagagcggggggggcctggtgcagcccggcggcagcctgaggctgagctgcgccgccagcgggagcatcgcccacatcgccgggatcaacgtgatggggtggtaccggcagacccccggcaagcagagggacttcgtggcccacatcttttacagcggcaccacaacatacgccgatagcgtgaagggcaggttcatcatcagccgggatagcgccaccaacaccatgagcctgcagatgaacaatctgaagcccgaggacacagccacatactactgctacgccaaccacaacatcgccaactattgggggcagggcacacaggtgaccgtgtcctcc

SEQ ID No.46:

gaagtgcaggtggtggagagcggcggcggcacagtgcagcccggcggcagcctgaggctgagctgcgccacaagccggagcatcctgagcttcagcgtgatggggtggtacaggcaggcccccggcaaccagcgggagtgggtggcccacatcttcaacggggggcacacaaactacgccgatagcgtgaaggggagggccaccatcagccgggataacgccaagaacacagtgtacctgcagatgagcagcctgaagcccgaggacaccgccgtgtactactgcaacgccaacgacctgggcttcctggcccagaactactgggggcaggggacacaggtgacagtgagcagc

SEQ ID No.47:

caggtgcagctggtggagagcggcggcgggagcgtgcagcccggcgggagcctgaggctgagctgcgccgcctccggccggatcttccgcattaacaacatgtactggtacaggcaggccgccgggaagcagcgggagttcgtggccggggtgagcctgggcggcaccaccaacgccgccgatagcgtgaagggccggttcacagtgagcagggataacgccaagaacaccatgtacctgcagatgagcgccatgaagcctgatgacaccgccgtctactactgtaacgctgacgtctatgagaccccctttagtagtaagaactactggggccagggcacacaggtgaccgtgagtagcSEQ ID No.48:

cagctgcagctggtggagagcggcgggggcctggtacagcccggcggcagcctccggctgtcctgcgccgccagcgggagaacatttgggaactacaatatggggtggtttaggcaggcccctggcaaggagcgggagttcgtcgccgggatcagctggagcggcagcatcaccgattacggcgactccgtgaaggggaggttcaccatcagccgggataacgccgagaacacactgtacctgcagatgaacaacctgaagcccgaggatacagccgtgtacttctgcgccgccgccagcttcgacctgaccagggacgccagcaggtacgcctactggggccagggcacacaggtgacagtgagcagc

SEQ ID No.49:

cagctgcagctggtggagagcgggggcggcctggtgcagcccggcgggagcctgaggctgagctgcgccgccagcgggttcaccctggactactactccatcgcctggttcaggcaggcccccggcaaggagcgggagggggtgagctgcatctccggcagcggggtgagcacaaagtacgccgatagcgtgaagggccggttcaccatcagcagggataacgccaagaacacagtgtacctgcagatgaacagcctgaagcccgaggatacagccatctactactgcatcgcccggcccatcgggaacagctggtactgcagcatcgcctacaccgacttcggcagctggggccaggggacacaggtgacagtgagcagc

In the present invention, 2) the recombinant expression vector comprises the nucleotide described in 1). The recombinant expression vector is a viral vector or a non-viral vector. For example, non-viral vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC), phages such as lambda phage or M13 phage, animal viruses, and the like. The viral vector comprises: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-vacuolated viruses (e.g., SV 40). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. The vector may also include components that assist in its entry into the cell, including, but not limited to, viral particles, liposomes, or protein shells. Preferably, the recombinant expression vector is selected from lentiviral vectors, retroviral vectors or adenoviral vectors, more preferably lentiviral vectors.

In the invention, 3) the recombinant expression vector of the bioengineering bacterium 1). Preferably, the bioengineering bacteria are prepared by transfecting mammalian cells, such as 293T cells, with the recombinant expression vector and helper plasmid.

Another aspect of the present application provides a cell expressing a polypeptide as described above.

In the present invention, the cells are selected from one or more of T lymphocytes, B lymphocytes, NK cells, mast cells and macrophages.

The invention utilizes the heavy chain antibody to prepare a chimeric antigen receptor and further prepares chimeric antigen receptor immune cells (CAR-T cells), wherein the chimeric antigen receptor immune cells can specifically identify CLDN18.2 positive tumor cells and kill the tumor cells with high efficiency, and simultaneously can release various cytokines including TNF-alpha, IFN-gamma factors and the like to play a role in cell killing.

Another aspect of the present application provides the use of a heavy chain antibody as described above or a polypeptide as described above or a biological material as described above or a cell as described above in the preparation of an in vitro assay product, in the preparation of a medicament for the prevention or treatment of a tumor.

Another aspect of the present application provides an assay product comprising a heavy chain antibody as described above or a polypeptide as described above or a biological material as described above or a cell as described above.

In the present invention, the product is used to detect CLND 18.2.

In the present invention, the detection product is selected from the group consisting of a kit, a chip and a membrane strip.

In the present invention, the detection product can be generally diagnosed against the CLND18.2 antigen serving as an action target, and CLND18.2 antigen is used as a biomarker. The product may also include a label against CLND18.2 antibodies, which may generally be used to label the anti-CLND 18.2 antibodies, optionally in the form of one or more of biotin, fluorescein, horseradish peroxidase, magnetic beads and agarose beads.

Another aspect of the present application provides a pharmaceutical composition comprising an antibody as described above or a polypeptide as described above or a biological material as described above or a cell as described above, and a pharmaceutically acceptable carrier.

In the present invention, the pharmaceutical composition is used for preventing or treating a tumor disease associated with CLND18.2 expression. Preferably, the tumor comprises a tumor expressing CLND18.2 antigen, non-limiting examples of cancer include bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, and thyroid cancer.

In the present invention, the pharmaceutically acceptable carrier refers to a carrier that does not cause allergic reactions or other untoward effects in the patient to whom it is administered. Pharmaceutically acceptable carriers include, for example, one or more of water, physiological saline, phosphate buffer, dextrose, glycerol, ethanol, and the like, as well as combinations of the foregoing.

In the present invention, the pharmaceutically acceptable carrier may further include a trace amount of auxiliary substances, such as a wetting or emulsifying agent, a preservative or a buffer, which can improve the shelf life or effect of the antibody.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Example 1 acquisition of heavy chain antibodies against CLDN18.2

In this example, a phage display technique was used to panning a CLDN18.2 immunized alpaca VHH immune library and screening by ELISA, thereby obtaining a high affinity heavy chain antibody against CLDN 18.2. The method comprises the following steps:

1.1 construction of phage nanobody library

Immunizing alpaca by CHO-Claudin 18.2 cells expressing CLDN18.2, and extracting peripheral blood after ELISA detection of serum titer; separating lymphocytes to obtain peripheral blood mononuclear lymphocyte precipitate, extracting total RNA, performing reverse transcription by taking the RNA as a template to obtain first-strand cDNA, and amplifying VHH genes by nest PCR; inserting VHH gene into pMECS phage display vector, electrically transforming TG1 competent cells, collecting bacterial liquid, performing library identification, uniformly coating all the rest cultures on LB/AMPGLU plate, collecting lawn after bacteria grow out, adding 1/3 volume of 50% glycerol, mixing, packaging, and preserving at-80deg.C to successfully construct a stock capacity of more than 10 ⁹ Is a phage display camelid VHH immune library.

1.2 panning and amplification of phage nanobody libraries

293T cells overexpressing the CLDN18.2 protein (293T-CLDN 18.2) were blocked with 500. Mu.L of 3% BSA.

Phage display camel VHH immune library constructed in step 1.1 was diluted with 5% serum-PBS and 3% BSA and blocked.

Finally, incubating the blocked 293T-CLDN 18.2 with a blocked phage library diluent, centrifuging to remove unbound phage, and panning; collecting specifically bound phage, and performing the next round of affinity panning; the panned phage library is amplified in culture for the next round of panning or analysis.

1.3 identification and analysis of specific phage clones

Dripping phage panned by the library in the step 1.2 on a flat plate, randomly picking a plurality of monoclonals by using a sterilized toothpick, inoculating the monoclonals into a 2YT culture medium, and carrying out cell: phage=1: 20 was added to M13K07 phage and incubated overnight, and the supernatant was used to identify the monoclonal by cell ELISA. Positive clones were sequenced.

6 candidate antibodies were co-screened and designated CLDN18.2-a93, CLDN18.2-a100, CLDN18.2-a102, CLDN18.2-a127, CLDN18.2-B123 and CLDN18.2-B142.

The amino acid sequences of the 6 candidate antibodies CLDN18.2-A93, CLDN18.2-A100, CLDN18.2-A102, CLDN18.2-A127, CLDN18.2-B123 and CLDN18.2-B142 are the sequences shown as SEQ ID No.38, SEQ ID No.39, SEQ ID No.40, SEQ ID No.41, SEQ ID No.42 and SEQ ID No.43 respectively.

The nucleotide sequences of the 6 candidate antibodies CLDN18.2-A93, CLDN18.2-A100, CLDN18.2-A102, CLDN18.2-A127, CLDN18.2-B123 and CLDN18.2-B142 are the sequences shown as SEQ ID No.44, SEQ ID No.45, SEQ ID No.46, SEQ ID No.47, SEQ ID No.48 and SEQ ID No.49, respectively.

Example 2

In this example, VHH Fc nanobody expression, purification, and antibody affinity measurement were performed on the 6 candidate antibodies obtained in example 1. The method comprises the following steps:

2.1VHH Fc nanobody expression and purification

The 6 candidate antibodies obtained in example 1 were cloned into eukaryotic expression plasmid PTT5-Human-FC with Human Fc tag, expressed in 293T cells and purified using Protein A/G affinity chromatography. After purification 6 anti-CLDN 18.2 VHH antibodies with human Fc tags were obtained, including anti-CLDN 18.2-a93 VHH Fc antibody, anti-CLDN 18.2-a100 VHH Fc antibody, anti-CLDN 18.2-a102 VHH Fc antibody, anti-CLDN 18.2-a127 VHH Fc antibody, anti-CLDN 18.2-B123 VHH Fc antibody, anti-CLDN 18.2-B142 VHH Fc antibody, absorbance of OD280 was detected by an enzyme-labeled instrument and concentration was calculated, purity and molecular weight were detected by SDS-PAGE gel.

The results of mass detection of the 6 human Fc tagged CLDN18.2VHH antibodies are shown in table 1.

TABLE 1 protein expression levels and other related information

Protein name	Concentration (mg/mL)	Reduced molecular weight Kda	Non-reducing molecular weight Kda	Purity (SDS-PAGE)
					CLDN18.2-A93-Fc	5.28	39Kda	78Kda	>95％
CLDN18.2-A100-Fc	4.92	38Kda	76Kda	>95％
					CLDN18.2-A102-Fc	1.3	39Kda	78Kda	>95％
CLDN18.2-A127-Fc	0.83	39Kda	78Kda	>95％
					CLDN18.2-B123-Fc	4.59	39Kda	78Kda	>95％
CLDN18.2-B142-Fc	3.47	39Kda	78Kda	>95％

2.2 antibody affinity assay

The affinity of the 6 anti-CLDN 18.2VHH antibodies with human Fc tag obtained in step 2.1 was determined by Biacore.

Biacore is a bioanalytical sensing technology developed based on surface plasmon resonance (surface Plasmon resonance, SPR), and can detect and track the whole change process of binding and dissociating molecules in a solution and molecules fixed on the surface of a chip, record the whole change process in the form of a sensor graph and provide kinetic and affinity data, solidifying an anti-CLDN 18.2VHH antibody purified in the step 2.1 on the surface of the chip in the measuring process, and solidifying the solution containing human Claudin-18.2 protein in a mobile phase, wherein the measuring results are shown in Table 2 and figures 1A to 1F.

Table 2 6 results of affinity test of human Fc tagged anti-CLDN18.2VHH antibodies with human Claudin-18.2 protein

Antibodies to	k _a (1/Ms)	k _d (1/s)	KD(M)
				CLDN18.2-A93-Fc	9.59E+05	4.99E-04	5.21E-10
CLDN18.2-A100-Fc	4.97E+05	6.41E-04	1.29E-09
				CLDN18.2-A102-Fc	8.30E+05	4.86E-04	5.86E-10
CLDN18.2-A127-Fc	1.29E+06	5.82E-04	4.53E-10
				CLDN18.2-B123-Fc	4.69E+05	6.39E-04	1.36E-09
CLDN18.2-B142-Fc	1.28E+06	4.45E-04	3.48E-10

K _a For the binding constant, the larger the value, the stronger the affinity, as opposed to Kd.

K _d The dissociation constant is expressed as the rate of dissociation reaction, K _d Larger represents faster dissociation, K _d Smaller represents slower dissociation.

KD is an equilibrium dissociation constant, KD represents the dissociation degree in an equilibrium state, and the larger KD indicates more dissociation and represents weaker affinity; smaller KD indicates less dissociation, representing stronger affinity.

Equilibrium dissociation constant K _D ＝Kd/Ka

As can be seen from table 2 and figures 1A to 1F, the affinity of these 6 anti-CLDN 18.2 VHH antibodies with human Fc tags all reached nM level.

Example 3

In this example 3, a flow assay was performed against the heavy chain antibody of CLDN 18.2.

293T cells (293T-claudin 18.2-GFP, preparation details see example 7) or 293T cells (293T-claudin 18.1-GFP, preparation details see example 7) stably overexpressing the CLDN18.2 protein were incubated with the purified 6 anti-CLDN 18.2 VHH antibodies (anti-CLDN 18.2-A93 VHH Fc antibody, anti-CLDN 18.2-A100 VHH Fc antibody, anti-CLDN 18.2-A102VHH Fc antibody, anti-CLDN 18.2-A127 VHH Fc antibody, anti-CLDN 18.2-B123 VHH Fc antibody and anti-CLDN 18.2-B142 VHH Fc antibody) obtained in step 2.1 of example 2, respectively, for 30min under an ice bath, and then incubated with the APC-labeled goat anti-mouse IgG antibody, and the results were detected by flow cytometry (FACS) as shown in FIGS. 2A and 2B.

A blank group was also established, with wild-type 293T cells as the blank group.

As can be seen from fig. 2A and 2B, it was shown that the heavy chain antibody against CLDN18.2 of the present application is capable of specifically recognizing the CLDN18.2 antigen on the cell surface, but not the CLDN18.1 antigen on the cell surface.

Example 4

In this example 4, lentiviral vectors were prepared that expressed chimeric antigen receptor of heavy chain antibody against CLDN18.2 VHH.

A schematic representation of the chimeric antigen receptor is shown in FIG. 4, comprising a promoter, signal peptide, anti-CLDN18.2 VHH, CD8 alpha hinge region, transmembrane region and immune receptor tyrosine activation motif.

The amino acid sequence of the CD8a signal peptide is: MALPVTALLLPLALLLHAARP (SEQ ID No. 50).

The amino acid sequences of the CD8a hinge region and the transmembrane region are:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYC(SEQ ID No.51)。

the amino acid sequence of the immunoreceptor tyrosine activation motif is:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID No.52)。

4.1 construction of lentiviral vectors

6 anti-CLDN18.2 VHH Fc antibody fragments (abbreviated as antibody fragments) containing a CD8a signal peptide were synthesized with the primers corresponding to table 3 using the anti-CLDN 18.2-a93 VHH Fc antibody, anti-CLDN 18.2-a100 VHH Fc antibody, anti-CLDN 18.2-a102 VHH Fc antibody, anti-CLDN 18.2-B123 VHH Fc antibody and anti-CLDN 18.2-B142 VHH Fc antibody obtained in example 2, respectively, and then the antibody fragments were ligated with the CD8a range-TM-41 BB-CD3Z fragment and the digested sinhd 03 CD19 CAR-KanaR plasmid, respectively, to obtain 6 chronic viral vectors including HD 03 a93 BBz, SIN03 a100 BBz, SIN03 a102 BBz, SIN 03B 41BBz, SIN 03B 123 BBz, 3812703B 41BBz, and 3803B 142 bb62.

The maps of lentiviral vectors HD SIN 03A 93 BBz, SIN 03A 100 BBz, SIN 03A 102 BBz, SIN 03A 12741BBz, SIN 03B 123 41BBz, SIN 03B 142 41BBz are shown in FIGS. 3A, 3B, 3C, 3D, 3E and 3F, respectively.

TABLE 3 Table 3

The specific construction method comprises the following steps:

4.1.1 preparation of fragment CD8a Signal-VHH

The primers corresponding to the antibodies in Table 3 (see Table 3) were used to obtain 6 PCR amplified fragments by PCR amplification using the nucleotide sequences of the 6 human Fc tagged anti-CLDN18.2VHH antibodies synthesized in example 2 as templates. A PCR reaction system was prepared according to Table 4, and a PCR reaction was performed according to the procedure of Table 7.

And (3) 1% agarose electrophoresis, and respectively recovering 6 PCR amplified fragments by using ZymocleanTM Gel DNA Recovery kit, wherein the length of each PCR amplified fragment is about 450bp, so as to obtain 6 anti-CLDN 18.2VHH antibody fragments containing CD8 alpha signal peptide.

TABLE 4 PCR reaction System

4.1.2 preparation of fragment CD8a range-TM-41 BB-CD3Z

PCR amplification was performed using two primers, CD8aH-F and Vector-R (see Table 5), and the HD SIN03 CD19 CAR-Kanar plasmid (plasmid map see FIG. 9) as a template. A PCR reaction system was prepared according to Table 6, and a PCR reaction was performed according to the procedure of Table 7. Wherein the HD SIN03 CD19 CAR-Kanar plasmid is based on 3G11-BBz plasmid (patent application No. 201710357213.3), the ampicillin resistance gene is replaced by the kana resistance gene, and the 5' LTR fragment is truncated to become a viral vector containing a terminal self-inactivating (SIN) structure (plasmid map is shown in FIG. 9).

1% agarose electrophoresis, recovering fragments with a length of 695bp using ZymocleanTM Gel DNA Recovery kit, yielded a CD8a range-TM-41 BB-CD3Z fragment.

TABLE 5

TABLE 6PCR reaction System

TABLE 7

4.1.3 preparation of the digestion vector

The HD SIN03 CD19 CAR-Kanar plasmid (plasmid map, see FIG. 9) was digested with BamHI-HF and EcoRI-HF, and the vector was recovered by 1% agarose electrophoresis ZymocleanTM Gel DNA Recovery kit, with a length of 7710bp.

The cleavage reaction system is shown in Table 8.

TABLE 8

4.2 recombination and transformation

4.2.1 the recovery vector of step 4.1.3 in this example and the CD8a signal-VHH fragment of step 4.1.1 in this example and the CD8a range-TM-41 BB-CD3Z fragment of step 4.1.2 in this example were subjected to a recombination reaction with ClonExpress II one step cloning kit, the reaction conditions: the recombinant product is obtained at 37 ℃ for 30 min. The recombination reaction system is shown in Table 9.

TABLE 9

Contents	Volume(μL)
		Step 4.1.3 HD SIN03 CD19 CAR-Kanar	200ng
Step 4.1.1 CD8a Signal-VHH fragment	18ng
		Step 4.1.2 CD8a range-TM-41 BB-CD3Z fragment	28ng
5×Buffer	4μL
		Recombinant enzyme	2μL
Total volume	Add ddH ₂ O to 20μL

4.2.2 Using 10. Mu.L of the recombinant product obtained in step 4.2.1 of the present example, the recombinant product was thermally transformed into E.coli stbl3 competent (100. Mu.L), and positive monoclonal strains were selected by kana resistance plate for the next sequencing.

4.3 PCR identification and sequencing identification

LV-F2 and LV-R are used as primers (see table 12 for details) and are prepared according to the reaction system of table 10, then PCR reaction is carried out according to the procedure of table 11 to obtain PCR products, and then 1% agarose electrophoresis and EB staining identification are carried out, wherein the length of the PCR products is 1200-1300bp.

Table 10

After preparing a PCR reaction system according to Table 10, PCR was performed according to the procedure of Table 11.

TABLE 11

After the PCR is finished, selecting and identifying correct clones, sending the clones to a company for sequencing (sequencing primers are LV-F2 and LV-R, and the primers are shown in table 12), and finally selecting correct clones, extracting plasmids by using a plasmid large extraction kit, and positive clones are constructed lentiviral vectors.

Table 12

Primer(s)	Sequence(s)	Sequence numbering
			LV-F2	tcttggttcattctcaagcctc	SEQ ID No.67
LV-R	gcaacatagttaagaatacc	SEQ ID No.68

Example 5

In this example, the lentiviral vector obtained in example 4 was subjected to lentiviral packaging comprising the steps of:

(1) At 1.6X10 ⁷ Cell count 293T cells were seeded in 15cm dishes at 37℃with 5% CO ₂ Overnight incubation preparing packaging virus, medium containing DMEM, 10% fetal bovine serum (fetal bovine serum, FBS) added;

(2) 14.5. Mu.g of the lentiviral vector obtained in example 4 was dissolved in 2mL of serum-free DMEM medium with 16.7. Mu.g of helper plasmid pMDlg-RRE, 16.7. Mu.g of helper plasmid pRSV-REV and 6.5. Mu.g of envelope plasmid VSVg, respectively, and mixed well;

(3) 163.2. Mu.g PEI (1. Mu.g/. Mu.L) was dissolved in 2mL serum-free DMEM medium, gently mixed (or vortexed at 1000rpm for 5 seconds), and incubated at room temperature for 5min to give a PEI mixture;

(4) Formation of transfection complexes: adding the PEI mixed solution obtained in the step (3) into the DNA mixed solution, immediately mixing by vortex or gently mixing, and incubating for 20min at room temperature to obtain a transfection complex;

(5) 4mL of the transfection complex obtained in the step (4) was added dropwise to a 15cm dish containing 25mL of DMEM medium, after 4-5 hours, the DMEM medium containing 2% FCS was changed, and after 48 hours of culture, the virus supernatant was collected.

Example 6

In this example, the packaged lentivirus obtained in example 5 was subjected to lentivirus concentration comprising the steps of:

filtering the virus supernatant obtained in the step (5) in the example 5 by using a 0.45 mu m filter membrane, collecting the filtered virus supernatant into a 50mL centrifuge tube, adding 1/4 PEG-NaCl virus concentrate, mixing the mixture upside down, and standing the mixture at 4 ℃ overnight; then centrifuging at 4 ℃ and 3500rpm for 30min; removing the supernatant, adding RPMI 1640 medium (containing 10% FBS) into the virus pellet to dissolve the resuspended virus pellet; the concentrated lentiviral suspension was split into 50. Mu.L portions each, stored in finished tubes and stored at-80 ℃.

Example 7

In this example, the construction of the overexpressing cell line includes the following:

claudin 18.2-GFP lentivirus acquisition: 14.5. Mu.g of claudin 18.2-GFP plasmid (purchased from Crohn), 16.7. Mu.g of helper plasmid pMDlg-RRE, 16.7. Mu.g of helper plasmid pRSV-REV and 6.5. Mu.g of envelope plasmid VSVg were obtained by co-transfection, in the same manner as in example 5.

claudin 18.1-GFP lentivirus acquisition: 14.5. Mu.g of claudin 18.1-GFP plasmid (purchased from Crohn's organism), 16.7. Mu.g of helper plasmid pMDlg-RRE, 16.7. Mu.g of helper plasmid pRSV-REV and 6.5. Mu.g of envelope plasmid VSVg were co-transfected and obtained, as in example 5.

Obtaining 293T cells overexpressing the CLDN18.2 protein (abbreviated as 293T-CLDN 18.2): 293T cells were grown according to 1X 10 ⁶ Each well was inoculated into a 6-well plate, and 1mL of claudin 18.2-GFP lentivirus was added to obtain the final product.

Obtaining 293T cells overexpressing the CLDN18.1 protein (abbreviated as 293T-CLDN 18.1): 293T cells were grown according to 1X 10 ⁶ Each well was inoculated into a 6-well plate, and 1mL of claudin 18.1-GFP lentivirus was added to obtain the final product.

Acquisition of HGC27 cells overexpressing the CLDN18.2 protein (HGC-27-18.2-GFP cells): HGC-27 cells were cultured according to 1X 10 ⁶ Each well was inoculated into a 6-well plate, and 1mL of claudin 18.2-GFP lentivirus was added to obtain the final product.

Obtaining of N87 cells (N87-18.2-GFP) overexpressing the CLDN18.2 protein: NCI-N87 cells were cultured according to 1X 10 ⁶ Each well was inoculated into a 6-well plate, and 1mL of claudin 18.2-GFP lentivirus was added to obtain the final product.

Example 8

In this example, lentivirus titer detection was performed on the concentrated virus obtained in example 6, comprising the steps of:

mu.L of Junkat cells (1X 10) ⁵ Individual cells) cells were seeded in 24-well plates to form a cell suspension; the concentrated lentiviruses obtained in example 6 were added to the cell suspension at 1. Mu.L, 0.2. Mu.L and 0.04. Mu.L, respectively, and polybrene was added to a final concentration of 5. Mu.g/mL; then at 37℃and 5% CO ₂ After overnight incubation, fresh medium was changed; infection withAfter 72h, cells were collected, centrifuged at 400g for 5min, the supernatant was discarded, and the flow buffer (PBS+2% neonatal calf serum) was washed once; mono Rab was added at a dilution ratio of 1:100 ^TM Rabbit Anti-Camelid VHH Cocktail[iFluor 488]Antibody, incubation on ice for 30min; then, after washing twice with 1mL of flow buffer, detecting by using a flow cytometer; taking a cell sample with a positive rate of 5-20%, and calculating the titer.

Titer (TU/mL) =cell number (10 ⁵ ) X positive rate/viral volume (mL).

Example 9

In this example, T lymphocytes were transduced with lentiviruses of example 8 titers satisfactory.

9.1 Resuscitation of T lymphocytes

Centrifuging resuscitated PBMC cells at 200g for 9min, discarding supernatant, resuspending the pellet with T cell culture medium, counting, and adjusting density to 1.5-2×10 ⁶ cells/mL, and inoculated into a 48-well cell culture plate, and allowed to stand for 2 hours for activation.

9.2 Activation of T lymphocytes

PBMC were density adjusted to 2X 10 with T cell medium (X-VIVO+10% FBS+IL-2 (300U/mL) ⁶ T cell trans-act was added at 1:100 ratio to cells/mL, inoculated into a suitable culture vessel and activated for 24-48 hours while maintaining the cell density at 2X 10 ⁶ cells/ml, activated T cells obtained.

9.3 T cell infection

Collecting the activated T cells obtained in step 9.2, and adjusting the cell density to 1×10 ⁶ Per mL, the lentivirus obtained in example 8 was added at a multiplicity of infection moi=10, polybrene was added to a final concentration of 5 μg/mL; at 37℃with 5% CO ₂ Fresh medium was changed after overnight incubation in the environment. Passaging was performed every 2-3 days.

9.4 chimeric antigen receptor expression

In this example, 3X 10 was taken 5 days after the infection of step 9.3 ⁵ Centrifuging at 4deg.C for 5min at 400g, discarding the supernatant, and washing with flow buffer (PBS+2% rabbit serum) once; mono Rab was added at a dilution ratio of 1:100 ^TM Rabbit Anti-Camelid VHH Cocktail (iFluor 488) antibody, incubation on ice 30min; then, after washing twice with 1mL of the flow buffer, the T lymphocyte chimeric antigen receptor infection efficiency was examined by flow cytometry, and the results are shown in FIG. 5.

As can be seen from fig. 5, the CAR-T cells after infection had a significant positive population, indicating that 6 CAR-T cells expressing chimeric antigen receptors of different structures were successfully constructed, the antigen binding domains of which contained CLDN18.2-a93, CLDN18.2-a100, CLDN18.2-a102, CLDN18.2-a127, CLDN18.2-B123 and CLDN18.2-B142 antibodies, respectively.

The CAR-T cell markers corresponding to CLDN18.2-a93, CLDN18.2-a100, CLDN18.2-a102, CLDN18.2-a127, CLDN18.2-B123 and CLDN18.2-B142 antibodies were CLDN18.2 (a 93), CLDN18.2 (a 100), CLDN18.2 (a 102), CLDN18.2 (a 127), CLDN18.2 (B123) and CLDN18.2 (B142), respectively.

Example 10

In this example, an in vitro toxicity assay was performed using the 6 CAR-T cells prepared in example 9, comprising the steps of:

10.1 target cell seeding

293T cells (claudin 18.2-), HGC-27-18.2-GFP cells (claudin 18.2+), N87-18.2-GFP cells (claudin 18.2+) were taken as target cells, centrifuged at 400g for 5min, resuspended in 1640 medium of 5% FBS, counted and the target cell concentration was adjusted to 1X 10 ⁵ /mL。

Among them, HGC-27-18.2-GFP cells (claudin 18.2+) and N87-18.2-GFP cells (claudin 18.2+) were prepared as described in example 7.

10.2 Effector cell seeding

6 CAR-T cells (CLDN 18.2 (a 93), CLDN18.2 (a 100), CLDN18.2 (a 102), CLDN18.2 (a 127), CLDN18.2 (B123) and CLDN18.2 (B142)) prepared in example 9 and control T cells (not lentivirally infected) were used as effector cells; effector cells 400g were centrifuged for 5min, resuspended in 1640 medium of 5% fbs, counted, and cell density adjusted to the desired concentration a (adjusted CAR-T density = 3 x 10 ⁵ cell/mL/infection efficiency);

10.3 dilution of effector cell ratios

3-fold dilution of CAR-T cells at a concentration to car+ T cells at B concentration, 3-fold dilution of CAR-T cells at B concentration to CAR-T cells at C concentration;

10.4 effector cells and target cells were added to 96-well plates containing medium at an effective target ratio of 0.3:1, 1:1 and 3:1 (appropriate 96-well plates were selected according to target cells), 3 multiple wells per group, total volume per well 200 μl;

after incubation at 37 ℃ for 18h, 20 μl of lysate (10×) was added to the wells; the cell lysis condition is not observed regularly, so that the cell lysis is ensured to be complete;

10.6 preparing a substrate, adding 12mL of assay buffer (lactic acid) into a substrate mix (tetrazolium salt)/bottle, uniformly mixing and keeping away from light (about the dosage of two 96-well plates) to obtain a substrate solution;

10.7 all wells treated in step 10.5 were sucked 6-8 times with a row gun and centrifuged at 500g for 5min, and 50. Mu.L/well supernatant was taken into a new ELISA plate (flat bottom 96 well plate).

10.8 adding the substrate solution prepared in the step 10.6 into the new ELISA plate prepared in the step 10.7 at a concentration of 50 mu L/hole, covering the plate, and incubating for about 30min at room temperature in a dark place;

10.9 add 50. Mu.L stop solution (acetic acid) per well and measure absorbance at 490nm over 30 min.

Data were derived and plotted analytically. The cytotoxicity calculation formula is (all minus blank medium control):

FIGS. 6A, 6B and 6C are graphs showing the killing effect of constructed CAR-T cells containing anti-CLDN 18.2 heavy chain antibody on 293T cells, HGC-27-18.2-GFP cells and N87-18.2-GFP cells, respectively.

As can be seen from fig. 6A, 6B and 6C, the CAR-T cells constructed in the present application (CLDN 18.2 (a 93), CLDN18.2 (a 100), CLDN18.2 (a 102), CLDN18.2 (a 127), CLDN18.2 (B123) and CLDN18.2 (B142)) have high killing activity against CLDN18.2 positive tumor cells (including HGC-27-18.2-GFP (claudin 18.2+) and N87-18.2-GFP (claudin 18.2+)), and have no killing effect against CLDN18.2 negative cells (293T (claudin 18.2-)), indicating that the constructed CAR-T cells have high specificity.

Example 11

In this example, the secretion of CAR-T cytokines was detected, including the following:

11.1 cell culture supernatant

The experiments were divided into three groups, one group of effector cells from example 9 were cultured alone at 37℃for 18h, one group of effector cells from example 9 and 293T cells were co-cultured at 37℃for 18h, one group of effector cells from example 9 and CLDN 18.2 positive 293T cells (293T-CLDN 18.2) were co-cultured at 37℃for 18h, with an effective target ratio of 1:1. While setting up Control T group (no lentiviral infection). CLDN 18.2 positive 293T cells (293T-CLDN 18.2) were obtained as detailed in example 7.

After the completion of the culture, the cell culture was centrifuged at 400 Xg for 10min to remove the precipitate, and the supernatant was stored at-80℃for examination.

11.2 detection of IFN- α and IFN- γ

11.2.1 reagents: the detection was carried out using a Union ELISA kit (product numbers: human gamma interferon ELISA kit: EK180-96; human tumor necrosis factor alpha (TNF-alpha) ELISA kit: EK 182-96), all reagents and samples were returned to 25℃before detection, and 1 Xwash solution, 1 Xdetection buffer solution, and detection antibody were prepared according to the instructions of use.

11.2.2 standard and sample preparation standard: standard stock was 2-fold diluted with 5%1640 medium for a total of 8 dilution gradients, including zero concentration.

Sample: samples were diluted in ratio using 5%1640 medium.

11.2.3 detection procedure

(1) Soaking the ELISA plate: adding 300 mu L of 1 Xwashing liquid, standing and soaking for 30s, removing the washing liquid, and then beating the micro-porous plate on water-absorbing paper;

(2) Adding a standard substance: standard wells were filled with 100 μl of 2-fold diluted standard and blank wells were filled with 100 μl of 5%1640 medium;

(3) Adding a sample: sample wells were added with 100 μl of the cell culture supernatant obtained in step 11.1 of this example;

(4) Adding a detection antibody: 50. Mu.L of diluted detection antibody (1:100 dilution) was added to each well;

(5) Incubation: sealing plates by using sealing plates, vibrating at 300rpm, and incubating at 25 ℃ for 2 hours;

(6) Washing: liquid was discarded, and 300. Mu.L of wash solution was added to wash the plate 6 times per well;

(7) And (3) enzyme adding and incubation: mu.L of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) was added to each well;

(8) Incubation: using a new sealing plate membrane sealing plate, oscillating at 300rpm, and incubating at 25 ℃ for 45min;

(9) Washing: repeating step (6);

(10) And (3) color development of the substrate: 100 mu L of chromogenic substrate TMB is added into each hole, and incubated for 15min at 25 ℃ in the dark;

(11) Adding a stop solution: adding 100 mu L of stop solution into each hole, and fully and uniformly mixing;

(12) Detecting and reading: the dual wavelength detection was performed using an enzyme-labeled instrument, and the OD at the 450nm maximum absorption wavelength and the 630nm reference wavelength was measured, and the OD after calibration was measured at 450nm minus the measured at 630 nm. The results are shown in FIGS. 7 and 8.

The IFN-alpha secretion results are shown in FIG. 7, where spontaneous is CAR-T cell culture alone, trace IFN-alpha was detected in CAR-T cell culture alone, as was trace IFN-alpha detected in CAR-T cell co-culture with 18.2 negative 293T cells, and higher levels of IFN-alpha were detected in CAR-T cell co-culture with 18.2 positive 293T-CLDN 18.2 cells (claudin 18.2+).

The results of FN-gamma factor secretion are shown in FIG. 8, where spontaneous CAR-T cell cultures alone, where almost no IFN-gamma factor was detected, as was the case in co-cultures of CAR-T cells and 18.2 negative 293T cells, whereas higher levels of IFN-gamma factor were detected in co-cultures of CAR-T cells and 18.2 positive 293T-CLDN 18.2 cells (claudin 18.2+).

From the combination of fig. 7 and fig. 8, it is shown that the CAR-T cell constructed in the present application can release cytokines to the CLDN18.2 positive tumor cells to perform a killing function, has high specificity, and has no cytokine secretion to the CLDN18.2 negative cells.

In summary, according to the application, through a transient transfection mode, a llama is immunized by using a CLDN18.2 recombinant protein, a phage display nano antibody library is constructed, and screening is carried out on anti-CLDN 18.2 antibodies according to the phage display nano antibody library, so that the heavy chain antibody of the anti-CLDN 18.2 with the CDR of a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 38-43 is obtained, and the determination of Ka, kd and KD of antibody affinity proves that the heavy chain antibody of the anti-CLDN 18.2 obtained by the application can specifically bind to the CLDN18.2 antigen and has high affinity. The heavy chain antibody against CLDN18.2 of the present application is capable of specifically recognizing CLDN18.2 but not CLDN18.1. The heavy chain antibody for resisting CLDN18.2 provided by the invention has better affinity, a chimeric antigen receptor is constructed by taking the heavy chain antibody as an antigen binding domain, the chimeric antigen receptor is utilized to prepare CAR-T cells, the constructed CAR-T cells have killing activity on CLDN18.2 positive tumor cells, and after the constructed CAR-T cells are co-cultured with the CLDN18.2 positive cells, cytokines TNF-alpha and IFN-gamma are secreted efficiently, so that the nano antibody, the chimeric antigen receptor constructed by the nano antibody and the CAR-T cells can improve the killing capacity on tumor cells, especially stomach cancer.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those of ordinary skill in the art without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications and variations which can be accomplished by persons skilled in the art without departing from the spirit and technical spirit of the present disclosure shall be covered by the claims of this application.

Claims

1. An anti-CLDN 18.2 heavy chain antibody, which is characterized in that the heavy chain antibody is composed of a heavy chain variable region, and the amino acid sequence of CDR1 of the heavy chain variable region is shown as one of sequences shown in SEQ ID nos. 1-6;

the amino acid sequence of CDR2 of the heavy chain variable region is shown as one of SEQ ID No.7-SEQ ID No. 12; the amino acid sequence of CDR3 of the heavy chain variable region is shown as one of SEQ ID No.13-SEQ ID No. 18.

2. The heavy chain antibody of claim 1, comprising one or more of the following features:

1) The amino acid sequence of CDR1 of the heavy chain variable region comprises a sequence shown as SEQ ID No.1, the amino acid sequence of CDR2 comprises a sequence shown as SEQ ID No.7, and the amino acid sequence of CDR3 comprises a sequence shown as SEQ ID No. 13;

2) The amino acid sequence of CDR1 of the heavy chain variable region comprises a sequence shown as SEQ ID No.2, the amino acid sequence of CDR2 comprises a sequence shown as SEQ ID No.8, and the amino acid sequence of CDR3 comprises a sequence shown as SEQ ID No. 14;

3) The amino acid sequence of CDR1 of the heavy chain variable region comprises a sequence shown as SEQ ID No.3, the amino acid sequence of CDR2 comprises a sequence shown as SEQ ID No.9, and the amino acid sequence of CDR3 comprises a sequence shown as SEQ ID No. 15;

4) The amino acid sequence of CDR1 of the heavy chain variable region comprises a sequence shown as SEQ ID No.4, the amino acid sequence of CDR2 comprises a sequence shown as SEQ ID No.10, and the amino acid sequence of CDR3 comprises a sequence shown as SEQ ID No. 16;

5) The amino acid sequence of CDR1 of the heavy chain variable region comprises a sequence shown as SEQ ID No.5, the amino acid sequence of CDR2 comprises a sequence shown as SEQ ID No.11, and the amino acid sequence of CDR3 comprises a sequence shown as SEQ ID No. 17;

6) The amino acid sequence of CDR1 of the heavy chain variable region comprises a sequence shown as SEQ ID No.6, the amino acid sequence of CDR2 comprises a sequence shown as SEQ ID No.12, and the amino acid sequence of CDR3 comprises a sequence shown as SEQ ID No. 18.

3. The heavy chain antibody of claim 1, wherein the heavy chain variable region further comprises framework regions FR1-FR4: the amino acid sequence of the framework region FR1 comprises a sequence shown as one of SEQ ID No.19-SEQ ID No.23, the amino acid sequence of the framework region FR2 comprises a sequence shown as one of SEQ ID No.24-SEQ ID No.29, the amino acid sequence of the framework region FR3 comprises a sequence shown as one of SEQ ID No.30-SEQ ID No.35, and the amino acid sequence of the framework region FR4 comprises a sequence shown as one of SEQ ID No.36 or SEQ ID No. 37.

4. The heavy chain antibody of claim 3, comprising one or more of the following features:

1) The amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.19, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.24, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.30, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 36;

2) The amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.20, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.25, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.31, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 37;

3) The amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.21, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.26, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.32, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 37;

4) The amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.22, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.27, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.33, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 37;

5) The amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.23, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.28, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.34, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 37;

6) The amino acid sequence of the framework region FR1 comprises a sequence shown as SEQ ID No.23, the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No.29, the amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No.35, and the amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 37.

5. The heavy chain antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region comprises A1) or A2):

a1 An amino acid sequence as shown in one of SEQ ID No.38-SEQ ID No. 43;

6. An isolated polypeptide comprising an antigen binding domain, a hinge region, and a transmembrane domain, wherein the antigen binding domain comprises the heavy chain antibody of any one of claims 1-5.

7. The polypeptide of claim 6, wherein the polypeptide is a chimeric antigen receptor;

And/or, the transmembrane domain comprises CD8 a, CD28 or DAP10;

and/or, the hinge region comprises CD8 a;

and/or the polypeptide comprises the heavy chain antibody, a hinge region and a transmembrane domain from the N-terminal to the C-terminal.

8. A biological material associated with the heavy chain antibody of any one of claims 1-5 or the polypeptide of claim 6 or 7, wherein the biological material comprises one or more of the following:

1) A nucleotide encoding the heavy chain antibody of any one of claims 1-5 or a nucleotide encoding the polypeptide of claim 6 or 7;

2) A recombinant expression vector comprising 1) said nucleotide;

9. The biomaterial of claim 8, wherein in 1) the sequence of nucleotides comprises a sequence as set forth in one of SEQ ID nos. 44-49;

and/or 2) the recombinant expression vector is selected from a lentiviral vector, a retroviral vector, or an adenoviral vector; preferably a lentiviral vector.

10. A cell expressing the polypeptide of claim 6 or 7.

11. The cell of claim 10, wherein the cell is selected from one or more of T lymphocytes, B lymphocytes, NK cells, mast cells, and macrophages.

12. Use of a heavy chain antibody according to any one of claims 1 to 5 or a polypeptide according to claim 6 or 7 or a biological material according to claim 8 or 9 or a cell according to claim 10 or 11 for the preparation of an in vitro assay product, for the preparation of a medicament for the prevention or treatment of a tumor.

13. An assay product comprising the heavy chain antibody of any one of claims 1-5 or the polypeptide of claim 6 or 7 or the biological material of claim 8 or 9 or the cell of claim 10 or 11; preferably, the product is used to detect CLND 18.2.

14. A pharmaceutical composition comprising the heavy chain antibody of any one of claims 1-5 or the polypeptide of claim 6 or 7 or the biological material of claim 8 or 9 or the cell of claim 10 or 11, and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is for use in the prevention or treatment of a neoplastic disease associated with CLND18.2 expression.