CN114539410A - CLDN 18.2-binding antibodies, probes and use in single cell sequencing of CLDN 18.2-expressing cells - Google Patents

Abstract

The present invention provides a novel antibody against CLDN 18.2. The above antibody can bind to CLDN18.2 with high affinity and specificity, can be simply bound to a fluorescent substance such as quantum dot, and the form can maintain its high affinity and specificity for CLDN18.2 at a high sorting speed. In addition, the present invention also provides a sorting method of CLDN 18.2-expressing cells based on the probe, which has better sorting efficiency and higher sorting accuracy than flow cell sorting generally performed using antibody probes, by which single cell sequencing of CLDN 18.2-expressing cells can be performed with extremely high reliability.

Description

CLDN 18.2-binding antibodies, probes and use in single cell sequencing of CLDN 18.2-expressing cells

Technical Field

The present invention relates to the field of cell sorting. In particular, the invention relates to antibodies having CLDN18.2 binding thereto and to the forms of probes binding to fluorescent labels, in particular to quantum dots, and to their use in the treatment of single cell sorting, in particular flow cytometric sorting.

Background

Upper gastrointestinal tumors, including gastric and esophageal cancers, are a common and poor prognosis malignancy worldwide. Gastric cancer ranks third in cancer-related mortality and is considered one of the most refractory cancers worldwide. Median overall survival (mOS) is not more than 10 months in patients with advanced or metastatic gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma. Therefore, the research on gastric cancer at a cellular level to obtain a new concept of targeted therapy is of great medical significance. In recent years, with the development of single cell analysis technology, there are more and more research means for gastric cancer molecular mechanism, but at the same time, this also requires the support of more accurate and higher throughput cell sorting method.

The tight junction protein (Claudin) family of proteins is a major component of the tight junction structure widely distributed in epithelial cells and functions to maintain tight junctions that control the exchange of molecules between cells. Claudin is widely distributed in stomach, pancreas and lung tissues and can be used for diagnosis and therapy. CLDN18.2 has a molecular weight of about 27.8kd, has four transmembrane regions, and has two shorter extracellular regions, EC1 and EC 2. Unlike other claudin family proteins, CLDN18.2 is very specifically expressed only in highly differentiated epithelial cells of the stomach in normal tissues (Tureci O et al, Gene, 2011). Among tumors derived from gastric epithelial cells, CLDN18.2 is expressed on the surface of a high proportion of tumor cells. High levels of CLDN18.2 expression can also be detected in lymph nodes of gastric carcinoma cells and other tissue metastases (Woll s.et al, int.j.cancer, 2013). In addition to gastric cancer, CLDN18.2 is expressed in a high proportion of tumor cells such as bile duct cancer, esophageal cancer and pancreatic cancer. Therefore, CLDN18.2 is also a target for cell sorting with great research and utilization value as a specific surface marker.

Quantum dots are an important low-dimensional semiconductor material, and the size of each of the three dimensions is not larger than twice the exciton bohr radius of the corresponding semiconductor material. Quantum dots are generally spherical or spheroidal, often with diameters between 2-20 nm. Common quantum dots are composed of IV, II-VI, IV-VI or III-V elements. Specific examples are silicon quantum dots, germanium quantum dots, cadmium sulfide quantum dots, cadmium selenide quantum dots, cadmium telluride quantum dots, zinc selenide quantum dots, lead sulfide quantum dots, lead selenide quantum dots, indium phosphide quantum dots, indium arsenide quantum dots, and the like.

Quantum dots are nano-scale semiconductors that emit light of a specific frequency by applying a certain electric field or light pressure to the nano-semiconductor material, and the frequency of the emitted light varies with the size of the semiconductor, so that the color of the emitted light can be controlled by adjusting the size of the nano-semiconductor, which is called quantum dots because the nano-semiconductor has a property of confining electrons and electron holes (Electro n holes), which is similar to atoms or molecules in the natural world.

Compared with fluorescent markers such as fluorescent protein, the quantum dots have the following advantages: 1. the same laser can excite more colors, so that the interference of the background caused by the protein can be effectively avoided; 2. the light stability is high; 3. the fluorescence leakage among the channels is small; 4. the brightness is high.

Disclosure of Invention

Technical problem to be solved by the invention

The antibody probe can detect cells expressing a specific target protein, and flow cytometry can separate the cells labeled with the antibody probe with high precision, and the combination of the two is one of the main means used when a specific cell population is to be separated from a sample, however, background fluorescence caused by protein can affect the accuracy of flow cytometry, and in addition, because the antibody is externally bound to the target cell, and the antibody probe is generally bound to a fluorescent label of a macromolecule, the binding activity of the antibody probe can be sometimes affected by the fluorescent label (such as steric hindrance and the like), so under the condition of high sorting rate, the recovery rate tends to be reduced.

Means for solving the problems

The present invention provides a novel antibody against CLDN 18.2. The above antibody can bind to CLDN18.2 with high affinity and specificity, can be simply bound to a fluorescent substance such as quantum dot, and the form can maintain its high affinity and specificity for CLDN18.2 at a high sorting speed. In addition, the present invention also provides a sorting method of CLDN 18.2-expressing cells based on the probe, which has better sorting efficiency and higher sorting accuracy than flow cytometric sorting generally using antibody probes.

In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof binding to CLDN18.2, comprising an antigen binding domain binding to CLDN18.2 having a heavy chain variable region (VH) sequence of SEQ ID NO:1 and a light chain variable region (VL) sequence of SEQ ID NO: 2.

In some embodiments of the antibodies or antigen binding fragments thereof of the invention, the antibody may be a monoclonal antibody. In other embodiments, the antibody can be a bispecific antibody or a multispecific antibody.

In some embodiments of the antibodies or antigen-binding fragments thereof of the invention, the antibody may be selected from IgG, IgA, IgM, IgE, and IgD isotypes. In some embodiments, the antibody may be selected from the IgG1, IgG2, IgG3, and IgG4 subtypes.

In any embodiment of the antibody or antigen binding fragment thereof of the present invention, the antigen binding fragment may be selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab')₂Fragments, Fd' fragments, Fv fragments, scFv fragments, ds-scFv fragments, dAb fragments, single chain fragments, diabodies and linear antibodies.

In one aspect, the invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the invention.

In another aspect, the invention relates to a vector comprising a nucleic acid molecule of the invention.

In one aspect, the invention relates to an antibody probe comprising an antibody or antigen-binding fragment thereof of the invention bound to a fluorescent substance.

In one aspect, the present invention relates to an antibody probe, wherein the fluorescent substance is a quantum dot.

In one aspect, the present invention relates to a flow cytometric sorting method for separating CLDN 18.2-expressing cells, wherein the CLDN 18.2-expressing cells are labeled with the antibody probe.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention provides a novel antibody specifically binding to CLDN18.2, which can effectively avoid background fluorescence caused by proteins when forming a probe with a quantum dot, and can still sort CLDN 18.2-expressing cells at a high sorting rate with high sorting purity and recovery rate.

Detailed description of the invention

The invention will be described below by means of specific embodiments, the advantages and features of which will become more apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Test 1 construction of the Natural phage library

Phage display technology is an in vitro screening technique for identifying proteins and other macromolecular ligands. Established by Simth in 1985. The principle is that the DNA sequence of exogenous polypeptide or protein is inserted into the proper position of a structural gene of the coat protein of the phage, under the condition that the reading frame is normal and the normal function of the coat protein is not influenced, the exogenous gene is expressed along with the expression of the coat protein, and finally the polypeptide or protein is displayed on the surface of the phage in a form of fusion with the coat protein. The displayed protein or polypeptide can maintain relative spatial structure and biological activity, and can be screened by using a target protein.

In this screening, total RNA of 200 mononuclear cells derived from peripheral blood of adult healthy individuals and spleen or umbilical cord blood of newborn was obtained and extracted in total, thereby avoiding bias of antibody library due to individual difference and improving diversity of library constructed as much as possible. The RNA was reverse transcribed to synthesize single-stranded cDNA, and a PCR reaction was performed using primers specific for the immunoglobulin heavy chain variable region family. The library was constructed by randomly cleaving the PCR product with restriction enzymes SfiI and XhoI and treating the MV1473 phagemid vector [ containing the gene encoding the common light chain (human kappa light chain IgV kappa 1-39 x 01/IGJ kappa 1 x 01 germ cell line type gene) ] with the same restriction enzymes, and inserting the PCR product into the phagemid vector.

Test 2. preparation of cell line having stable expression of CLDN18.2

For phage display screening, a HEK293T cell strain (HEK293T-CLDN18.2) stably expressing CLDN18.2 was prepared. The specific experimental process is as follows: the cDNA sequence for human CLDN18.2 was cloned into a pCDH lentiviral vector and then co-transfected with a lentiviral packaging vector into 293t cells. After 48 or 72 hours of culture, the cell supernatants enriched with lentiviral particles were collected and directly infected with HEK293T cells, CT26 cells or SNU601 cells. Lentivirus infected cells were screened with puromycin and flow cytometry using CLDN18.2 specific antibodies (IMAB362, Ganymed) was used to detect CLDN18.2 expression 2-3 weeks later.

The above results indicate that HEK293T cells (designated HEK293T-CLDN18.2 cell line), CT26 cells (CT26-CLDN18.2) and SNU601 cells (SNU601-CLDN18.2) stably expressing CLDN18.2 were obtained by lentivirus transfection and puromycin screening.

Test 3 screening of anti-CLDN 18.2 antibodies from antibody phage display libraries

Panning of phage display libraries was performed using a HEK293T-CLDN18.2 stably expressing cell line.

The specific process is briefly described as follows: the phage library was incubated with HEK293T-K1 cells and the supernatant was removed after centrifugation. The supernatant was then incubated with HEK293T-CLDN18.2 cells to allow binding of phage specific for CLDN18.2 to the cells. After centrifugation, the cell pellet was collected, and the cells were washed and collected. The phage library bound to the cells was then eluted using 75mM sodium citrate buffer. After neutralization of the phage library, the 100-fold screened phage library was amplified using M13K07 helper phage, and then subjected to a second and third round of screening in a manner similar to the first round of screening. Enrichment of phage was monitored by initial phage dose per round of screening and phage titers collected after screening, and flow cytometric assays were performed using phage libraries and HEK293T or HEK293T-CLDN18.2 stably expressing cell lines.

After three rounds of screening, an enriched phage library was obtained. After infecting the bacteria with this library, it was spread on an agarose plate for culture. The monoclonal colonies were picked and placed in a 96-well deep-well plate and shake-cultured at 37 ℃ using 2YT medium containing ampicillin and kanamycin, to obtain a supernatant containing the monoclonal phage. Subsequently, monoclonal phages binding to HEK293T-CLDN18.2 cells but not to HEK293T control cells were screened for CLD1-CLD3 using flow cytometry to detect binding of the phages to the cells.

Test 4 preparation of recombinant antibody

The cDNA sequences of the heavy chain and light chain variable regions of the monoclonal phage were cloned into pcDNA3.4 vector (Invitrogen) already containing the antibody constant regions, respectively, to obtain heavy chain and light chain expression plasmids of 3 monoclonal antibodies in total. The plasmids were transfected into EXPI-293 cells (Invitrogen) using the PEI method, transiently transfected for 7-10 days, centrifuged and the supernatant harvested. The supernatant was purified by protein a to obtain purified antibody. The 3 monoclonal antibodies are designated as CLD-1, CLD-2 and CLD-3. The amino acid sequences and coding sequences of the variable regions of the heavy and light chains of these antibodies are shown in table 1 below.

TABLE 1

Test 5 detection of binding Activity of recombinant monoclonal antibody

The binding activity of the recombinant monoclonal antibodies to CLDN18.2 was examined. Specifically, a well-grown cell line stably expressing CT26-CLDN18.2 was obtained. After washing the cells with PBS, they were mixed with antibody diluted in multiple proportions and incubated for 1 hour at 4 ℃. Cells were washed once with flow cytometric buffer, PE-labeled anti-human IgG antibody was added, and incubated at 4 ℃ for 30 minutes. The supernatant was removed by centrifugation, washed with flow cytometry buffer, and the fluorescence intensity of the cell surface was measured by a flow cytometer. The average fluorescence intensity was used to calculate the relative binding activity of each antibody. The results are shown in table 2 below.

TABLE 2

	CLD-1	CLD-2	CLD-3
				EC50	1.312	0.712	0..693

Experiment 6. preparation of Quantum dot-conjugated antibody Probe

The antibody is combined with the quantum dot, and the specific process is briefly described as follows: (1) quantum dots were washed (using QBC620(CdSe/ZnS quantum dots) purchased from kunza organisms, size 100 nm): and washing the quantum dots by using a phosphate buffer solution with the pH value of 7.2 until the original buffer solution for storing the quantum dots is completely removed, storing the quantum dots by using a phosphate buffer solution with the pH value of 7.2, and performing ultrafiltration concentration. The concentration of the phosphate buffer solution at pH 7.2 was 10 mmol/L. The ultrafiltration concentration is carried out by centrifuging for 6min at 20 ℃ and 7000r/min by using a 100K ultrafiltration tube. (2) And (3) activation: respectively weighing EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) according to the proportion, and directly adding EDC and NHS into the quantum dot solution obtained in the step (1)Mixing for 5 times by using an oscillator, and reacting for 50-70min at 23 ℃ to activate the quantum dots; the mass ratio of EDC, NHS and quantum dots is 25000: 5000:1. (3) And (3) secondary cleaning of the quantum dots: washing the activated quantum dots obtained in step (2) with borate buffer at pH 8.3 until EDC, NHS and phosphate buffer are completely removed, preserving the activated quantum dots with borate buffer at pH 8.3 and concentrating by ultrafiltration for use. The concentration of the borate buffer solution with the pH value of 8.5 is 50 mmol/L; the ultrafiltration concentration was carried out at 20 ℃ and 7000r/min and centrifuged for 6min using a 100K ultrafiltration tube. (4) Antibody cleaning: washing the antibody with borate buffer solution with pH 8.3, ultrafiltering, and concentrating to remove NaN in stock solution₃The antibody was stored in a borate buffer at pH 8.3 for use. The concentration of the borate buffer solution with the pH value of 8.3 is 50 mmol/L; the ultrafiltration concentration is carried out by centrifuging for 10min by using an ultrafiltration tube of 50K at the temperature of 4 ℃ and the speed of 3000 r/min. (5) Coupling: uniformly mixing the activated quantum dot solution obtained in the step (3) with the antibody solution obtained in the step (4), and reacting for 7 hours at 23 ℃ under continuous shaking to obtain a protein-quantum dot conjugate reaction solution; the molar ratio of the activated quantum dots to the antibody is 1:10, and the concentration of the activated quantum dots is 0.8 mu mol/L. (6) Separation: separating the reaction solution obtained in the step (5) by exclusion chromatography to obtain a protein-quantum dot conjugate solution and an antibody recovery solution; adding the protein-quantum dot conjugate solution into a confining liquid after ultrafiltration and concentration, and storing at 4 ℃; and the antibody recovery solution is subjected to ultrafiltration concentration and then is recycled. The exclusion column used in the exclusion chromatography was Sephacryl300, the flow rate was 1.5ml/min and the column length was 60 cm. The confining liquid is Gly confining liquid, the concentration of Gly is 1mg/mL, and the solvent is borate buffer solution with the concentration of 50mmol/L, pH ═ 8.3. Adding NaN into the concentrated protein-quantum dot conjugate solution in the step (6)₃Adding NaN in the protein-quantum dot conjugate solution₃Is 0.05mg/mL, so that the protein-quantum dot conjugate solution can be stored for a long time.

In this example, 100nm CdSe/ZnS quantum dots were bound to antibodies using the edc-nhs reaction, but the quantum dots are not limited to CdSe/ZnS quantum dots, and the connection method is not limited to the edc-nhs reaction.

Test 7 evaluation of the above probes by flow cytometry

Using the above-prepared CLDN 18.2-expressing HEK293T cell strain, a CLDN 18.2-expressing HEK293T cell strain (hereinafter also referred to as HEK293T-CLDN18.2-GFP) having a GFP knock-in at AAVS1 site was prepared. The effect of the above prepared probes was tested using HEK293T-CLDN18.2-GFP, the brief procedure was as follows: (1) after trypsinization of normal HEK293T cells and HEK293T-CLDN18.2-GFP, centrifugation, cell resuspension in PBS buffer (2% FBS containing), single cell suspension preparation, counting, and 1:1 ratio 2 cells were mixed to serve as test cells. (2) The amount of antibody was determined from the counting results, and each probe was incubated with the test cells in ice bath for 30min, protected from light. (3) Centrifuge at 800rpm for 5min, and discard the supernatant. (4) The cells were resuspended in 500. mu.L of PBS buffer (containing 2% FBS), centrifuged at 800rpm for 5min, and the supernatant was discarded. (5) The cell concentration of each sample was adjusted to 10 with PBS buffer (containing 2% FBS)⁶One per ml. (6) And (3) performing flow cytometry sorting by using a BD FACSCalibur flow cytometer and using 475nm as excitation light wavelength and 625nm as emission light wavelength, wherein the flow rate is set to 5000/s, so as to obtain a recovered group cell and a non-recovered group cell. (7) The cells were seeded in a 96-well plate, cultured for one day, and washed out of dead cells for the following evaluation. The results are shown in the following table.

And (3) evaluating the recovery rate:

the non-recovery group of cells was observed under a fluorescence microscope, and the ratio of GFP-expressing cells was counted and subtracted from 100% to obtain the recovery rate.

Evaluation of recovery purity:

the recovered cells were observed under a fluorescent microscope, and the proportion of cells expressing GFP was counted as the recovery purity.

The test results are shown in table 3 below.

TABLE 3

	Recovery rate	Recovery of purity
			CLD-1 quantum dot probe	99.8％	99.1％
CLD-2 quantum dot probe	93.3％	90.3％
			CLD-3 quantum dot probe	96.2％	93.1％

Test 8 sorting evaluation at high flow

In the case of the quantum dots having a large size, the flow rate was high, and therefore, the possibility of elution of the antibody was also high, and therefore, the recovery rate and the recovery purity of the CLD-1 quantum dot probe were further evaluated under the conditions of 7000/sec, 10000/sec and 12000/sec, and the results are shown in table 4 below.

TABLE 4

	Recovery rate	Recovery of purity
			7000 pieces/second	99.8％	99.1％
10000 pieces/second	99.2％	98.9％
			12000 pieces/second	99.0％	99.3％

From the above results, it can be seen that although the binding activity of CLD-1 is at position 3 in the candidate, the CLD-1 quantum dot probe has the highest sorting purity and recovery rate of HEK293T-CLDN18.2-GFP of 99% or more, and still can maintain the sorting performance at a high flow rate of 12000/s, and it can be seen that the binding activity of the antibody is not affected after binding to a larger quantum dot, and the CLDN 18.2-expressing cells can still be sorted at a high sorting purity and recovery rate at a high sorting throughput.

Experiment 9 application in Single cell sequencing

10 single cells were sorted under the 12000/sec conditions described in example 8, and after overnight culture, sequencing was performed on the AAVS1 site, confirming that all the cells obtained by separation were HEK293T-CLDN18.2-GFP cells with 100% reliability.

Possibility of industrial utilization

The antibody provided by the invention can be combined with quantum dots to prepare a probe which can still sort CLDN18.2 expression cells with high sorting purity and recovery rate even at high sorting rate, and has great practical value in scientific research and clinic.

Sequence listing

<110> Suzhou quantitative cell Biotechnology Ltd

<120> CLDN18.2 binding antibodies, probes and use in single cell sequencing of CLDN18.2 expressing cells

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aatcagcagc tctctgggca cacaaacata catttgcgac aagactcaca cttccagcaa 1020

cacaaaagtg gatcgaagcg gtcattactg tcagccatgg agcagtcgaa gaagctggat 1080

tcagatggca gtttctttct gtacagtcaa ctcaccgttc acaagagtcg gtggcagcag 1140

ggtcacgtgt tctcctgtcg tgtcatgcac gaagcactgc atcatcacta cactcagaag 1200

tctctcagtc tgtctcctgg aaagtgc 1227

<210> 6

<211> 588

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gatattcacg tggaaagaga actgggtgca gttcgcagct ccatccgtgg tactcaaatc 60

tggctcaatc agccctccga tgctctcacg gaggggctcc gagctcatca cccgttagtg 120

gcacagcctc cgtcgtctga cagggtctcg tgctctctga ggtggaacca ggacacaact 180

ctaagagatg aggacaagaa ccaacactgc gatggttcag atgggtgttc ggttcatctt 240

tcccgaggca caaacacgag caaccgaaac ctggaggcac cgttacaccg acctgtcgct 300

cttccactgg cgccgtcacg gctaacgcga gtctggaccc aagcagctcc agggactcct 360

gtctcagaac atggagcagg tcaagtgact ctggttgatc gagatcaagc cttccaacta 420

tcacaagcac caagtctttc aacaggggag acgggaggcc aaggtccagg cgcaaagatc 480

agtccggaaa tagccaactg ggtctctgat cggtgggaag gcagctctga caatcactgg 540

agtgcaaccc gaggaacaat ttctaccctt agctcccaca tgctcgtg 588

<210> 7

<211> 434

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Ser Leu Glu Trp Val Ala Arg Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

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

65 70 75 80

Ala Tyr Leu Gln Met Asn Asn Leu Lys Ser Ala Ser Gln Leu Ser Gly

85 90 95

His Thr Asn Ile His Leu Arg Gln Asp Ser His Phe Gln Gln His Lys

100 105 110

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

115 120 125

Val His Leu Ala Gln His Pro Lys Leu Pro Ala Val Gln Asp Leu Ser

130 135 140

His Ser Ser Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

145 150 155 160

His Gln Asp Ser Phe Phe Leu Tyr Ser Asn Lys Cys Lys Val Ser Asn

165 170 175

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

180 185 190

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Leu Cys Thr Ile Ala Cys

195 200 205

Gly Lys Cys Lys Val Ser Asn Thr Met Ser Val Gly Leu Pro Thr Gly

210 215 220

Asp Arg Glu Leu Ser Leu Leu Ser Ala Pro Pro Val Pro Arg Ala His

225 230 235 240

Pro Phe Ser Leu Trp His Pro Pro Pro Asn Gln Gln Ala Ala Gly Leu

245 250 255

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

260 265 270

Ser Val Gly Thr Val Ala Pro Ser Val Ala Gln Gly His Pro Asp Asp

275 280 285

Leu Ser Pro Cys Gly Trp Arg Gly Gly Ser Gln Arg Gln Asn Ser Ala

290 295 300

Lys Arg Gly Ala Val Tyr Leu Trp Ser Pro Tyr Leu Ser Ser Arg Pro

305 310 315 320

Pro Cys Pro Ala Pro Val Phe Pro Leu Gln Cys Gly Asp Pro Thr Ser

325 330 335

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

340 345 350

Pro Cys Cys Thr Lys Thr Lys Gly Phe Tyr Pro Ser Arg Gly Tyr Arg

355 360 365

Glu Gln Trp Ser Ala Arg Glu Gln Leu Gln Asn His Ser Glu Glu Ala

370 375 380

Gly Phe Arg Trp Gln Phe Leu Ser Val Gln Ser Thr His Arg Ser Gln

385 390 395 400

Glu Ser Val Ala Ala Gly Ser Arg Val Leu Leu Ser Cys His Ala Arg

405 410 415

Ser Thr Ala Ser Ser Leu His Ser Glu Val Ser Gln Ser Val Ser Trp

420 425 430

Lys Val

<210> 9

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Asp Ile Gln Cys Ser Leu Val Leu Lys Ser Gly Ser Ser Arg Ser Asp

1 5 10 15

Trp Arg His Lys Gln Glu Ser Ser Arg Asp Ser Cys Leu Arg Ser Arg

20 25 30

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

35 40 45

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

50 55 60

Gln Ser Gly Asn Ser Gln Glu Met Arg Thr Arg Thr Asn Thr Ala Arg

65 70 75 80

Asn Leu Leu Leu Ser Arg Thr Leu Arg Ile Gln Pro Thr Ala Ser Pro

85 90 95

Ala Pro Lys Leu Ser Ala Arg Gln Ile Ile Trp Phe Ser Gly Trp Val

100 105 110

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Pro Leu Pro Thr Met Gln

115 120 125

Thr Gly Cys Ser Ser Gln Leu His Pro Cys Ser Ser Phe His Pro Pro

130 135 140

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

145 150 155 160

Ala Trp Thr Val Ser Asp Arg Trp Glu Gly Ser Ser Asp Asn His Trp

165 170 175

Ser Ala Thr Arg Gly Thr Ile Ser Thr Leu Val His Lys Leu Thr Thr

180 185 190

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

195 200 205

Val Thr Ala Thr Val Cys Ser Cys

210 215

<210> 10

<211> 389

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Glu Val Gln Ser Ile Pro Thr Phe Lys His Arg Ser Gln Gly Trp His

1 5 10 15

Asp Gly Val Asp Ser Arg Ser Thr Thr Ser Met Glu Ile Ser Gln Ser

20 25 30

His Pro Gln Pro Asp Ala Asp Ala Lys Thr Gln Pro Arg Arg Val Leu

35 40 45

Leu His Pro Tyr Ser Leu Ser Ala Met Ser Ser Phe Leu Glu Ser Pro

50 55 60

Ser Asp Arg Ser Leu Leu Val Asn Leu Pro Arg Asp Gln Glu Gln Ile

65 70 75 80

Gln Gln Leu Arg His Ile Pro Arg Lys Val Gly Phe Arg Trp Gln Phe

85 90 95

Leu Ser Val Gln Ser Thr His Arg Ser Pro Leu Leu Gln Ile Asn Lys

100 105 110

Ala Lys Glu Leu Arg Gln Ala Trp Arg Leu Trp Ser Ser Gln Ser Val

115 120 125

Cys Ala Ala Lys Leu Pro Ala Val Gln Asp Phe Gln Gln Thr Thr Ser

130 135 140

Cys Thr Gln Pro Ser Thr Phe Val Arg Thr Leu Ser Gly Phe Thr Phe

145 150 155 160

Asn Thr Tyr Ala Ser Pro Lys Cys Phe Pro Trp Phe Lys Arg Ser Phe

165 170 175

Gln Glu Tyr Trp Asn Gly Trp Gln Ser Arg Val Leu Leu Ser Cys His

180 185 190

Ala Arg Ser Thr Ala His Pro Arg Phe Tyr Pro Val Gln Gln Val Glu

195 200 205

Gly Ser Lys Met Gly Leu Leu His Gly Thr Asn Ser Gln Arg Asn Phe

210 215 220

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

225 230 235 240

Pro Pro Glu Asp Thr Glu Ser Asn Gly Gln Lys Val Glu Ile His Thr

245 250 255

Cys Pro Pro Cys Ser Ala Ala Ser Ile Trp His Ala Ser Gly Gln Gly

260 265 270

Thr Leu Lys Glu Arg Arg Arg Gln Ser Ala Ala Leu Trp Ala His Lys

275 280 285

Gln Gly Ser Thr Cys Glu Val Cys Asn Cys Asp Pro Thr Ser Arg Glu

290 295 300

Pro His Gly Gly Thr Ala Ala Leu Gly Tyr Ser Pro Lys Lys Thr Ile

305 310 315 320

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

325 330 335

Cys Lys Pro Cys Cys Thr Lys Thr Val Arg Gln Tyr Gln Gly Pro Ile

340 345 350

Arg Phe Ser Asp Arg Ala Ala Pro Gly Pro Ala Ser Thr Leu Lys Leu

355 360 365

Arg Thr Pro Ser Lys Ala Arg Ala Ile Leu Cys Gln Leu Val Leu Leu

370 375 380

Ser Val Glu Ser Ala

385

<210> 10

<211> 183

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Asp Ile His Glu Phe Ala Asn His Trp Arg Val Arg Asp Ser Cys Leu

1 5 10 15

Arg Thr Trp Ser Asn Arg Arg Lys Glu Ser Gly Thr Thr Arg Gly Gly

20 25 30

Gly Asp Val Pro Asn Ser Asn Thr Ala Met Val Gln Met Gly Val Arg

35 40 45

Phe Ile Phe Pro Glu Ala Gln Thr Ala Ser Ile Ser Pro Lys Ser Glu

50 55 60

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

65 70 75 80

Thr Val Ala Thr Leu Gln Pro Ser Arg Asn Val Arg Asp Ile Trp Leu

85 90 95

Asn Gln Pro Ser Asp Ala Leu Thr Glu Gly Leu Arg Lys Gly Gly Thr

100 105 110

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

115 120 125

Leu Ser Thr Gly Glu Thr Gly Gly Pro Glu Asn Cys Ser Ser Trp Thr

130 135 140

Ile Ser His Ser Leu Arg Arg Leu Thr Gly Ser Arg Ala Leu Arg Gly

145 150 155 160

Gly Thr Ser Leu His Ser Val His Ser Trp Pro Arg Ser Ser Leu Thr

165 170 175

Leu Ser Ser His Met Leu Val

180

<210> 11

<211> 409

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Glu Val Gln Leu Glu Phe Pro Glu Met Ile Pro Arg Ile Leu Leu Ile

1 5 10 15

Trp His Pro Pro Pro Asn Gln Gln Gly Gln Arg Val Ala Thr Ser Leu

20 25 30

Glu Ala Leu Glu Ser Ile Pro Leu Tyr Ala Pro Pro Pro Val Pro Arg

35 40 45

Ala His Pro Phe Ser Leu Val His Asn Ala Lys Thr Gln Pro Arg Arg

50 55 60

Val Cys Ala Tyr Trp Ser Thr Tyr Ser Glu Asp Asn Gln Ser Ser Gln

65 70 75 80

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

85 90 95

Gly Ile Leu Ser Leu Gln Arg Ile Gln Arg Ala Met Val Ser Pro Arg

100 105 110

Thr Ile Thr Met Ile Ser Arg Pro Gln Val Asp Gly Val Asp Ser Arg

115 120 125

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

130 135 140

Thr Gln His Thr Gly Ser Tyr Ala Cys Lys Pro Cys Cys Thr Lys Thr

145 150 155 160

Val Ser Phe Cys Thr Val Ser Gly Gly Val His Pro Ala Met Tyr Tyr

165 170 175

Cys Val Arg Gln Val Gln Ser Glu Gln Tyr Tyr Val Ser Trp Phe Ser

180 185 190

Val Ala Pro Leu Pro Val Leu His Ser Thr Leu Met Pro Ala Trp Asn

195 200 205

Gly Trp His Arg Phe Thr Leu Val His Leu Ala Gln His Pro Lys Leu

210 215 220

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

225 230 235 240

Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr

245 250 255

Tyr Tyr Ala Asp Ser Val Tyr Leu Thr His Pro Ala Val His Leu Ala

260 265 270

Gln His Pro Tyr Ser Leu Ser Ala Ile Ser Val Val Thr Arg His Arg

275 280 285

Ser Lys Met Gly Leu Leu His Gly Thr Asn Ser Gln Arg Ile Ser Asp

290 295 300

Arg Ala Ala Pro Gly Pro Ala Ser Thr Leu Lys Leu Lys Gly Glu Asp

305 310 315 320

Asn Gln Gln Leu Ser Gly His Thr Asn Ile His Leu Arg Gln Asp Ser

325 330 335

His Phe Gln Gln His Lys Ser Gly Ser Lys Arg Ser Leu Leu Ser Ala

340 345 350

Met Glu Gln Ser Lys Lys Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

355 360 365

Ser Gln Leu Thr Val His Lys Ser Arg Trp Gln Gln Gly His Val Phe

370 375 380

Ser Cys Arg Val Met His Glu Ala Leu His His His Tyr Thr Gln Lys

385 390 395 400

Ser Leu Ser Leu Ser Pro Gly Lys Cys

405

<210> 13

<211> 196

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Asp Ile His Val Glu Arg Glu Leu Gly Ala Val Arg Ser Ser Ile Arg

1 5 10 15

Gly Thr Gln Ile Trp Leu Asn Gln Pro Ser Asp Ala Leu Thr Glu Gly

20 25 30

Leu Arg Ala His His Pro Leu Val Ala Gln Pro Pro Ser Ser Asp Arg

35 40 45

Val Ser Cys Ser Leu Arg Trp Asn Gln Asp Thr Thr Leu Arg Asp Glu

50 55 60

Asp Lys Asn Gln His Cys Asp Gly Ser Asp Gly Cys Ser Val His Leu

65 70 75 80

Ser Arg Gly Thr Asn Thr Ser Asn Arg Asn Leu Glu Ala Pro Leu His

85 90 95

Arg Pro Val Ala Leu Pro Leu Ala Pro Ser Arg Leu Thr Arg Val Trp

100 105 110

Thr Gln Ala Ala Pro Gly Thr Pro Val Ser Glu His Gly Ala Gly Gln

115 120 125

Val Thr Leu Val Asp Arg Asp Gln Ala Phe Gln Leu Ser Gln Ala Pro

130 135 140

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

145 150 155 160

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

165 170 175

Asp Asn His Trp Ser Ala Thr Arg Gly Thr Ile Ser Thr Leu Ser Ser

180 185 190

His Met Leu Val

195

Claims (8)

1. An antibody or antibody fragment comprising the heavy chain sequence set forth in SEQ ID NO. 1 and the light chain sequence set forth in SEQ ID NO. 2.

2. The antibody or antibody fragment of any one of claim 1, wherein the antibody fragment is selected from the group consisting of a Fab fragment, a Fab ' fragment, a Fd fragment, an Fd ' fragment, an Fv fragment, a dAb fragment, a F (ab ')2 fragment, and a scFv fragment.

3. An antibody probe comprising the antibody or antibody fragment of any one of claims 1-2 and a fluorescent label.

4. The antibody probe of claim 3, wherein the fluorescent label is a quantum dot.

5. A method for sorting cells expressing CLDN18.2 using the antibody probe of claim 3.

6. The method of claim 5, which is a flow cytometric sorting method.

7. The method of claim 6, wherein the flow rate is 10000/s to 12000/s.

8. A method for single cell sequencing of CLDN18.2 expressing cells using the sorting method of claim 5.