CN106866820A

CN106866820A - A kind of monoclonal antibody and its application for capturing the anti-human Keratin 18 of tumour cell

Info

Publication number: CN106866820A
Application number: CN201710088383.6A
Authority: CN
Inventors: 王朝晖; 侯冰; 孟逊
Original assignee: Abby Mart (shanghai) Co Ltd Pharmaceutical Technology
Current assignee: Abby Mart (shanghai) Co Ltd Pharmaceutical Technology
Priority date: 2017-02-17
Filing date: 2017-02-17
Publication date: 2017-06-20
Anticipated expiration: 2037-02-17
Also published as: CN106866820B

Abstract

The invention discloses a kind of anti-CK18 monoclonal antibodies for capturing tumour cell, amino acid sequence of its weight chain variabl area sequence comprising the CDR3 shown in SEQ ID NO.8；With amino acid sequence of the light-chain variable sequence comprising the CDR3 shown in SEQ ID NO.15.The present invention is with the extracellular region polypeptide of human keratin 18 (CK18) as immunogene, immune mouse obtains lymphocyte, and fused cell is obtained using hybridoma cell fusion technology, obtain being enriched with the cell line of the monoclonal antibody of the anti-human Keratin 18 (CK18) of tumor living cell.Antibody maximum feature of the invention is to target CK18 extracellular regions, there is provided the lung carcinoma cell of enough affinity capture expression CK18；And be the CK18 antibody that living cells enrichment capture is can apply to known to first, being allowed to have is used for the potentially possible of circulating tumor cell (CTC) capture.

Description

A kind of monoclonal antibody for capturing the anti-human Keratin 18 of tumour cell and its Using

Technical field

The invention belongs to monoclonal antibody biological technical field, more particularly to a kind of anti-human angle for capturing tumour cell The monoclonal antibody of protein 18 and its application.

Background technology

Keratin 18 (CK18) is one kind of a keratin like protein, and size is about 48kd；Inside human body simple epithelium tissue Wide expression, general and CK8 occurs in pairs.The mutation of CK8/CK18 can cause various disease (Gastroenterology 2005；129:885).CK18 is the blood serum designated object of kinds of tumors diagnosis, while being also blood serum designated object (the Am J of hepatic injury Clin Pathol 2005；123:66, Clin Bi ochem 2002；35:327, J Histochem Cytochem 2005； 53:229)；And low expressions of the CK18 in breast cancer is mark (the Clin Cancer Res 2004 of poor prognosis；10: 2670)。

Into human peripheral blood tumour cell be referred to as circulating tumor cell (circulating tumor cell, CTC).Its capture, identification are played an important role in diagnosing tumor and accurate medical treatment guidance medication.CK18 is clinical wide The CTC appraisal mark things of general application.There is large-tonnage product to be based on antibody capture circulating tumor cell in existing market, and major part is Tumour cell is enriched with using antibody coupling other magnetic beads etc..But have no the product using CK18 antibody captures CTC.Market Upper existing CK18 antibody, whole no as shown by data can be used for tumor cell enrichment of living, so as to limit the anti-of in the market Body captures the application of this key areas in CTC.

The content of the invention

The first object of the present invention is to provide a kind of list for capturing the anti-human Keratin 18 (CK18) of tumour cell Clonal antibody, recognizes and combines the high-affinity mouse monoclonal antibody of human tumor cells film surface marker CK18 extracellular segments, resists this Body can be applied to for the living cells enrichment of CK18 albumen, sorting, flow cytometry, immunofluorescence, co-immunoprecipitation and Western Blotting。

The second object of the present invention is to provide a kind of above-mentioned anti-human Keratin 18 (CK18) for capturing tumour cell Application of the monoclonal antibody in tumor living cell is enriched with, be especially applicable to lung carcinoma cell etc. of capture enrichment expression CK18 Circulating cells.

Technical scheme is as follows：

The extracellular region polypeptide of present invention synthesis human keratin 18 (CK18), using it as immunogene, immune mouse is drenched Bar cell, and fused cell is obtained using hybridoma cell fusion technology, through Western blot, limiting dilution assay and ELISA method, Obtain being enriched with the cell line of the monoclonal antibody of the anti-human Keratin 18 (CK18) of tumor living cell, and by the cell line point The monoclonal antibody secreted, and through enzyme-linked, protein blot, immunofluorescence is immunized, co-immunoprecipitation adds mass spectrum and living cells hybridization to catch The checking such as obtain, determine most effective antibody.

A kind of anti-CK18 monoclonal antibodies for capturing tumour cell, the heavy chain of described anti-CK18 monoclonal antibodies can Become amino acid sequence of the region sequence comprising the CDR3 shown in SEQ ID NO.8；SEQ ID are included with light-chain variable sequence The amino acid sequence of the CDR3 shown in NO.15.

Preferably, amino acid sequence, SEQ of the described weight chain variabl area sequence comprising the CDR1 shown in SEQ ID NO.4 The amino acid sequence of the CDR3 shown in the amino acid sequence and SEQ ID NO.8 of the CDR2 shown in ID NO.6；Described light chain can The amino of the CDR3 shown in amino acid sequence of the change region sequence comprising the CDR1 shown in SEQ ID NO.11 and SEQ ID NO.15 Acid sequence.

Preferably, amino acid sequence, SEQ of the described weight chain variabl area sequence comprising the CDR1 shown in SEQ ID NO.4 The amino acid sequence of the CDR3 shown in the amino acid sequence and SEQ ID NO.8 of the CDR2 shown in ID NO.6；Described light chain can Change region sequence includes the amino acid of the CDR2 shown in the amino acid sequence of the CDR1 shown in SEQ ID NO.11, SEQ ID NO.13 The amino acid sequence of the CDR3 shown in sequence and SEQ ID NO.15.

Preferably, the framework region of described weight chain variabl area sequence is successively comprising the ammonia shown in SEQ ID NO.3,5,7 and 9 Base acid sequence, and described light-chain variable sequence framework region successively comprising the ammonia shown in SEQ ID NO.10,12,14 and 16 Base acid sequence.

Preferably, described weight chain variabl area sequence is the amino acid sequence shown in SEQ ID NO.1；Described light chain can Become amino acid sequence of the region sequence shown in SEQ ID NO.2.

Lived carefully in enrichment tumour for capturing the anti-CK18 monoclonal antibodies of tumour cell the invention also discloses above-mentioned Application in born of the same parents, particularly expresses the application on the circulating cells such as the lung carcinoma cell of CK18 in capture enrichment.

Compared with prior art, beneficial effects of the present invention are as follows：

By experimental verification, a kind of monoclonal for capturing the anti-human Keratin 18 (CK18) of tumour cell of the invention The features such as antibody has high-affinity, high specific, many application scenarios, maximum feature is to target CK18 extracellular regions, and is carried For the lung carcinoma cell of enough affinity capture expression CK18, and it is that living cells enrichment capture is can apply to known to first CK18 antibody, being allowed to have is used for the potentially possible of circulating tumor cell (CTC) capture.

Certainly, implement any product of the invention to it is not absolutely required to while reaching all the above advantage.

Brief description of the drawings

Fig. 1 is the schematic diagram of recombinant protein SDS-PAGE；

Fig. 2 is that PC9 human lung carcinoma cells verify schematic diagram in the intrinsic protein marking WB of clone's 1P1anti-CK18 antibody；

Fig. 3 is that PC9 human lung carcinoma cells verify schematic diagram in the immunofluorescence IF of clone's 1P1anti-CK18 antibody；

Fig. 4 is the Hybridization principle schematic diagram of PC9 human lung carcinoma cells and clone's 1P1anti-CK18 antibody；

Fig. 5 is the capture enrichment schematic diagram of PC9 human lung carcinoma cells and clone's 1P1anti-CK18 antibody.

Specific embodiment

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate this hair It is bright, rather than limit protection scope of the present invention.Those skilled in the art are according to changing that the present invention makes in actual applications Enter and adjust, still fall within protection scope of the present invention.

The not special explanation of the present invention, technological means used is well known to those skilled in the art in following examples Conventional technical means, and raw material, reagent used is also commercially available commercially available commodity.

A kind of preparation for capturing the anti-CK18 monoclonal antibody hybridoma cells of tumour cell of embodiment 1 and anti-CK18 The preparation and purification of monoclonal antibody

The preparation of 1.1 antigens

Synthesis people's CK18 extracellular regions polypeptide ' LEDGEDFNLGDALD ' used as immunogene, the polypeptide has been coupled VLP and biography The immunogenicity enhancer of KLH systems of uniting.

1.2 immune mouses

Every group of antigen will be for being immunized 6 Balb/c mouse (8-12 week old), and it is optimal to determine to monitor its serum titer Immune time.Each 6 immunogene polypeptide is mixed into one group.Each solvable fragment immunogen is individually for one group.The assistant for optimizing Agent and immunization method can produce the antibody (IgG hypotypes) of the high-affinity for most of antigen polypeptides.Can be through after initial immunity Cross the reinforcement of 3 to 4 times, mice serum will be taken after reinforcement and detects titre (recombinant protein is used as anti-antigen coat).Titre is qualified Mouse will impact once and be used to merge, and underproof mouse will continue one to strengthening twice, to titre highest after fusion.

CK18 recombinant proteins are by Shanghai Communications University systems generation research institute oncogene and related gene state key Laboratory provides, and protein sequence takes from UniProt P05783：

MSFTTRSTFSTNYRSLGSVQAPSYGARPVSSAASVYAGAGGSGSRISVSRSTSFRGGMGS

GGLATGIAGGLAGMGGIQNEKETMQSLNDRLASYLDRVRSLETENRRLESKIREHLEKKG

PQVRDWSHYFKIIEDLRAQIFANTVDNARIVLQIDNARLAADDFRVKYETELAMRQSVEN

DIHGLRKVIDDTNITRLQLETEIEALKEELLFMKKNHEEEVKGLQAQIASSGLTVEVDAP

KSQDLAKIMADIRAQYDELARKNREELDKYWSQQIEESTTVVTTQSAEVGAAETTLTELR

RTVQSLEIDLDSMRNLKASLENSLREVEARYALQMEQLNGILLHLESELAQTRAEGQRQA

QEYEALLNIKVKLEAEIATYRRLLEDGEDFNLGDALDSSNSMQTIQKTTTRRIVDGKVVS

ETNDTKVLRH

DNA sequence dna takes from NCBI CCDS31809.1：

ATGAGCTTCACCACTCGCTCCACCTTCTCCACCAACTACCGGTCCCTGGGCTCTGTCCAGGCGCCCAGC T

ACGGCGCCCGGCCGGTCAGCAGCGCGGCCAGCGTCTATGCAGGCGCTGGGGGCTCTGGTTCCCGGATCT C

CGTGTCCCGCTCCACCAGCTTCAGGGGCGGCATGGGGTCCGGGGGCCTGGCCACCGGGATAGCCGGGGG T

CTGGCAGGAATGGGAGGCATCCAGAACGAGAAGGAGACCATGCAAAGCCTGAACGACCGCCTGGCCTCT T

ACCTGGACAGAGTGAGGAGCCTGGAGACCGAGAACCGGAGGCTGGAGAGCAAAATCCGGGAGCACTTGG A

GAAGAAGGGACCCCAGGTCAGAGACTGGAGCCATTACTTCAAGATCATCGAGGACCTGAGGGCTCAGAT C

TTCGCAAATACTGTGGACAATGCCCGCATCGTTCTGCAGATTGACAATGCCCGTCTTGCTGCTGATGAC T

TTAGAGTCAAGTATGAGACAGAGCTGGCCATGCGCCAGTCTGTGGAGAACGACATCCATGGGCTCCGCA A

GGTCATTGATGACACCAATATCACACGACTGCAGCTGGAGACAGAGATCGAGGCTCTCAAGGAGGAGCT G

CTCTTCATGAAGAAGAACCACGAAGAGGAAGTAAAAGGCCTACAAGCCCAGATTGCCAGCTCTGGGTTG A

CCGTGGAGGTAGATGCCCCCAAATCTCAGGACCTCGCCAAGATCATGGCAGACATCCGGGCCCAATATG A

CGAGCTGGCTCGGAAGAACCGAGAGGAGCTAGACAAGTACTGGTCTCAGCAGATTGAGGAGAGCACCAC A

GTGGTCACCACACAGTCTGCTGAGGTTGGAGCTGCTGAGACGACGCTCACAGAGCTGAGACGTACAGTC C

AGTCCTTGGAGATCGACCTGGACTCCATGAGAAATCTGAAGGCCAGCTTGGAGAACAGCCTGAGGGAGG T

GGAGGCCCGCTACGCCCTACAGATGGAGCAGCTCAACGGGATCCTGCTGCACCTTGAGTCAGAGCTGGC A

CAGACCCGGGCAGAGGGACAGCGCCAGGCCCAGGAGTATGAGGCCCTGCTGAACATCAAGGTCAAGCTG G

AGGCTGAGATCGCCACCTACCGCCGCCTGCTGGAAGATGGCGAGGACTTTAATCTTGGTGATGCCTTGG A

CAGCAGCAACTCCATGCAAACCATCCAAAAGACCACCACCCGCCGGATAGTGGATGGCAAAGTGGTGTC T

GAGACCAATGACACCAAAGTTCTGAGGCATTAA

Protein expression vector uses Novagen pET28a expression vectors.

Expression system is E.coli Rosetta cells, IPTG induced expression systems.

Protein purification system is purified for Ni-NTA (Invitrogen), and it is qualified that SDS-PAGE check tables reach, as shown in Figure 1.

1.3 Virus monitories and screening

Immune mouse eye socket takes blood, and detects serum titer with ELISA (recombinant protein is used as antigen coat).Serum titer 10K need to be more than, otherwise continue booster immunization.

1.4 fusions and screening

Full spleen and 1/2 lymph node are taken, is merged with myeloma SP2/0 cell lines.Technique is the PEG fusions for optimizing.Melt Close cell and be taped against on 4 piece of 384 orifice plate (per hole cell 102 to 104), cultivated.The supernatant in all holes is collected, with ELISA pairs Polypeptide detection original is screened, and the positive hole that microscopy has cell goes to 96 orifice plates and continues to cultivate.Growth after a few days, collects all holes Supernatant detected with ELISA and solvable fragment detects former reaction.Further detect the solvable fragment of different dilution factors in positive hole Detection is former to be combined, to carry out affinity sequence.20 Parental clones of each polypeptide immunogen affinity highest enter subclone. 60 Parental clones of each solvable fragment immunogen affinity highest enter subclone.

1.5 subclones and screening

Screened by limiting dilution assay and ELISA and be subcloned, obtain monoclonal hybridoma.Cell spreads 96 holes Plate, and cultivate to the bottom of covering about 1/6.ELISA detects that each hole supernatant is former for the detection of solvable fragment and corresponding polypeptide is examined Former reaction is surveyed, OD values height is taken and two good holes of cell state is subcloned into lower whorl.In steps be repeated alternatively until hole Cell line positive rate 100%.Now we obtain monoclonal cell strain.After last wheel is subcloned, all positive cells Amplification Culture immediately a, part freezes after being provided with and uses, and another part carries out supernatant or prepared by ascites.

1.6 antibody supernatants are prepared and purified

Finally we obtain 8 plants of monoclonal cell strains, and are used for antibody producing by abdomen injection to F1 mouse.Produce Ascites Protein A/G are purified, and for subsequent detection.

The checking of the anti-CK18 monoclonal antibodies of embodiment 2

8 plants of cell strain of monoclonal antibody to obtaining carry out that enzyme-linked, protein blot, immunofluorescence, co-immunoprecipitation is immunized Plus mass spectrum, the checking such as living cells hybrid capture, determine most effective antibody.

Elisa (immune enzyme-linked) pair verification of 2.1 antibody and antigen polypeptide

Take ascites antibody to be paired and be coated with 96 hole elisa plates, be incubated, skim milk is overnight closed after washing, PBS washings, 4 DEG C preservation is stand-by.Antigen polypeptide is incubated, PBS washings, while setting control.HRP marks detection antibody, and being added to incubation has foregoing Elisa plate in.TMB chromogenic reactions, ELIASA reading.We screen and obtain 8 potency of cell line as shown in Table 1：

Table one

The intrinsic protein marking (WB) checking of 2.2 antibody

Use the full cell pyrolysis liquid of PC9 human lung cancer cell lines, antibody diluted concentration 1：1000 and 1：2000 carry out WB checkings. Experimental result shows, anti-CK18 (clone 1P1) in the WB checkings of PC-9, can with specific identification 48KD bands, with Expected size is consistent, as shown in Figure 2.

2.3 antibody mediated immunity fluorescence (IF) are verified

PC9 cells are fixed using PFA, and primary antibody incubation is carried out under conditions of not permeabilized cells, therefore antibody can directly be combined In memebrane protein extracellular region.The working concentration of anti-CK18 (clone 1P1) is 1：1000.Sheep anti mouse secondary antibody is coupled using FITC Developed the color, as a result as shown in figure 3, this antibody can apply to the immunofluorescence experiment of CK18.

Co-immunoprecipitation (IP) plus the mass spectrum checking of 2.4 antibody

PC9 LMP-1 mg are extracted, immunoprecipitation is carried out using anti-CK18 (clone 1P1), it is big that IP products cut 48kd Small band carries out Mass Spectrometer Method.As shown in Table 2, CK18 is largely enriched with mass spectral results in IP samples, illustrates this antibody to CK18 The specificity of identification is high.Also, this antibody can apply to IP experiments.

Table two

2.5 tumor living cell hybrid captures are tested

Anti-CK18 (clone 1P1) antibody and control antibodies point are formed on NC films as matrix using chip point sample instrument Sheet glass, form the antibody point of a diameter of 100um.By nucleus fluorescent color (dyestuff：Syto14 PC-9 cell suspensions) With 10⁷The density of/ml is incubated on antibody chip, is incubated at room temperature half an hour.PBS softly cleaning three times.Use GenePix fluorescence Chip scanner scans chip.Hybridization principle is illustrated as shown in Figure 4.

Experimental result as shown in figure 5, PC-9 cells are largely enriched with anti-CK18 (clone 1P1) antibody point, and Entirely without combination in control antibodies.Result illustrates that this antibody can apply to express the tumor living cell capture enrichment of CK18.

Embodiment 3

The variable region sequencing of anti-CK18 (clone 1P1) antibody is using Novagen Mouse Ig-Primer Set# 69831-3.RNA is extracted, reverse transcription cDNA, PCR amplification are according to Novagen User Protocol TB326Rev.C 0308. Extension increasing sequence clone is using Thermo Fisher TOPO TA Cloning Kit, Cat:450641.The carrier for building by GENEWIZ provides sequencing and Analysis Service.Sequence carries out variable region sequences analysis on www.igmt.org simultaneously.

Present invention disclosed above preferred embodiment is only intended to help and illustrates the present invention.Preferred embodiment is not detailed All of details is described, it is only described specific embodiment that the invention is not limited yet.Obviously, according to the content of this specification, Can make many modifications and variations.This specification is chosen and specifically describes these embodiments, is to preferably explain the present invention Principle and practical application so that skilled artisan can be best understood by and utilize the present invention.The present invention is only Limited by claims and its four corner and equivalent.

SEQUENCE LISTING

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Claims

1. a kind of anti-CK18 monoclonal antibodies for capturing tumour cell, it is characterised in that described anti-CK18 monoclonals resist Amino acid sequence of the weight chain variabl area sequence of body comprising the CDR3 shown in SEQ ID NO.8；Included with light-chain variable sequence The amino acid sequence of the CDR3 shown in SEQ ID NO.15.

2. anti-CK18 monoclonal antibodies for capturing tumour cell according to claim 1, it is characterised in that described Weight chain variabl area sequence includes the ammonia of the CDR2 shown in the amino acid sequence of the CDR1 shown in SEQ ID NO.4, SEQ ID NO.6 The amino acid sequence of the CDR3 shown in base acid sequence and SEQ ID NO.8；Described light-chain variable sequence includes SEQ ID The amino acid sequence of the CDR3 shown in the amino acid sequence and SEQ ID NO.15 of the CDR1 shown in NO.11.

3. anti-CK18 monoclonal antibodies for capturing tumour cell according to claim 1, it is characterised in that described Weight chain variabl area sequence includes the ammonia of the CDR2 shown in the amino acid sequence of the CDR1 shown in SEQ ID NO.4, SEQ ID NO.6 The amino acid sequence of the CDR3 shown in base acid sequence and SEQ ID NO.8；Described light-chain variable sequence includes SEQ ID The amino acid sequence and SEQ ID NO.15 of the CDR2 shown in the amino acid sequence of the CDR1 shown in NO.11, SEQ ID NO.13 The amino acid sequence of shown CDR3.

4. anti-CK18 monoclonal antibodies for capturing tumour cell according to claim 1, it is characterised in that described The framework region of weight chain variabl area sequence is successively comprising the amino acid sequence shown in SEQ ID NO.3,5,7 and 9, and described light chain The framework region of variable region sequences is successively comprising the amino acid sequence shown in SEQ ID NO.10,12,14 and 16.

5. anti-CK18 monoclonal antibodies for capturing tumour cell according to claim 1, it is characterised in that described Weight chain variabl area sequence is the amino acid sequence shown in SEQ ID NO.1；Described light-chain variable sequence is SEQ ID NO.2 Shown amino acid sequence.

6. described in claim 1 for capture the anti-CK18 monoclonal antibodies of tumour cell in tumor living cell is enriched with should With.

7. the anti-CK18 monoclonal antibodies for capturing tumour cell according to claim 6 are in tumor living cell is enriched with Application, it is characterised in that described tumor living cell be express CK18 lung carcinoma cell.