CN104292334B - A kind of anti-CTLA-4 monoclonal antibody of full source of people, preparation method and application - Google Patents
A kind of anti-CTLA-4 monoclonal antibody of full source of people, preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of anti-CTLA-4 monoclonal antibody of full source of people, preparation method and application, belong to that antibody drug is open and production technical field.By constructing the natural Large human naive scFv phage library of large capacity, filter out a kind of anti-CTLA-4 monoclonal antibody of full source of people, there is high-affinity and low dissociation rate (low immunogenicity) to people's CTLA-4 molecule, chimeric or humanization antibody is compared, can avoid many side effects when applying human body.The preparation method of the complete anti-CTLA-4 monoclonal antibody of source of people is simple in the present invention, the high efficient expression in zooblast, is suitable for scale industrial production.In addition, the complete anti-CTLA-4 monoclonal antibody of source of people can significantly inhibit growth of tumour cell, there is wide application prospect in terms of antitumor, anti-infective and treatment autoimmune disease.
Description
Technical field
The present invention relates to the preparation sides of a kind of complete anti-CTLA-4 monoclonal antibody of source of people and anti-CTLA-4 monoclonal antibody
Method and application belong to antibody drug opening and production technical field.
Background technique
The activation of T cell needs two to come from extracellular signal stimulus, i.e. the dual signal effect of T lymphocyte activation.T is thin
1st signal of born of the same parents' activation is tied mainly from T cell antigen identification receptor (TCR) and MHC molecule-antigenic peptide complexes specificity
It closes, this process is antigen recognizing.2nd signal of T cell activation is also known as costimulatory signal, is by antigen presenting cell (APC) and T
The adhesion molecule of cell surface is to offer.These adhesion molecules are referred to as costimulatory molecules, most important of which is that T cell surface
The surface CD28, CTLA-4 and APC respective ligand B7 (including B7-1 and B7-2).
(cytotoxicT lymphocyte associated antigen-4, Cytotoxic T lymphocytes are related by CTLA-4
Antigen -4) albumen contactin, in CD4+ the and CD8+T lymphocyte of activation and the bone-marrow-derived lymphocyte of activation
Surface expression.After cell-stimulating, CTLA-4, which relies on CD28 and expresses in cell surface, to be enhanced, and peak is reached after 48-72h.CTLA-4 with
CD28 has identical ligand B7-1 and B7-2, although the number of the T cell surface C TLA-4 of activation only has the 3%~5% of CD28,
But the affinity ratio CD28 of CTLA-4 and B7 is 10-50 times strong.
Early stage research prompt CTLA-4 provides costimulatory signal for the immune starting of T cell and development, to supplement CD28 biography
The signal led.But discovery CTLA-4 was opposite with CD28 effect later.In vitro and in vivo experiment prompt CTLA-4 are as CD28's
Antagonist participates in lowering t cell responses.Using the Fab segment of anti-CTLA-4mAbs, can promote T cell hyperplasia, promote cell because
Sublist reaches.It blocks CTLA-4 that T cell also can be improved to the reactivity of common antigen, reinforces the toxic reaction that super antigen mediates, mention
High body is antitumor, anti parasitic ability.The positive evidence that CTLA-4 lowers t cell responses is the small of CTLA-4 gene knockout
2-3 weeks lymphoproliferative disorder after mouse birth, the institutional framework for the T cell and autoimmunity sample that largely activate occur are destroyed,
3-4 weeks after birth, mouse is all dead.CTLA-4 can not only lower the reactivity of T cell, can also adjust the differentiation of T cell.
The CD4+T cell of CTLA-4 deficient mice is characterized by Th2 cell effect.Experiment in vitro discovery CTLA-4 can promote naivety
CD4+T cell to Th1 cell development.But it has been found that causing Th1 cell-mediated with anti-CTLA-4 antibody blocking CTLA-4PB7
Autoimmunity disease generation.Therefore, although CTLA-4 participates in the differentiation of regulatory T-cell, which kind of causes as a result, there is no at present
Final conclusion.Somebody find CTLA-4 can mediate T cell tolerance, block CTLA-4 can prevent tolerance generation.
Since CTLA-4 not only plays negative regulation to T cell activation, autoimmune tolerance can be also mediated, induction T is thin
Born of the same parents' differentiation maintains body cell P humoral immunity balance.So CTLA-4 may with T cell mediate autoimmune disease,
Graft-rejection and body are antitumor, anti-infective, antiallergy ability is related.It is expected to effective target as immunotherapy of tumors
Molecule, also the treatment for HIV and HBV chronic diseases poison infectious diseases and autoimmune disease provides a new strategy.
Summary of the invention
The object of the present invention is to provide a kind of complete anti-CTLA-4 monoclonal antibodies of source of people.
Meanwhile the present invention also provides a kind of preparation methods of complete anti-CTLA-4 monoclonal antibody of source of people.
Finally, the present invention also provides a kind of applications of complete anti-CTLA-4 monoclonal antibody of source of people.
In order to achieve the goal above, the technical scheme adopted by the invention is that：
A kind of anti-CTLA-4 monoclonal antibody of full source of people, containing heavy chain and light chain, (heavy chain and light chain pass through disulfide bond and connect
Connect), heavy chain variable region contains tri- hypervariable regions CDR1, CDR2 and CDR3, and light chain variable region contains CDR1 ', CDR2 ' and CDR3 '
Three hypervariable regions, the amino acid sequence in the area CDR1 such as SEQ ID NO:Shown in 1, the amino acid sequence in the area CDR2 such as SEQ ID NO:
Shown in 2, the amino acid sequence in the area CDR3 such as SEQ ID NO:Shown in 3, the amino acid sequence in the area CDR1 ' such as SEQ ID NO:4 institutes
Show, the amino acid sequence in the area CDR2 ' such as SEQ ID NO:Shown in 5, the amino acid sequence in the area CDR3 ' such as SEQ ID NO:Shown in 6.
Specifically, the anti-CTLA-4 monoclonal antibody of full source of people, the amino acid sequence of heavy chain variable region such as SEQ ID NO:7 institutes
Show, the amino acid sequence of light chain variable region such as SEQ ID NO:Shown in 8.
More specifically, in the anti-CTLA-4 monoclonal antibody of full source of people, heavy chain constant region is isotype people CHConstant region,
Constant region of light chain behaviour CLConstant region.
A kind of encoding gene of such as above-mentioned complete anti-CTLA-4 monoclonal antibody of source of people.
A kind of expression vector of the anti-CTLA-4 monoclonal antibody encoding gene containing full source of people.
A kind of recombinant cell for the expression vector converting the anti-CTLA-4 monoclonal antibody encoding gene containing full source of people, Su Zhuxi
Chinese hamster ovary cell (CHO), small hamster kidney cell (BHK), COS cell can be used in born of the same parents, and mouse NSO thymoma etc. is fed
Newborn zooblast.
A kind of antibody fragment obtained by digesting the anti-CTLA-4 monoclonal antibody of full source of people, such as Fab, Fab ', F (ab ') 2
Or Fv segment etc..
A kind of encoding gene of such as above-mentioned complete anti-CTLA-4 monoclonal antibody enzymatic fragment of source of people.
A kind of preparation method of the anti-CTLA-4 monoclonal antibody of full source of people, includes the following steps：
(1) Large human naive scFv phage library is constructed；
(2) from the anti-CTLA-4 single-chain antibody of the full source of people of Phage Antibody Library；
(3) the complete anti-CTLA-4 complete antibody molecule carrier for expression of eukaryon of source of people is constructed；
(4) the anti-CTLA-4 antibody molecule of the full source of people of host cell expression is converted, purifying obtains monoclonal antibody.
It is anti-in preparation to specifically include anti-CTLA-4 monoclonal antibody for a kind of application of the anti-CTLA-4 monoclonal antibody of full source of people
Application in terms of tumour, anti-infective and treatment autoimmune disease drug.
The tumour is liver cancer, breast cancer, squamous cell carcinoma, oophoroma, the colorectal cancer, stomach of high expression CTLA-4
The diseases such as cancer, gastrointestinal stromal tumor, bladder cancer, thyroid cancer, neck cancer, prostate cancer, prostate cancer, melanoma, epithelioma.
The infection includes HIV, hepatitis A virus, hepatitis B, hepatitis C virus, influenza virus, Respiratory Syncytial Virus(RSV), colyliform disease
Disease caused by poison, papillomavirus, poliovirus, rabies viruses etc. infect.
Beneficial effects of the present invention：
The present invention filters out a kind of anti-CTLA-4 Dan Ke of full source of people by the building natural Large human naive scFv phage library of large capacity
Grand antibody has high-affinity and low dissociation rate (low immunogenicity) to people's CTLA-4 molecule, compares chimeric or humanization
Antibody, can avoid many side effects when applying human body, such as side effect caused by HAMA and HACA reacts.Test proof,
The complete anti-CTLA-4 monoclonal antibody of source of people will not stimulate human blood cell discharge cell factor (IFN-γ, TNF-α, IL-2, IL-6 and
IL-10), the interaction that can significantly inhibit PD-L1 and CTLA-4 does not have an impact t cell proliferation.
The preparation method of the complete anti-CTLA-4 monoclonal antibody of source of people is simple in the present invention, the high efficient expression in zooblast,
Suitable for scale industrial production.In addition, the complete anti-CTLA-4 monoclonal antibody of source of people can significantly inhibit growth of tumour cell, anti-
There is wide application prospect in terms of tumour, anti-infective and treatment autoimmune disease.
Detailed description of the invention
Fig. 1 is full expression and purifying of the anti-CTLA-4 monoclonal antibody of source of people in Chinese hamster ovary celI in the embodiment of the present invention 1；
Fig. 2 is the binding characteristic of full source of people anti-CTLA-4 monoclonal antibody and people CTLA-4 in test example；
Fig. 3 is the binding characteristic of the complete anti-CTLA-4 monoclonal antibody of source of people and CTLA-4 on T cell plasma membrane in test example.
Specific embodiment
Only invention is further described in detail for following embodiments, but does not constitute any limitation of the invention.
Embodiment 1
(1) preparation of cDNA
Each 10 milliliters of mixing of peripheral blood of liver cancer, kidney and melanoma and Healthy People totally 50 people are collected, heparin sodium is anti-
Coalescence carries out external sensitization with CTLA-4 antigen using density-gradient centrifugation method separation mononuclearcell (PBMC).PBMC is external
After sensitization, by 4 × 1061ml EBV culture supernatant is added in PBMC, in 37 DEG C of incubation 3h, inhales and abandons supernatant, is added and contains 100ml/L
The RPMI1640 complete medium of FCS uses the RPMI1640 complete medium of the NBS containing 200ml/L instead after culture 2 weeks.Transformed cells
The screening of culture supernatant uses indirect elisa method, uses CTLA-4 as tumor-cell antigen solid phase plate, the B cell culture of collection
Supernatant is secondary antibody as primary antibody, goat anti-human igg/M (HRP-GAH-IgG/M) of HRP label.By the cell expansion culture in positive hole
To 106~107A cell.
(2) PCR amplification and connection of VH and VL gene
The primer of design synthesis amplification human immunoglobulin gene.Using the first chain of cDNA as template, PCR amplification VH and VL base is carried out
Cause.And VH and VL gene is set to be linked to be ScFv gene by encoding link peptide (Gly4Ser) 3.
(3) building of primary phage antibody library
With VHAnd VLGenetic fragment is template, according to the frequency of use of each subgroup of normal person, in appropriate proportions by amplified production
Mixing, is spliced by over-lap PCR, is expanded ScFv gene, is cloned into pComb3XSS carrier, Electroporation Transformation XL1-Blue, is counted
Number colony assay storage capacity is 107, phage antibody library is obtained through helper virus superinfection after bacterial multiplication, measures drop after precipitation concentration
Degree is 1013CFU/ml。
(4) Large phage library and elutriation are constructed
5 × 10 are taken out from primary phage antibody library12The phage particle of CFU is with (MOI=100/l) infection at high proportion
BS1365 bacterium, the positioning recombination mediated by loxp-ere exchange the light/heavy chain between same intracellular different carriers at random
Pairing, low ratio (MOI≤1) infects XL1-Blue bacterium to obtained bacteriophage supernatant again, and obtaining storage capacity is 1 × 1010Large capacity people
Source phage antibody library.Using CTLA-4 as antigen, the 4 affine screening processes of wheel are carried out to phage antibody library, measurement is thrown
Enter the number with the bacteriophage of output, output/input ratio increases about 500 times, illustrates to specifically bind CTLA-4 albumen
ScFv has obtained effective enrichment.
(5) screening combines the positive bacteriophage antibody monoclonal of CTLA-4
Every wheel selects 500 or so at random and separates good monoclonal, and picking 3-4 takes turns totally 1500 grams of output after elutriation
Grand progress bacteriophage monoclonal redemption, phage antibody are detected through ELISA, obtain the phage single-chain of specific binding CTLA-4
Antibody cloning.The bacteriophage monoclonal antibody positive by ELISA identification is passed through into streaming using straight mark FITC-AntiM13
It is detected in conjunction with CTLA-4, the phage single-chain antibody clone of specific binding CTLA-4 is obtained, to the positive colony of acquisition
Sequencing is carried out, and to its further analysis of amino acid sequence and CDR region.
(6) construction of eukaryotic expression vector of bacteriophage complete antibody
Using the plasmid of the 3 kinds of positive monoclonal antibodies screened as template, designed primer amplification light chain variable is utilized
Area VLGene, about 300bp.With amplification human antibody light chain constant region CLGene, then with VLAnd CLFor template, overlop PCR is utilized
L chain gene is amplified, is cloned into pMD19-T carrier, is built into pMD19-L, by pMD19-L with Hindlll and EcoRI enzyme
It cuts, is attached with the plasmid PcDNA3.1 (+) of same digestion, is built into carrier for expression of eukaryon.
Total serum IgE is extracted from Healthy Individuals, synthesizes cDNA with RT-PCR, then expand the area heavy chain CH gene, about
1000bp is cloned into pMD19-T carrier, is built into pMD19-CH.Using bacteriophage monoclonal antibody DNA as template, amplification weight
Chain variable region VHGene, about 300bp are cloned into pMD19-T carrier, are built into pMD19-VH.By pMD19-CH and pMD19-
VH uses Hindlll and Nhel double digestion respectively, and VH segment is connected in pMD19-CH carrier.Positive colony passes through again
Hindlll and EcoRI digestion is connected in pcDNA3.I (+), constructs carrier for expression of eukaryon.
(7) expression of the antibody in Chinese hamster ovary celI
3 × 10 are inoculated in 3.5cm tissue culture dishes5Chinese hamster ovary celI, cell culture to 90-95% are transfected when merging,
Above-mentioned carrier is linearized, transfection CHO cell.Transfection finishes, and cell is selected under G418 pressure, every concentration 1 time
Culture 2 weeks.96 orifice plate cultures to 70% coverage are then transferred to, Selective agar medium is changed into and (containing 5% dialysis fetal calf serum and fits
As horizontal G418) it is screened, and its growing state is monitored, until the cell of untransfected is die, leave behind the cell of transfection.
The plate with transfection cell is grown about 4 weeks, until forming cell mass.The cell mass that microscopy observation generates, until appropriate big
Small (greater than the > 60% of hole floor space), and confirm only one cell mass of every hole.According to the IgG κ ELISA inspection to cell conditioned medium
It surveys as a result, to filter out a expression more than 80 from 960 transfectants relatively high, and has carried out static culture mirror to it
It is fixed.Suspension culture is carried out in EX302 serum free medium to the cell line selected, with ELISA method into-step calibrating its express water
It puts down and cell growth rate is observed.According to the antibody concentration and acceptable growth characteristics of harvest supernatant, two are had selected
A best cell line carries out batch shaking flask culture in EX302 serum free medium.
(8) purifying of antibody
Purified with the Hi Trap Protein A chromatographic column of GE Healthcare to recombinant antibodies.As a result such as Fig. 1
It is shown.
Test example
(1) full influence of the anti-CTLA-4 monoclonal antibody of source of people to cytokine release in human blood
The anti-CTLA-4 monoclonal antibody of the full source of people purified in embodiment 1 is mixed with human whole blood, with determination
Whether individual CTLA-4 antibody stimulates human blood cell to discharge certain cell factors.The human whole blood of 500 μ l test tube of hepari is added
It is added in each hole.10 μ g or the anti-CTLA-4 human antibody of 100 μ g are added into each hole.There are some holes and anti-cd 3 antibodies-to rise
It incubates and is used as positive control, use human IgG1's antibody as negative control.Cell is incubated 6 in 37 DEG C.Cell is centrifuged simultaneously
The measurement that blood plasma is used for cell factor IFN-γ, TNF-α, IL-2, IL-6 and IL-10 is collected, the measurement utilizes cell factor
Cytolytic dose pearl array measuring method.The result shows that individually people will not be stimulated using the anti-CTLA-4 monoclonal antibody processing of full source of people
Haemocyte discharges cell factor (IFN-γ, TNF-α, IL-2, IL-6 and IL-10).
(2) full influence of the anti-CTLA-4 monoclonal antibody of source of people to t cell proliferation
Test is dyed using annexin V to measure the anti-CTLA-4 monoclonal antibody of full source of people to induction of T cell apoptosis
It influences.T cell is cultivated in mixed lymphocyte reaction (MLP), the anti-CTLA- of full source of people that concentration is 25 μ g/ml is added into test tube
4 monoclonal antibodies.Use non-specific antibody as control.Annexin V and propidium iodide are added according to standard scheme.It will mix
Object is closed in the dark in incubation at room temperature 15 minutes, is then analyzed using flow cytometer.The result shows that full source of people is anti-
CTLA-4 monoclonal antibody does not influence t cell proliferation.
(3) blocking effect of the complete anti-CTLA-4 monoclonal antibody of source of people to CTLA-4/PD-L signal
The anti-human anti-CD3mAb of excitated type mouse (0.5 μ g/ml), anti-CD3mAb (0.5 μ g/ml)+PD-L1 (1 μ g/ml)
96 well culture plates are coated with, every hole adds 100 μ l, and 4 DEG C overnight, sucks coating buffer, and PBS is washed 2 times.With RPMI1640 complete medium
T cell is adjusted to 1 × 105/ ml is inoculated in 96 orifice plate (100 hole μ l/), and various concentration (2 μ g/ml, 4 μ g/ml and 8 are added
μ g/ml) monoclonal antibody, while the anti-human CD28 monoclonal antibody of mouse (1 μ g/ml) is added and is placed in 37 DEG C, 5%CO2 culture.3 are set for every group in experiment
A multiple holes, and set human IgG and mouse IgG Isotype control group.Countingkit-8 is added after culture 4d, 10 holes μ l/ continue to train
Support 6h.Cell plates are collected to measure the light absorption value of each culture hole cell in 450nm wavelength and carry out statistical analysis.Resist as the result is shown
CTLA-4 antibody can significantly inhibit the interaction of PD-L1 and CTLA-4.
(4) the complete anti-CTLA-4 monoclonal antibody of source of people is in conjunction with people CTLA-4
The complete anti-CTLA-4 monoclonal antibody of source of people is gone out by ELiSA using standard method and program display in conjunction with people CTLA-4
Come (see Fig. 2).Then the CTLA-4 coating of microtiter plate purifying, the anti-human CTLA-4 antibody incubation with varied concentration are used
Coupling has the goat anti-human igg of alkaline phosphatase to develop the color.The anti-CTLA-4 monoclonal antibody of dose-dependent full source of people is illustrated in tables of data
In conjunction with it is higher that the half maximum combined at 14.5ng/m reflects that the anti-CTLA-4 monoclonal antibody of full source of people and CTLA-4 have
Binding ability, observed at about 0.1 μ g/ml in conjunction with saturation.
(5) the complete anti-CTLA-4 monoclonal antibody of source of people with express in conjunction in the CTLA-4 on T cell plasma membrane
The complete anti-CTLA-4 monoclonal antibody of source of people and the combination for expressing the CTLA-4 on T cell plasma membrane use gravity flow cell
Instrument analysis, is as a result shown in Fig. 3.Flow cytometry analysis is used to express the T cell system of high-caliber people CTLA-4 transfection.Varied concentration
Fluoresceinated anti-human CTLA-4 antibody and CTLA-4 positive cell incubate.The relevant fluorescence of cell is surveyed by flow cytometer
It is fixed.The complete anti-CTLA-4 monoclonal antibody of source of people in a dose-dependent manner with CTLA-4 expressivity cell combination, half maximum combined
For 200ng/ml, reach saturation at 2 μ g/ml.The anti-CTLA-4 monoclonal antibody of full source of people is not negative with any CTLA-4 of test
Property cell line combine, show the anti-CTLA-4 monoclonal antibody of full source of people to the specificity of people CTLA.
Claims (6)
1. a kind of anti-CTLA-4 monoclonal antibody of full source of people, it is characterised in that：The amino acid sequence of heavy chain variable region such as SEQ ID
NO:Shown in 7, the amino acid sequence of light chain variable region such as SEQ ID NO:Shown in 8.
2. a kind of encoding gene of the anti-CTLA-4 monoclonal antibody of full source of people as described in claim 1.
3. a kind of expression vector containing the anti-CTLA-4 monoclonal antibody encoding gene of full source of people as claimed in claim 2.
4. a kind of conversion expression vector of the anti-CTLA-4 monoclonal antibody encoding gene containing full source of people as claimed in claim 3
Recombinant cell.
5. a kind of enzymatic fragment of the anti-CTLA-4 monoclonal antibody of full source of people as described in claim 1, it is characterised in that：It is described
Enzymatic fragment is Fab, Fab ', F (ab ') 2 or Fv segment.
6. a kind of encoding gene of the anti-CTLA-4 monoclonal antibody enzymatic fragment of full source of people as claimed in claim 5.
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| CN107406504B (en) * | 2015-11-19 | 2021-04-30 | 蔡则玲 | CTLA-4 antibodies and uses thereof |
| EP4374926A3 (en) * | 2015-12-15 | 2024-08-07 | OncoC4, Inc. | Chimeric and humanized anti-human ctla4 monoclonal antibodies and uses thereof |
| CN106883298B (en) * | 2015-12-16 | 2021-12-17 | 上海康岱生物医药技术股份有限公司 | Bispecific conjugated antibodies, methods of making and uses thereof |
| US11542332B2 (en) | 2016-03-26 | 2023-01-03 | Bioatla, Inc. | Anti-CTLA4 antibodies, antibody fragments, their immunoconjugates and uses thereof |
| US11319372B2 (en) * | 2017-02-21 | 2022-05-03 | Remd Biotherapeutics, Inc. | Cancer treatment using antibodies that bind cytotoxic T-lymphocyte antigen-4 (CTLA-4) |
| CN108948194B (en) * | 2017-05-19 | 2023-02-17 | 上海药明生物技术有限公司 | Novel CTLA-4 monoclonal antibody |
| CN108003238B (en) * | 2017-11-30 | 2021-02-02 | 常州费洛斯药业科技有限公司 | Fully human monoclonal antibody or antibody fragment capable of specifically recognizing CTLA-4, and method and application thereof |
| KR102476887B1 (en) * | 2017-12-20 | 2022-12-13 | 하버 바이오메드 (상하이) 컴퍼니 리미티드 | Antibodies that bind CTLA-4 and uses thereof |
| CN110272490B (en) * | 2018-03-14 | 2021-05-14 | 上海开拓者生物医药有限公司 | Targeted CTLA-4 antibody, preparation method and application thereof |
| CN110760002A (en) * | 2018-07-25 | 2020-02-07 | 南京金斯瑞生物科技有限公司 | Humanized anti-human CTLA4 monoclonal antibody and preparation method and application thereof |
| EP3873940A4 (en) * | 2018-10-31 | 2022-11-30 | BioAtla, Inc. | Anti-ctla4 antibodies, antibody fragments, their immunoconjugates and uses thereof |
| CN110256563A (en) * | 2019-07-05 | 2019-09-20 | 石河子大学 | CTLA-4 nano antibody, preparation method and applications |
| CN110498856B (en) * | 2019-09-17 | 2020-08-11 | 浙江玉安康瑞生物科技有限公司 | CTLA-4 monoclonal antibody 5H7 and application thereof in tumor resistance |
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