CN106928361A - A kind of monoclonal antibody of anti-human PD-1 and its preparation method and application - Google Patents
A kind of monoclonal antibody of anti-human PD-1 and its preparation method and application Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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Abstract
The invention belongs to antibody art, more specifically, the invention discloses a kind of monoclonal antibody of anti-human PD-1 and its preparation method and application.Anti-human PD-1 monoclonal antibodies of the invention have good bioactivity, can effectively with reference to the extracellular region of people's PD-1 protein receptors, and can effectively close PD-1 albumen in protein level and cellular level, the combination of PD-1 albumen and part PD-L1 and PD-L2 is prevented, and effectively strengthens immunologic function.The monoclonal antibody can be individually or with other antineoplastic use in conjunction in the diagnosis and examination of immunotherapy of tumors and PD-L1 positive tumor patients, also with the good prospect for preparing the medicines such as treatment tumour, anti-autoimmune disease.
Description
Technical field
The present invention relates to biomedicine field, and in particular to a kind of monoclonal antibody of anti-human PD-1 and its preparation
Methods and applications.
Background technology
People's apoptosis acceptor -1 (PD-1) be it is a kind of have 288 I type memebrane proteins of amino acid, be
One of known principal immune checkpoint (Immune Checkpoint) (Blank et al, 2005, Cancer
Immunotherapy,54:307-314).PD-1 is expressed in the T lymphocytes for having activated, it and part
PD-L1 (programmed death receptor-ligand 1, programmed cell death-Ligand 1) and PD-L2 (journeys
Sequence death receptor-part 2, programmed cell death-Ligand 2) can to suppress T lymphs thin for combination
The active and related internal cell immune response of born of the same parents.PD-L2 is mainly expressed in macrophage and BMDC,
And PD-L1 then wide expression in B, T lymphocyte and peripheral cells such as microvascular endothelial cells, lung, liver,
In the histocytes such as the heart.Numerous studies show that the interaction of PD-1 and PD-L1 does not maintain still to exempt from vivo
Epidemic disease system balancing institute is necessary, is also to cause PD-L1 to express the main machine that positive tumor cell evades immunosurveillance
System and reason.By blocking negative regulation of the cancer cell to PD-1/PD-L1 signal paths, activating immune system,
The tumor specific cell immune response that T cell can be promoted related, controls so as to open the new tumour of a fan
The gate for the treatment of method --- tumour immunotherapy.
PD-1 (being encoded by gene Pdcd1) be the immunoglobulin superfamily relevant with CD28 and CTLA-4 into
Member.Achievement in research shows, can negative regulator antigen when PD-1 and its part (PD-L1 and/or PD-L2) are combined
Receptors signal transduction.The cocrystallization knot of mouse PD-1 structures and mouse PD-1 and human PD-L 1 has been understood fully at present
Structure (Zhang, X. etc., Immunity 20：337-347(2004)；Lin etc., Proc.Natl.Acad.Sci.USA 105：
3011-6(2008)).PD-1 and similar family member are I type transmembrane glycoproteins, and it contains responsible ligand binding
Ig changeable types (v-shaped) domain and responsible binding signal transduction molecule cytoplasmic tail.PD-1 cytoplasmic tails
Containing two based on tyrosine signal transduction die body ITIM (immunity receptor tyrosine inhibitory action die body) and
ITSM (immunity receptor tyrosine transformation die body).
PD-1 plays an important role in the immune evasion mechanism of tumour.Tumour immunotherapy, that is, utilize
The immune system of human body itself resists cancer, is a kind of breakthrough tumor therapeuticing method, but tumor microenvironment
Tumour cell can be protected from effective immune destruction, therefore how to break tumor microenvironment as antitumor research
Emphasis.Existing achievement in research has determined that effects of the PD-1 in tumor microenvironment：PD-L1 is many small
(and can be induced by IFN γ in most of PD-L1 negative tumor cell lines) is expressed in mouse and human tumour, and quilt
It is estimated as important target spot (Iwai Y. etc., Proc.Natl.Acad.Sci.U.S.A.99 of mediate tumor immune evasion：
12293-12297(2002)；Strome S.E. etc., Cancer Res., 63：6501-6505(2003).By exempting from
Epidemic disease histochemistry assesses biopsy, finds PD-1 (in tumour leaching in many primary tumors of people
On profit lymphocyte) and/or expression of the PD-L1 on tumour cell.It is such tissue include lung cancer, liver cancer,
Oophoroma, cervical carcinoma, cutaneum carcinoma, colon cancer, glioma, carcinoma of urinary bladder, breast cancer, kidney, esophagus
Cancer, stomach cancer, OSCC, urothelial cell cancer and cancer of pancreas and H/N tumors etc..Thus may be used
See, blocking the interaction of PD-1/PD-L1 can improve the immunocompetence of tumor specific T cells, help
Tumour cell is removed in immune system, therefore PD-1 turns into the popular target spot of exploitation immunotherapy of tumors medicine.
However, also there is the selectivity not relatively low defect of strong, affinity in existing anti-PD-1 monoclonal antibodies.
Therefore, the monoclonal antibody of new anti-PD-1 is researched and developed, and applies it to treatment tumour, anti-LADA
The preparation of the related drugs such as disease, as technical problem urgently to be resolved hurrily.
The content of the invention
The technical problems to be solved by the invention are to overcome anti-human PD-1 monoclonal antibodies at present to there is selection
Property weaker, the relatively low defect of affinity, there is provided a kind of monoclonal antibody of new anti-human PD-1, while also
There is provided the preparation method and application of the monoclonal antibody, so as to complete the present invention.
Therefore, first purpose of the invention is to provide a kind of monoclonal antibody of anti-human PD-1.
Second object of the present invention is the nucleic acid molecule for providing the coding anti-human PD-1 monoclonal antibodies.
Third object of the present invention is to provide the expression vector comprising the nucleic acid molecule.
Fourth object of the present invention is to provide the host cell comprising the expression vector.
5th purpose of the invention is to provide a kind of preparation method of the anti-human PD-1 monoclonal antibodies.
6th purpose of the invention is to provide the composition comprising the anti-human PD-1 monoclonal antibodies.
7th purpose of the invention is to provide the anti-human PD-1 monoclonal antibodies answering in medicine is prepared
With.
To achieve these goals, this invention takes following technical scheme：
One of technical scheme that the present invention takes is：A kind of monoclonal antibody of anti-human PD-1 is provided, it is described
Monoclonal antibody is included：
(1) complementary determining region of heavy chain CDR1, CDR2, CDR3, the amino acid sequence of the CDR1 is such as
SEQ ID NO：Shown in 1, the amino acid sequence such as SEQ ID NO of the CDR2：Shown in 2, the CDR3
Amino acid sequence such as SEQ ID NO：Shown in 3, and
(2) complementary determining region of light chain CDR1 ', CDR2 ', CDR3 ', the amino acid sequence of the CDR1 '
Such as SEQ ID NO：Shown in 4, the amino acid sequence such as SEQ ID NO of the CDR2 '：It is described shown in 5
The amino acid sequence of CDR3 ' such as SEQ ID NO：Shown in 6.
In this area, a light chain variable district and a weight chain variable district are typically each contained in the land of antibody, often
Contain tri- domains of CDR1, CDR2 and CDR3 in one variable region.Single-chain antibody of the present invention
It is conventional single-chain antibody, it includes weight chain variable district, light chain variable district and 15~20 small peptides of amino acid.
Preferably, the amino acid sequence such as SEQ of the weight chain variable district of the monoclonal antibody of anti-human PD-1 of the present invention
ID NO：Shown in 8, the amino acid sequence such as SEQ ID NO of its light chain variable district：Shown in 10.
Monoclonal antibody of the present invention can be prepared by this area routine techniques, including hybridoma technology, phagocytosis
Body display technology, single lymphocyte gene clone technology etc., preferably by hybridoma technology from wild type or
Transgenic mice prepares monoclonal antibody.
The two of the technical scheme that the present invention takes are：A kind of nucleic acid molecule, the nucleic acid molecule coding is as above
Described anti-human PD-1 monoclonal antibodies.
The nucleotide sequence of wherein described nucleic acid molecule encoding heavy chain variable region is preferably such as SEQ ID NO：7
Shown, the nucleotide sequence of coding light chain variable region is preferably such as SEQ ID NO：Shown in 9.
The preparation method of nucleic acid molecule of the present invention is the conventional preparation method in this area, be preferably comprised with
Lower preparation method：By gene clone technology such as PCR method etc., coding said monoclonal antibody is obtained
Nucleic acid molecule, or obtain encoding the nucleotides of said monoclonal antibody by the method that artificial complete sequence synthesizes
Molecule.
Those skilled in the art know, the nucleotide sequence for encoding the amino acid sequence of said monoclonal antibody can be with
Replacement, missing, change, insertion or increase is suitably introduced into provide a homologue for polynucleotide.This hair
The homologue of bright middle polynucleotide can be by one or more bases to encoding the monoclonal antibody gene
It is obtained keeping being replaced, lack or increasing in the range of antibody activity.
The three of the technical scheme that the present invention takes are：A kind of expression vector, the expression vector contains as described above
Nucleic acid molecule.
Wherein described expression vector is the conventional expression vector in this area, refers to comprising appropriate regulating and controlling sequence, example
Such as promoter sequence, terminator sequence, polyadenylation sequences, enhancer sequence, marker gene and/or sequence
The expression vector of row and other appropriate sequences.The expression vector can be virus or plasmid, as appropriate
Bacteriophage or phasmid, more ins and outs refer to such as Sambrook etc., Molecular Cloning：
A Laboratory Manual, the second edition, Cold Spring Harbor Laboratory Press, 1989.It is many
Known technology and scheme for nucleic-acid manipulation refer to Current Protocols in Molecular Biology,
The second edition, Ausubel etc. write.
Expression vector of the present invention is preferably pDR1, pcDNA3.1, pDHFF, GM-CSF or pCHO
1.0, more preferably it is pcDNA3.1.
The four of the technical scheme that the present invention takes are：A kind of host cell, the host cell contains as described above
Expression vector.
Host cell of the present invention is the conventional various host cells in this area, if can meet make it is above-mentioned heavy
Group expression vector is stably voluntarily replicated, and entrained described nucleotides can be by effective expression.Wherein institute
Stating host cell includes prokaryotic expression nucleus eukaryotic expression cell, and the expression vector is preferably comprised：COS、
CHO (Chinese hamster ovary, Chinese H amster Ovary), NS0, sf9, sf21, DH5 α, BL21 (DE3)
Or E.coli TG1, more preferably for E.coli TG1, BL21 cell (expression single-chain antibody or Fab antibody) or
Person CHO-K1 cells (expression total length IgG antibody).Foregoing expression vectors are converted into host cell, i.e.,
Currently preferred recombinant expression transformants can be obtained.Wherein described method for transformation is this area conventional transformation methods,
Preferably chemical transformation, heat shock method or electric robin.
The five of the technical scheme that the present invention takes are：A kind of monoclonal antibody of anti-human PD-1 as described above
Preparation method, the preparation method is comprised the following steps：
A) under expression condition, host cell of the present invention is cultivated, so as to express the Dan Ke of anti-human PD-1
Grand antibody；
B) monoclonal antibody of anti-human PD-1 simultaneously described in purification step a) is separated.
The cultural method of host cell of the present invention, the separation of the anti-human PD-1 monoclonal antibodies and pure
Change method is this area conventional method, and concrete operation method refer to corresponding cell culture technology handbook and list
Clonal antibody separating and purifying technology handbook.
The six of the technical scheme that the present invention takes are：A kind of composition, the composition contains as described above anti-
The monoclonal antibody and pharmaceutically acceptable carrier of people PD-1.
The anti-human PD-1 monoclonal antibodies that the present invention is provided, can be with pharmaceutically acceptable carrier together group
Into pharmaceutical preparations composition so as to more stably play curative effect, these preparations can ensure disclosed by the invention anti-human
The conformation integrality of the amino acid core sequence of PD-1 monoclonal antibodies, while going back the multifunctional of protected protein matter
Group prevents its degraded (including but not limited to cohesion, deamination or oxidation).Under normal circumstances, for liquid preparation,
Can generally be preserved under the conditions of 2 DEG C -8 DEG C and at least stablized 1 year, for lyophilized formulations, at 30 DEG C at least six
Keep stabilization within individual month.The anti-human PD-1 monoclonal antibody formulations can be pharmaceutical field commonly use suspension, liquid drugs injection,
It is lyophilized to wait preparation, preferably liquid drugs injection or lyophilized formulations,
For the liquid drugs injection or lyophilized formulations of anti-human PD-1 monoclonal antibodies disclosed by the invention, can pharmaceutically connect
The carrier received is preferably comprised but is not limited to：Surfactant, solution stabilizer, isotonic regulator and buffer solution
One or a combination set of.Wherein surfactant is preferably comprised but is not limited to：Nonionic surface active agent is as gathered
Oxygen ethene Span (polysorbas20 or 80)；Poloxamer (such as poloxamer 188)；Triton；
Lauryl sodium sulfate (SDS)；Sodium Laurylsulfate；Myristyl, sub- oil base or octadecyl methyl amimoacetic acid；
Pluronics；MONAQUATTMDeng its addition should make the granulating of anti-human PD-1 monoclonal antibodies become
Gesture is minimum.Solution stabilizer one or a combination set of is preferably comprised but is not limited to be exemplified below：Carbohydrate, for example,
Reducing sugar and nonreducing sugar；Amino acids, for example, monosodium glutamate or histidine；Alcohols, for example：
Trihydroxylic alcohol, senior sugar alcohol, propane diols, polyethylene glycol etc., the addition of solution stabilizer should make to eventually form
Preparation those skilled in the art think to reach stable state kept in the time of stabilization.Isotonic regulator compared with
One or a combination set of sodium chloride, mannitol are included but is not limited to goodly.Buffer solution is preferably comprised but is not limited to：
One or a combination set of Tris, histidine buffering liquid, phosphate buffer.
The seven of the technical scheme that the present invention takes are：The monoclonal antibody of anti-human PD-1 of the present invention is in system
Application in standby medicine.
Medicine of the present invention is preferably antitumor, treatment autoimmune disease, treatment infectious diseases
And/or the medicine of resisting transplant rejection reaction, it is more preferably antineoplastic, treats autoimmune disease medicine,
Preferably antineoplastic.The monoclonal antibody of anti-human PD-1 of the present invention can be used alone or with it is anti-
PD-L1 monoclonal antibodies or other antineoplastics are used in combination.
The targeted tumour of wherein described antineoplastic is preferably comprised but is not limited to：Lung cancer, liver cancer, ovum
Nest cancer, cervical carcinoma, cutaneum carcinoma, colon cancer, glioma, carcinoma of urinary bladder, breast cancer, kidney, cancer of the esophagus,
One kind or many in stomach cancer, OSCC, urothelial cell cancer, cancer of pancreas and/or H/N tumors
Kind.
Antineoplastic alleged by the present invention, refers to the medicine for suppressing and/or treating tumour, can include companion
The delay and/or the reduction of these severity of symptom developed with tumor-related symptoms, further comprise and have deposited
Tumour simultaneous phenomenon mitigation and prevent the appearance of other symptoms, also including reducing or preventing the transfer of tumour
Deng.
Anti- PD-L1 monoclonal antibodies and combinations thereof are to animal including people when being administered in the present invention,
Age and body weight of the dosage because of patient, disease traits and seriousness, and method of administration and different, Ke Yican
The result and a variety of situations of zoopery are examined, total dosage is no more than certain limit.Specifically it is injected intravenously
Dosage is 1-1800mg/ days.
On the basis of common sense in the field is met, above-mentioned each optimum condition can be combined, and obtain final product of the invention each
Preferred embodiments.
Agents useful for same of the present invention and raw material are commercially available.
Positive effect of the invention is：The anti-human PD-1 monoclonal antibodies that the present invention is provided have good
Bioactivity, can effectively with reference to the extracellular region of people's PD-1 protein receptors, and can be in protein level and thin
Born of the same parents' level effectively closes PD-1 albumen, prevents the combination of PD-1 albumen and part PD-L1 and PD-L2.Should
Monoclonal antibody can be individually or with other antineoplastic use in conjunction in immunotherapy of tumors and PD-L1 sun
Property tumour patient diagnosis and examination in, i.e., can effectively apply to treatment tumour, infectious diseases, itself exempt from
In the preparation of medicine such as epidemic disease disease and anti-immunity repulsion.
Brief description of the drawings
Fig. 1 is pcDNA3.1 plasmid construct schematic diagrames.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but is not therefore limited the present invention to described
Scope of embodiments among.The experimental technique of unreceipted actual conditions in the following example, conventionally and
Condition, or selected according to catalogue.Room temperature described in embodiment is the conventional room temperature in this area, typically
It is 10~30 DEG C.
The preparation method of the anti-human PD-1 monoclonal antibodies disclosed in the present invention includes：Under expression condition, training
Above-mentioned host cell is supported, so as to express anti-human PD-1 monoclonal antibodies；Separate and the described anti-human PD-1 of purifying
Monoclonal antibody.Can be substantially uniform material by recombinant protein purification using the above method, for example, exist
It is single band on SDS-PAGE electrophoresis.
Anti-human PD-1 monoclonal antibodies disclosed by the invention can be separated using the method for affinity chromatography
Purifying, according to the characteristic of the affinity column for being utilized, it is possible to use conventional method such as high-salt buffer, change
Monoclonal antibody of the method elution of bound such as PH on affinity column.The present inventor PD-1 anti-human to gained
Monoclonal antibody carried out test experience, test result indicate that the monoclonal antibody can well with people PD-1
With reference to affinity higher.
The preparation of the PD-1 antigens of embodiment 1
In order to produce the antibody of anti-human PD-1 (hPD-1), encoding human source PD-1 total length ammonia is obtained by PCR method
The cDNA of the open read frame of base acid sequence, and by gained cDNA be subcloned into carrier pcDNA3.1 (Invitrogen,
Carlsbad, CA) obtain hPD-1 carriers.The structure of wherein described carrier pcDNA3.1 is as shown in figure 1, described
CDNA sequence such as SEQ ID NO:Shown in 11, the GenBank numberings of the sequence are：BC074740.2.
With gained hPD-1 carrier stable transfection CHO-K1 cells (being purchased from Life technologies companies), use
Flow cytometry (FACS) monitors the expression of PD-1, separates CHO-K1 grams in its film upper table intelligent PD-1
It is grand, and CHO-hPD1 is named as, concrete operation method refers to the cell technology handbook of correlation.
By the golden bullet (BioRad) with Helios particle guns (BioRad) and coating DNA, according to manufacturer's usage
Illustrate to carry out Gene gun immunization Mice Inoculated.In short, with the gained hPD-1 carriers of 2: 1: 1 ratios and city
Sell mouse Flt3L expression vectors and mouse GM-CSF carriers (the two is all bought from Aldevron, Fargo ND) are applied
1 μm of golden particulate is covered, the golden bullet of 500 μ g is coated with the DNA that gross mass is 1 μ g.
Specifically, to 6~8 week old Harbour human antibodies transgenic mices (purchased from Beijing company of dimension tonneau China)
Immunity inoculation is carried out, mouse is raised under the conditions of SPF.With particle gun in Harbour human antibody transgenic mices
Impact immunity inoculation is carried out on ear, 2,3 or 4 bombardments in cycle is received on ears, wherein a mouse exists
Abdominal cavity receives 5 × 106The last booster immunization inoculation of individual CHO-hPD1 cells.By using cell ELISA method
Detection CHO-hPD-1 clones and CHO-K1 mother cells, find after result is contrasted：DNA immunization
The titre of antibody is more than 1: 1000 in postvaccinal mice serum.
The method of the cell ELISA is comprised the following steps：In 50 μ L culture mediums (DMEM/HAMShi F12/10%
FBS CHO-hPD-1 cell culture is converged to 80-100% in).It is subsequently added into 50 μ L and contains various concentration
The culture medium of mAb is purified, is incubated 1 hour at 37 DEG C.After washing 3 times with PBS-Tween, 100 μ L are added
Goat anti-mouse-HRP (Southern Biotech, catalog number 1030-05) is (dilute with 1: 1000 in the medium
Release), it is incubated 1 hour at 37 DEG C.After washing 3 times again with PBS-Tween, with colour developing peroxidase substrate
TMB (BD Biosciences) manifests the immunoglobulin of immobilization.Read in microtiter plate reader
The value added (450nm) of absorbance.
The preparation of the hybridoma of embodiment 2, screen and build storehouse
Four days after last immunity inoculation, put to death mouse, according to existing document (Steenbakkers etc., 1992,
J.Immunol.Meth.152：69-77；Steenbakkers etc., 1994, Mol.Biol.Rep.19：125-134),
The splenocyte group of removal red blood cell is prepared, gained splenocyte is merged.Take the good hybridoma of growth conditions
Sp2/0 cells (to be derived from the American Type Culture Collection committee of Chinese Academy of Sciences cell bank) are in 37 DEG C, 5%CO2
Cultivated in incubator, fusion the previous day changes liquid.Fusion is as follows with screening process：Mouse spleen is taken, after grinding washing
Count.According to splenocyte:Sp2/0 cell=10:1 two kinds of cells of mixing, 1500rpm is centrifuged 7 minutes.Wash away supernatant
Liquid.Add 1ml PEG (1450), jog 90 seconds that serum-free DMEM was added in 2.5 minutes in 1 minute
Nutrient solution (be purchased from Gibco companies) 5ml, again property add 5ml serum-free medium terminating reactions, stand 5
Minute, 1280rpm is centrifuged 8 minutes.Uniform amount according to one piece of 96 orifice plate, 2,000,000 sp2/0 cells is inoculated with
Enter 96 orifice plates per the μ l of hole 200, first with containing hypoxanthine (hypoxanthine, H), MTX (aminopterin,
A) and thymidine (thymidine, T) HAT Screening of Media, every 3~4 days half amounts change liquid,
Use HT culture mediums instead within 10 days.After 14 days cell is screened with ELISA and Acumen (microwell plate cell assay)
Fusion plate supernatant, by OD in ELISA450nm>MFI values in 1.0 and Acumen>100 positive colony amplification is arrived
24 orifice plates, containing 10% (w/w) HT hyclones, DMEM (invitrogen) is in 37 DEG C, 5% (v/v)
CO2Under the conditions of Amplification Culture.
Wherein described ELISA method step is：The immunogene PD-1 or other immune detection point albumen that will be purified
(buying from Southern Biotech) is diluted to the μ g/mL of final concentration 1.0 with PBS respectively, then with 100 μ l per hole
It is added to 96 hole elisa plates.4 DEG C of overnight incubations are sealed with plastic foil, second day with board-washing liquid [PBS+0.01% (v/v)
Tween20] board-washing 2 times, add confining liquid [PBS+0.01% (v/v) Tween20+1% (w/w) BSA] room
Temperature closing 2 hours.Confining liquid, plus PD-1 antibody (buying from Southern Biotech) 100 μ l are outwelled per hole.
After 37 DEG C are incubated 2 hours, with board-washing liquid [PBS+0.01% (v/v) Tween20] board-washing 3 times.Add HRP (peppery
Root peroxidase) mark secondary antibody (be purchased from Sigma), after 37 DEG C are incubated 2 hours, with board-washing liquid [PBS+0.01%
(v/v) Tween20] board-washing 3 times.The μ l of tmb substrate 100 are added per hole, after being incubated at room temperature 30 minutes, is added
The μ l of terminate liquid (1.0N HCl) 100 are per hole.Read with ELISA plate reading machines (being purchased from Molecular Device)
A450nm numerical value.
Culture takes Amplification Culture in 24 orifice plates nutrient solution after 3 days is centrifuged, and supernatant is collected, to supernatant
Antibody subtype analysis is carried out, the combination to PD-1 albumen and PD-1 positive cells is determined using above-mentioned ELISA method
Activity.According to 24 orifice plate the selection results, OD in ELISA experiments is selected450nm>1.0th, MFI in FACS experiments
Value>50 and be qualified positive colony, and be subcloned by limiting dilution assay, obtain stabilization expression
The hybridoma cell strain of the PD-1 monoclonal antibodies.Gained hybridoma cell strain is carried out into conservation and builds storehouse, and
Can be used for follow-up antibody producing and purifying.
The production and purifying of the anti-human PD-1 monoclonal antibodies of embodiment 3
The AC that hybridoma is produced is relatively low, and only about 1-15 μ g/ milliliters, change in concentration is larger.And
Hyclone composition contained by multiple protein and culture medium in culture medium produced by cell culture is to many biological living
Property analysis method have different degrees of interference, it is therefore desirable to carry out small-scale (1-5 milligrams) antibody producing pure
Change.
The hybridoma of the gained of embodiment 2 is inoculated into T-75 Tissue Culture Flasks and uses production medium
(Hybridoma serum free medium, purchased from Invitrogen companies) domestication 3 generations of passage.Treat that it grows
It is in good condition, inoculating cell culture rolling bottle.500 milliliters of production mediums are added in each 2 liters culture rolling bottle,
Inoculating cell density is 1.0 × 105/ milliliter.Bottle cap is covered tightly, on the Rotary Machine that rolling bottle is placed in 37 DEG C of incubators,
Rotating speed 5RPM.Continuous rotating and culturing collects cell culture fluid, filtering removal cell, and use 0.45 after 16 days
The membrane filtration of micron to culture supernatant is clarified.The culture supernatant of clarification can be purified or -30 DEG C at once
Freeze.
Monoclonal antibody 3mL Protein G posts in the culture supernatant (500mL) of the hybridoma of clarification
(being purchased from GE Healthcare) purifying.Protein G post first uses level pad (PBS phosphate buffers, pH7.5)
Balance, is then loaded to Protein G post by the culture supernatant of clarification, and coutroi velocity was at 5mL/ minutes.Loading is complete
Protein G post is cleaned after finishing with level pad, the volume of level pad is 5 times of Protein G post bed volumes.With
Eluent (the sweet ammonia salt acid buffers of 0.1M, pH3.0) the PD-1 antibody of elution of bound on Protein G post, uses
UV-detector monitors elution profile (A280 ultraviolet absorption peaks).The antibody of wash-out is collected, 10%1.0M is added
In Tris-HCl buffer solutions and pH value, the percentage is percent by volume, and PBS phosphoric acid buffers are used immediately after
Liquid dialysed overnight, changes liquid 1 time for second day and continues dialysis 6 hours.The PD-1 antibody after dialysis is collected, with 0.22
The filter of micron is sterile filtered, and Preservation in sterile condition obtains final product the PD-1 monoclonal antibodies of purifying.
The PD-1 antibody of purifying is carried out into protein concentration (A280/1.4), purity, endotoxin (Lonza reagents
Box) etc. detection and analysis, testing result shows：The endotoxin concns of gained monoclonal antibody finished product are in 1.0EU/
Within milligram.
The affinity analysis of the anti-human PD-1 monoclonal antibodies of embodiment 4
The monoclonal antibody of anti-human PD-1 and the binding affinity of people PD-1/Fc are determined using ELISA method.
People hPD-1/Fc (R＆D Systems) is immobilized in by being incubated at room temperature 4 hours (or at 4 DEG C overnight)
On Maxisorp 96- orifice plates (Nunc).By with containing 3%BSA PBST be incubated at room temperature 1 hour it is non-to block
Specific binding site.After coating, the plate is washed with PBST 3 times.(contain 0.1% with combination buffer
The PBS of Tween 20 and 0.3%BSA) prepare anti-PD-1 monoclonal antibodies of the present invention and commercially available
The dilution of positive control PD-1 antibody, gained dilution and the fusion protein of immobilization are incubated at 25 DEG C
1 hour.With reference to rear, the plate is washed with PBST 3 times, with containing the mistake for being diluted to 1/4,000 at 25 DEG C
The combination buffer of the secondary antibody (Southern Biotech) of oxide enzyme mark is incubated 1 hour, washs again, uses
TMB develops the color.With half maximum combined concentration (EC50) represent RA value, testing result
As shown in table 1.
Table 1, the monoclonal antibody of anti-human PD-1 are to the affinity of people PD-1
| Antibody | EC50(pM) | Kd(1/s) | KD(pM) |
| The monoclonal antibody of the anti-human PD-1 of the present invention | 76 | 1.35E+06 | 2.34E-11 |
| Positive control PD-1 antibody | 420 | 8.42E+04 | 1.78E-09 |
The result of table 1 can illustrate that the affinity of the anti-human PD-1 monoclonal antibodies of present invention gained is higher than the positive
Control PD-1 antibody, effectively can be combined, with affinity higher with PD-1.
The measure of the anti-human PD-1 monoclonal antibody genes sequence of embodiment 5
The total serum IgE of the gained hybridoma cell strain of embodiment 2 is extracted using Trizol (giving birth to work purchased from Shanghai biological),
With Reverse Transcriptase kit (being purchased from Takara companies) by mRNA reverse transcriptions into cDNA, by PCR method
The light chain and heavy chain variable region gene of anti-human PD-1 monoclonal antibodies are expanded, then PCR primer is cloned into
PMD18-T carriers, transfer to Sheng Gong companies to be sequenced and analyze variable region gene sequence.As a result：Gained PD-1 is mono-
The heavy chain variable region gene sequence 369bp of clonal antibody, encodes 123 amino acid residues, its nucleotides
Sequence such as SEQ ID NO：Shown in 7, coding gained amino acid sequence such as SEQ ID NO：Shown in 8；Light chain
Variable region gene sequence total length 336bp, encodes 112 amino acid residues, nucleotide sequence such as SEQ ID NO：
Shown in 9, amino acid sequence such as SEQ ID NO：Shown in 10.Amino acid sequence is carried out in GeneBank
Analysis is compared, the two meets the feature of people's PD-1 variable region genes.
Embodiment 6 detect anti-human PD-1 monoclonal antibodies blocks PD-1 albumen and its part PD-L1 and
The combination of PD-L2
PD-1 antibody blocking PD-1 albumen and its part are detected by the receptor ligand binding assay of PD-1 albumen
The combination of PD-L1 and PD-L2.
Prepare PD-1 extracellular region proteins (PD1-hFc)
Nucleotide sequence containing encoding human source PD-1 protein extracellulars amino acid sequence is cloned into band someone
PCpC carriers (be purchased from Invitrogen) of IgG Fc fragments (hFc) and by the standard molecular biological set up
Method prepares plasmid, and specific method is referring to Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989)
Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:
Cold Spring Harbor Laboratory Press).Wink is carried out to HEK293 cells (being purchased from Invitrogen)
When transfection (PEI, Polysciences) and use FreeStyleTM293 (Invitrogen) are carried out at 37 DEG C
Amplification Culture.Cell culture fluid is collected after 4 days, centrifugation removal cell component obtains protein extracellular containing PD-1
Culture supernatant.Culture supernatant is loaded to protein A affinity chromatography post (purchased from GE Healthcare),
The change of ultraviolet absorption value (A280nm) is monitored with ultraviolet (UV) detector simultaneously.PBS is used after loading
Phosphate buffer (pH7.2) cleans protein A affinity chromatography post until ultraviolet absorption value returns to baseline, then
Eluted with 0.1M glycine hydrochlorides (pH2.5), the band that collection is eluted from protein A affinity chromatography post
The PD-1 albumen (PD1-hFc) of hFc labels, it is saturating in 4 DEG C of refrigerators with PBS phosphate buffers (pH7.2)
Analysis is overnight.Albumen after dialysis is sub-packed in -80 DEG C of preservations after being sterile filtered through 0.22 micron, that is, obtain purifying
PD-1 extracellular region proteins (PD1-hFc) albumen.
The preparation of PD-L1 extracellular region proteins (PD-L1-hFc) and PD-L2 extracellular region proteins (PD-L2hFc)
And biotin labeling
The preparation method of PD-L1 extracellular region proteins (PD-L1-hFc) and the preparation method phase of above-mentioned PD1-hFc
Together, the wherein amino acid sequence information of PD-L1 extracellular region proteins is referring to Uniprot databases, numbering
Q9NZQ7.1.The biotin labeling method of PD-L1-hFc is：Gained PD-L1-hFc and biotinylation are tried
Agent reaction was both obtained.The biotinylation reagent is purchased from Sigma, article number B3295；It is anti-with biotinylation reagent
The operating procedure answered refers to the specification of the biotinylation reagent.The PD-L1 of gained biotin labeling is extracellular
Area's albumen is named as：PD-L1-ECD.
The preparation method and biotin labeling method of PD-L2 extracellular region proteins (PD-L2-ECD) with it is described
The preparation method of PD-L1-ECD is identical, wherein the amino acid sequence information of PD-L2 extracellular region proteins referring to
Uniprot databases, numbering Q9BQ51.The PD-L2 extracellular region proteins of gained biotin labeling are named as：
PD-L2-ECD。
Gained PD-1 extracellular region proteins (PD1-hFc) is diluted to the μ g/mL of final concentration 1.0 with PBS, then
With 100 μ l 96 hole elisa plates are added to per hole.4 DEG C of overnight incubations are sealed with plastic foil, board-washing is used within second day
Liquid [PBS+0.01% (v/v) Tween20] board-washing 2 times, adds confining liquid [PBS+0.01% (v/v) Tween20
+ 1% (w/w) BSA] room temperature close 2.5 hours.Confining liquid is outwelled, the gained of embodiment 3 is first added per hole
The μ l of PD-1 monoclonal antibodies testing sample 50 of purifying, then the PD-L1 extracellular regions for being separately added into biotin labeling
The PD-L2 extracellular region proteins (PD-L2-ECD) of albumen (PD-L1-ECD) and biotin labeling are per hole
100 microlitres, after mixing 37 DEG C be incubated 2 hours after, washed with board-washing liquid [PBS+0.01% (v/v) Tween20]
Plate 3 times.Avidin (being purchased from Sigma) dilution of HRP (horseradish peroxidase) marks is added per hole
100 microlitres, after 37 DEG C are incubated 2 hours, with board-washing liquid [PBS+0.01% (v/v) Tween20] board-washing 3 times.
The μ l of tmb substrate 100 are added per hole, after being incubated at room temperature 30 minutes, terminate liquid (1.0N HCl) 100 μ l is added
Per hole.A450nm is read with ELISA plate reading machines (SpectraMax 384plus, Molecular Device)
Numerical value.
Experimental results show that the anti-human PD-1 monoclonal antibodies that the present invention is provided can effectively close PD-1
The combination of acceptor and its part PD-L1 and PD-L2, with preferable bioactivity and selection blocking property,
With the potentiality for preparing antineoplastic.
The LS of embodiment 7 experiment detection PD-1 antibody blocking PD-L1 or PD-L2 drenches to T
The suppression of bar cell
LS experiment detection PD-1 antibody blocking PD-1 albumen and its part PD-L1 and PD-L2
Combination so as to release its suppression to T lymphocyte activities.
(1) Ficoll separates whole blood and obtains peripheral blood mononuclear lymphocyte PBMC.
By the whole blood of fresh acquisition with phosphate buffer PBS with 1:1 volume ratio dilutes the whole blood after must diluting,
Gently the whole blood after dilution is paved in Ficoll liquid levels (purchased from GE Healthcare), Ficoll with aseptic straw
It is 3 with the volume ratio of the whole blood after dilution:4, it is to avoid concussion is mixed, with 400g rotating speed room temperatures gradient centrifugation 45
Minute, the centrifuge tube after centrifugation is divided into three layers, and upper strata is blood plasma, and it is thin that middle milky layering is monokaryon lymph
Born of the same parents, with aseptic straw gentle aspiration intermediate layer cell, collect to new centrifuge tube, use PBS phosphate buffers
Three times volume is diluted to, 150g rotating speeds room temperature is centrifuged 8 minutes, abandons supernatant.By lymphocyte PBS phosphoric acid
Buffer solution is resuspended to 15mL, repeats preceding step and takes out blood platelet, finally that lymphocyte is resuspended to 15mL
Many component RPMI1640 culture mediums (being purchased from Invitrogen) containing 10% hyclone are standby, as outer
All blood monokaryon lymphocyte PBMC, the percentage is mass percent.
(2) PBMC stimulation tests
To build PDL1 and anti-CD3 the single-chain antibodies CHOK1 that is co-expressed on cell membrane or 293F
Stable cell line, constructs the plasmid of the nucleotide sequence containing coding PD-L1 full-length proteins
pIRES-puro-PD-L1；To make anti-CD3 (OKT3) (referring to Kipriyanov et al.1997, PEDS
10:445-453) chimeric ScFv can be anchored on cell membrane, by the ScFv and mouse CD8a (NCBI
Accession No:NP_001074579.1 C-terminal 113-220 amino acid sequences connection), is built into plasmid
pIRES-OS8.(preparation method of Plasmid pIRES-puro-PD-L1 and Plasmid pIRES-OS8 is referring to Joe
Sambrook, Molecular Cloning:A Laboratory Manual).By above-mentioned plasmid
PIRES-puro-PD-L1 and Plasmid pIRES-OS8 transfect CHOK1 and 293F cells jointly, by foregoing
The method of plasmid-transfected cells prepares stabilization passage cell strain CHOK1-PDL1/OS8 and stablizes passage cell strain
293F-PDL1/OS8, and by both as T lymphocyte stimulating factors.Using preceding, by T lymphocytes thorn
Swash 10 μ g/ml mitomycins of factor CHOK1-PDL1/OS8 and 293F-PDL1/OS8,37 DEG C for the treatment of 3
Hour.The PD-1 antibody of the purifying of isometric gained of embodiment to be measured 3 than dilution is prepared simultaneously, obtains to be measured
Sample solution.
By peripheral blood mononuclear lymphocyte PBMC with 8 × 104150 microlitres of individual cell is per hole Pu Zhi96 holes cell
Culture plate, then adds culture plate by described testing sample solution, and incubated at room temperature 20 minutes is eventually adding
T lymphocyte stimulating factor CHOK1-PDL1/OS8 or 293F-PDL1/OS8,50 are contained in every reacting hole
Microlitres 6 × 103Individual T lymphocyte stimulating factors, it is ensured that each μ L volume of reacting hole 200, by reaction plate in
37 DEG C, 5%CO2Incubator culture collects supernatant after 48 hours, obtains cell supernatant, is frozen in -20 DEG C, institute
Percentage is stated for percent by volume.
(3) cell factor gamma interferon (IFN-γ) or interleukin I L-2 enzyme linked immunologicals in cell conditioned medium
Absorption detection.
Cell factor gamma interferon (IFN-γ) or interleukin I L-2 Enzyme-linked Immunosorbent Assays are examined in cell conditioned medium
Survey using R＆D system coherent detections kit Human IFN-gamma Quantikin (SIF50) and
Quantikine ELISA human IL-2 detection kits (S2050), and operate to specifications.Except to be measured
All detection reagents outside sample are provided by detection kit.
Cell factor gamma interferon (IFN-γ) or interleukin I L-2 Enzyme-linked Immunosorbent Assays are examined in cell conditioned medium
Survey using double crush syndrome kit (being purchased from R＆D Systems).Experimental implementation is in strict accordance with kit
Specification requirement, all detection reagents are provided by kit.Specific experiment is summarized as follows：By IFN-γ or
IL-2 polyclonal antibodies are coated on ELISA microwell plates, on the cell that the present embodiment step (2) is obtained
Clear liquid adds standard items and testing sample to be incubated at room temperature 3 hours as testing sample.400 microlitres are added per hole
Washing lotion, repeats board-washing 6 times；Add the horseradish peroxidase labeling antibody of anti-human IFN-γ or IL-2, room
The IFN-γ or IL-2 that temperature is incubated on 2 hours, with microwell plate form immune complex, clean micropore；Add
Substrate is developed the color, and lucifuge room temperature 45 minutes ultimately joins terminate liquid, and A450nm absorbances are determined with ELIASA.
Detection PD-1 monoclonal antibodies are in PBMC stimulation tests to the influence of IFN-γ secretion.Acquired results
Show：PD-1 monoclonal antibodies obtained by the present invention can make PBMC in PBMC lymphocyte stimulation tests
IFN-γ secretion enhancing, effectively strengthen immunologic function, reduce the possibility of immune escape, this is anti-human
PD-1 monoclonal antibodies have the potentiality for preparing antineoplastic.
It should be understood that after the above of the invention has been read, those skilled in the art can make to the present invention
Various changes or modification, these equivalent form of values equally fall within the application appended claims limited range.
Claims (10)
1. a kind of monoclonal antibody of anti-human PD-1, it is characterised in that the monoclonal of the anti-human PD-1 resists
Body is included：
(1) complementary determining region of heavy chain CDR1, CDR2, CDR3, the amino acid sequence of the CDR1 is such as
SEQ ID NO：Shown in 1, the amino acid sequence such as SEQ ID NO of the CDR2：Shown in 2, the CDR3
Amino acid sequence such as SEQ ID NO：Shown in 3, and
(2) complementary determining region of light chain CDR1 ', CDR2 ', CDR3 ', the amino acid sequence of the CDR1 '
Such as SEQ ID NO：Shown in 4, the amino acid sequence such as SEQ ID NO of the CDR2 '：It is described shown in 5
The amino acid sequence of CDR3 ' such as SEQ ID NO：Shown in 6.
2. the monoclonal antibody of anti-human PD-1 as claimed in claim 1, it is characterised in that described anti-human
The amino acid sequence of the weight chain variable district of the monoclonal antibody of PD-1 such as SEQ ID NO：Shown in 8, its light chain
The amino acid sequence of variable region such as SEQ ID NO：Shown in 10.
3. a kind of nucleic acid molecule, it is characterised in that the nucleic acid molecule coding such as claim 1 or 2
The monoclonal antibody of described anti-human PD-1.
4. nucleic acid molecule as claimed in claim 3, it is characterised in that the nucleic acid molecule coding is anti-
The nucleotide sequence of the weight chain variable district of the monoclonal antibody of people PD-1 such as SEQ ID NO：Shown in 7, coding
The nucleotide sequence of its light chain variable district such as SEQ ID NO：Shown in 9.
5. a kind of expression vector, it is characterised in that the expression vector contains as described in claim 3 or 4
Nucleic acid molecule.
6. a kind of host cell, it is characterised in that the host cell contains table as claimed in claim 5
Up to carrier.
7. a kind of preparation method of the monoclonal antibody of anti-human PD-1 as claimed in claim 1 or 2, its
It is characterised by, the preparation method is comprised the following steps：
A) under expression condition, host cell as claimed in claim 6 is cultivated, so as to express anti-human PD-1
Monoclonal antibody；
B) monoclonal antibody of anti-human PD-1 simultaneously described in purification step a) is separated.
8. a kind of composition, it is characterised in that the composition contains as claimed in claim 1 or 2 anti-
The monoclonal antibody and pharmaceutically acceptable carrier of people PD-1.
9. application of the monoclonal antibody of anti-human PD-1 as claimed in claim 1 or 2 in medicine is prepared.
10. application as claimed in claim 9, it is characterised in that described medicine is antitumor, treats certainly
The medicine of body immunity disease, treatment infectious diseases and/or resisting transplant rejection reaction.
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