CN106046162A - Preparation and application of anti-human programmed death factor 1 (PD-1) monoclonal antibody - Google Patents
Preparation and application of anti-human programmed death factor 1 (PD-1) monoclonal antibody Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C07K—PEPTIDES
- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K2317/565—Complementarity determining region [CDR]
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Abstract
The invention discloses preparation, a variable-region sequence and application of an anti-human programmed death factor 1 (PD-1) monoclonal antibody. The invention provides the anti-human PD-1 monoclonal antibody which comprises gene sequences of heavy-chain variable regions of the monoclonal antibody and gene sequences of light-chain variable regions of the monoclonal antibody. The amino acid sequences of the heavy-chain variable regions are as shown in site 20th to site 134th of SEQ ID NO.3; the amino acid sequences of the light-chain variable regions are shown in site 20th to site 131th of SEQ ID NO.4. The invention discloses the monoclonal antibody 189-H-1 capable of blocking a human PD-1 function and a coding gene thereof. The monoclonal antibody 189-H-1 can be specifically combined with human PD-1 antigen, has median effective concentration EC50 being 1.0667 nM, can specifically block a PD-1/PD-L inhibition signal, and therefore, the monoclonal antibody 189-H-1 can be used as a blocker of a PD-1 access, so that the anti-human PD-1 monoclonal antibody becomes a novel drug for tumor immune therapy, chronic virus infective disease treatment and autoimmune disease treatment.
Description
Technical field
The present invention relates to biomedicine technical field, particularly relate to preparation and the variable region sequence of anti-human PD-1 monoclonal antibody
Row and application.
Background technology
PD-1 gene is found and clone in 1992 by Tasuku Honjo and colleague thereof the earliest, and its extracellular region has 1
GeIgVYang district, with the homology that CTLA-4 has 23%, the immunoglobulin superfamily I type cross-film being made up of 288 aminoacid
Glycoprotein, is initially considered programmed death-1 relevant and named to apoptosis (programmed death-1, PD-1).
(Ishida, Y. etc., Induced expression of PD-1, a novel member of the
Immunoglobulingene superfamily, upon programmed cell death.EMBO J, 1992,11:
3887).But PD-1 albumen for want of mediates the MYPPPY sequence that CD28/CTLA-4 with B7.1/B7.2 combines, and
The mediation FDPPPF sequence that combines of ICOS with ICOS-L, thus structurally with CD28, CTLA-4 and ICOS
There is notable difference.Therefore, PD-1 is the most special with the combination of part, and the B7 family molecule with other does not produces cross knot
Close.
PD-1 has two known parts, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), and they are
The member of the cell surface expression of B7 family.PD-1 be mainly expressed in CD4+T cell, CD8+T cell, NKT cell, B cell and
The onthe surface of monocytes of activation, mainly by φt cell receptor (TCR) or the abduction delivering of B-cell receptor (BCR) signal, TNF can increase
Strong PD-1 is at expression (Francisco etc., the The PD-1pathway in tolerance and of these cell surfaces
Autoimmunity.Immunol Rev, 2010,236:219-242).This receptor and its part PD-L1 and PD-L2 combine
(Okazaki etc., PD-1 and PD-1 ligands:from discovery to clinical
Application.International Immunology, 2007,19 (7): 813-824).Give PD-1 or PD-L1 antibody
To block the interphase interaction of PD-1/PD-L1, then can break or eliminate the immunosuppressant or immunologic tolerance caused because of tumor, extensive
The identification of T lymphocyte and the ability of attack tumor target cell in complex, and then reach the good result of neoplasm growth and transfer
(HiranoF. etc., Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates
Cancer therapeutic immunity [J]. Cancer Res, 2005,65 (3): 1089-1096.).
It is by TasukuHonjo and colleague thereof the earliest that PD-1 participates in these material facts of negative regulation vivo immunization function
Research PD-1 knock out mice is observed.They find that the mice of PD-1 gene knockout is at C57BL/6 genetic background
Under, there is lupoid acne glomerulonephritis and arthritis (Nishimura H etc., Developmentof lupus-like
autoimmune diseases by disruption of the PD-1gene encoding an ITIM motif-
Carrying immunoreceptor.Immunity, 1999,11:141);And under Balb/c genetic background, then produce height and drip
Degree anti-cardiac muscular tissue antibody and thus cause serious autoimmune cardiomyopathy.
PD-1 and PD-L1 interacts and has obtained a large amount of in tumor and virus infect with the activation of regulation control T cell
Checking.PD-L1 is expressed in kinds of tumor cells surface, and these tumor cells include: pulmonary carcinoma, hepatocarcinoma, ovarian cancer, cervical cancer, skin
Skin cancer, bladder cancer, colon cancer, breast carcinoma, glioma, renal carcinoma, gastric cancer, esophageal carcinoma, oral squamous cell carcinoma, head and neck cancer.
And the CD8+T cell of great expression PD-L1 is have also discovered at these cancer peripheries.Clinical statistics shows, PD-L1 is thin in tumor
High expression level on born of the same parents is relevant to cancer patient's poor prognosis.Interaction between PD-1 and PD-L1 causes penetrating into tumor
Lymphopenia, φt cell receptor mediation propagation reduce and immune evasion (Dong H. etc., the B7-of cancerous cells
H1 pathway and its role in the evasion of tumor immunity . J Mol Med, 2003 ,
81: 281 7).Research finds the Local Phase interaction reversible immunosuppressant by suppressing PD-1 Yu PD-L1, and works as PD-
The interaction of 1 and PD-L2 has cumulative effects (Brown JA etc., Blockade of programmed when being also blocked
death-1 ligands on dendritic cells enhances T cell activation and cytokine
production. J Immunol. 2003;170:1257 1266).
In sum, the responsiveness cell of anergy in body can be made by specific inhibition PD-1/PD-L suppression signal
Recover biological function, promote the activation and proliferation of tumor and virus-specific CD8+T cell and the secretion of cytokine, strengthen
Lymphocyte to tumor antigen, the lethality of the virus etc. of exotic invasive, improves immunity of organisms, remove in time tumor cell and
Virus.Therefore, PD-1/PD-L is expected to become effective target molecule of immunotherapy of tumors, is also that HIV chronic diseases poison infects
Property disease and autoimmune disease treatment provide a new strategy.
Summary of the invention
It is an object of the invention to provide the antibody having very high-affinity to hPD-1 (people PD-1), Antibody Designation is 2-
189-H-1.This antibodies block hPD-1 receptor is combined with its part B7-H1, can apply to treat antitumor, infection and
In the medicines such as autoimmune disease.
The present invention provides 1 strain stably to express the hybridoma cell strain of antibody protein, and is deposited in Wuhan University's Chinese Typical Representative
Culture collection center.Depositary institution address be No. 299 Wuhan Universitys of Wuchang District, Wuhan City, Hubei Province Bayi Road in the school, Wuhan is big
Learn preservation center.Preservation date is JIUYUE in 2015 28, and deposit number is CCTCC C2015160.Classification And Nomenclature is hybridoma
Cell strain 2-189-H-1.
The present invention provides the DNA molecule of a kind of separation, encodes heavy chain and or the light chain of described anti-hPD-1 human antibody
Variable region or full length amino acid.
The nucleotide sequence of antibody heavy chain variable region is the 58th to 402 the shown nucleotide sequences of SEQ ID NO:1
;The nucleotide sequence of its antibody chain variable region is the 58th to 393 the shown nucleotide sequences of SEQ ID NO:2.
The aminoacid sequence of antibody heavy chain variable region is SEQ ID NO:3 or its conservative series of variation;Its antibody is light
The aminoacid sequence of chain variable region is SEQID NO:4 or its conservative series of variation.
SEQ ID NO.1 and 2 the 1st is to the nucleotide sequence that 57 bit base sequences are coding signal peptide sequences.
SEQ ID NO.3 and 4 the 1st is signal peptide sequence to 19 amino acids sequences.
Described anti-hPD-1 monoclonal antibody can be the full length sequence of antibody, it is also possible to be the sheet of anti-PD-1 antibody
Section, above-mentioned albumen and antibody include: recombiant protein, recombinant antibodies, ScFv antibody, humanized antibody, chimeric antibody, bispecific
Antibody, single domain antibody and ADC coupled antibody and albumen.
Described antibody can also further provide for the derivant of described anti-hPD-1 antibody, and described derivant is that hPD-1 resists
The fragment of body, antibody/antibody fragment-factor fusion protein, antibody/antibody fragment-chemical coupling thing.
New discovery of the present invention a kind has monoclonal antibody 2-189-H-1 of significantly high affinity to hPD-1.This Dan Ke
Grand antibody can not only be combined with hPD-1 antigenic specificity, and medium effective concentration is near with reference to condition, and can block hPD-1
The ability being combined with its part.
The present invention obtains the gene order of purpose antibody from monoclonal cell strain, may be used to build carrier for expression of eukaryon,
The activity of antibody can be rebuild, it is thus achieved that anti-hPD-1 monoclonal antibody after expression.
Anti-hPD-1 monoclonal antibody of the present invention can be used to prepare antitumor (include high expressed hPD-1 pulmonary carcinoma,
Hepatocarcinoma, breast carcinoma, squamous cell carcinoma, ovarian cancer, colorectal cancer, gastric cancer, gastrointestinal stromal tumor, bladder cancer, thyroid carcinoma, black
Element tumor, neck cancer, carcinoma of prostate) medicine, infection (infectious disease includes that the virus such as HIV, HBV, HCV infects the disease caused)
Medicine and be used for prepare autoimmune disease (include systemic lupus erythematosus (sle), rheumatoid arthritis, SV,
Psoriasis, multiple sclerosis, ulcerative colitis) medicine treated, its preparation method is to be with anti-PD-1 monoclonal antibody
Main component, adds acceptable adjuvant and/or additive on pharmacopedics, through frozen dried, is prepared as pharmaceutically acceptable
Medicament.
It is an advantage of the current invention that this antibody has the highest affinity to hPD-1.High efficient expression in zooblast, available
In industrialized production.It is demonstrated experimentally that the anti-hPD-1 monoclonal antibody blocks hPD-1 receptor of the present invention is tied with its part B7-H1
Close.
So, the treatment of antibodies on tumor of the present invention, infectious disease and autoimmune disease has widely
Application prospect.
Accompanying drawing explanation
Fig. 1 is the affinity capacity experimental of anti-PD-1 antibody.
Fig. 2 is the blocking test of anti-PD-1 antibody.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, protection scope of the present invention is not limited to down
State specific specific embodiments.
Embodiment 1.
The stable film expression of hPD-1.
The cell strain of hPD-1 albumen is expressed as immunogen, purchase encoding human PD-1 total length in order to obtain surface of cell membrane
CDNA clone (Sino Biological Inc. HG10377-CF, Genbank accession number NM-of open reading frame
005018.2), directing it and be inserted into pCDNA3.1(+) in (invitrogen company) carrier, order-checking determines that hPD-1 gene is compiled
After code frame is correct.Being transformed in CHO-DG44 cell by plasmid electricity, pressurization screening, clone obtains the CHO-of stable expression hPD-1
DG44 cell.Named hPD-1/DG44.
Embodiment 2.
Animal immune.
Select 5 6-8 week old A/J mices, carry out 5 immunity, each immunization interval 14 days altogether.Immunity 10 every time7Individual
HPD-1/DG44 cell, subcutaneous and abdominal cavity multiple spot immunity.First immunisation uses equal-volume Freund's complete adjuvant and cell suspension to mix
Closing, remaining 4 immunity is incomplete Freund's adjuvant and cell suspension mixing.
The configuration of cell culture medium.
MD6 serum-free medium is used when cultivating sp2/0 cell.After fusion, cell uses HAT culture medium culturing, specifically becomes
Divide as follows: MD6 serum-free medium adds hyclone and the HAT of 10%.
Cell merges.
Exempting from eventually latter 3 days, 3 mouse selecting titer high carry out cell fusion.Aseptic taking-up mouse spleen and preparing in advance
Good sp2/0 cell, after cell counting, is mixed in the ratio of 10:1, adds PEG3350 and carries out cell fusion.Cell after fusion
Use HAT culture medium culturing in 96 porocyte culture plates.
The detection of fused cell.
After fusion the 10th day, in 96 orifice plates, in Tissue Culture Plate, each hole major part became yellow, and cell clone colony accounts for
20%-30% at the bottom of whole hole, carries out ELISA screening positive clone.Specific as follows: to use the people's PD-1-Fc recombiant protein bought
(RD) being coated 96 hole elisa plates, the cell supernatant after addition fusion resists as one hatches, and concurrently sets sp2/0 cell conditioned medium
Liquid is negative control, and addition mountain sheep anti mouse IgG-Fc-HRP resists as two hatches, and adds nitrite ion, OD450 readings.Positive colony
It is defined as: cell detects 2 times more than sp2/0 supernatant OD450 value of supernatant OD450 value after merging.
The sub-clone screening of positive fused cell.
Positive fused cell after detection is incubated in 6 porocyte culture plates, treats that cell state is good, cell counting,
It is laid in 96 porocyte culture plates, average every 1, hole cell.After cultivating 10 days, the cell selecting single clonal population is carried out
ELISA detects, and OD450 value detects the highest cell and carries out second time sub-clone, after 3-5 sub-clone, until detection knot
Till fruit is 100% positive, determine the stability of cell.
The preparation of antibody.
The monoclonal cell of acquisition ties up to carry out in MD6 culture medium shake-flask culture, and expression time is usually 7-14 days,
The harvesting culture fluid supernatant when viable cell density is less than 50%.Protein A affinity column is used to cultivate from cell
Isolated and purified purpose antibody in supernatant.
Example 3.
Anti-hPD1 monoclonal antibody Function Identification.
Affinity is identified.
Use hPD-L1-Fc(RD) it is coated 96 hole elisa plates;Blocking antibody anti-carries out 3 times of dilutions, totally 12 ladders as one
Degree, joins hPD-L1-Fc and is coated in 96 hole elisa plates.Add mountain sheep anti mouse IgG-Fc-HRP(SANT Cruz
BIotechnology) resist as two, add nitrite ion, after termination, read OD450 value.Use Graphpad Software Create EC50
Concentration.The EC50 of this antibody is 1.0667nM, and the EC50 of positive control is 0.5344nM, from the results, it was seen that screening obtains
Antibody PD-1 is had obvious affinity, and (see figure 1) close with positive reference substance.
The Function Identification that antibody blocking hPD-1 with hPD-L1 is combined.
Use hPD-L1-Fc(RD) it is coated 96 hole elisa plates;The hPD-1 antibody of purification is carried out 4 times of dilutions, totally 4 ladders
Degree, respectively with hPD-1-Fc effect.The mixed liquor of the hPD-1 antibody of variable concentrations Yu hPD-1-Fc effect is joined hPD-
In the coated 96 hole elisa plates of L1-Fc.Add rabbit anti-hPD-1 antibody (Yi Qiao Divine Land, Beijing) to resist as one, resist adding goat
Rabbit igg-Fc-HRP(SANT Cruz BIotechnology) resist as two, add nitrite ion, read OD450 value.Filter out
Antibody has the function that blocking-up hPD-1 with HPDL1 is combined.
The blocking effect of antibody is detected further by Biocore.HPD-1-Fc is coupled on chip, by purification
HPD-1 antibody carries out 2 times of dilutions, totally 7 gradients, respectively with hPD-1-Fc effect.Upper machine testing.Use Graphpad software raw
Becoming EC50 concentration, the EC50 of this antibody is 64.42nM, and the EC50 of positive control is 26.59nM,.From the results, it was seen that screening
The combination of the antibodies block PD-1 obtained and its part B7-H1, with positive reference substance at the same order of magnitude (see figure
2).
Example 4.
The subgroup identification of antibody and stability test.
Subgroup identification, result weight is carried out with reference to murine antibody hypotype identification kit (Pierce, 37503) description antagonist
Chain is IgG1, and light chain is Kappa.
After hybridoma cell strain In vitro culture continuous passage 3 months, measure supernatant antibody titer, and by cell strain
Recover after frozen 4 months, detect supernatant antibody titer.All do not change.Show to obtain the hybridoma that secretory antibody is stable
Cell strain.
Example 5.
The acquisition of antibody gene sequences.
With reference to SMARTer RACE test kit (clonetech, 634859) operation instructions, according to Mus heavy chain IgG1 with light
The Fc fragment conserved regions design downstream primer of chain Kappa, utilizes RACE round pcr, to have blocking effect heavy chain of antibody and
Chain variable region gene expands and checks order.
In sum, the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.
The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any ripe
Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage knowing this technology.Cause
This, have usually intellectual such as complete with institute under technological thought without departing from disclosed spirit in art
All equivalences become are modified or change, and must be contained by the claim of the present invention.
[0001]
Sequence table
<110>grand celebration Dong Zhuming Bioisystech Co., Ltd
<120>anti-human programmed death factor 1(PD-1) preparation of monoclonal antibody and application
<160>10
<210>1
<211>402
<212>DNA
<213>mice (mus musculus)
<220>
<221>CDS
<222>(1)......(402)
<223>2-189-H-1 variable region of heavy chain
<400>1
atggctgtcc tggcactgct cctctgcctg gtgacattcc caagctgtgt cctgtcccag 60
gtgcagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcaca 120
tgcactgtct cagggttctc attaaccacc tatggtgtaa gctgggttcg ccagcctcca 180
ggaaagggtc tggagtggct gggagtaata tggggtgacg ggagcacaga ttatcattca 240
gttctcatat ccagactgag tatcactaag gatatctcca agagtcaagt tttcttaaaa 300
ctgaacagtc tgcaaagtga tgacacagcc acgtactact gtgcccaaca ggggagtagg 360
ggtgactact ggggccaagg caccactctc acagtctcct ca 402
[0002]
<210>2
<211>393
<212>DNA
<213>mice (mus musculus)
<220>
<221>CDS
<222>(1)......(393)
<223>2-189-H-1 variable region of light chain
<400>2
atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 60
gttttgctga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120
tcttgcagat ctagtcagac cattgtacat agtaatggaa tcacctattt agaatggtac 180
ctgcagaaac caggccagtc tccaaaggcc ctgatctcca aagtttccaa ccgattgtct 240
ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300
agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcaca tattccgtac 360
acgttcggag gggggaccaa gctggaaata aaa 393
[0003]
<210>3
<211>134
<212>PRT
<213>mice (mus musculus)
<223>2-189-H-1 variable region of heavy chain
<400>3
Met Ala Val Leu Ala Leu Leu Leu Cys Leu Val Thr Phe Pro Ser Cys
1 5 10 15
Val Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala
20 25 30
Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Thr Thr Tyr Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
Glu Trp Leu Gly Val Ile Trp Gly Asp Gly Ser Thr Asp Tyr His Ser
65 70 75 80
Val Leu Ile Ser Arg Leu Ser Ile Thr Lys Asp Ile Ser Lys Ser Gln
85 90 95
Val Phe Leu Lys Leu Asn Ser Leu Gln Ser Asp Asp Thr Ala Thr Tyr
100 105 110
Tyr Cys Ala Gln Gln Gly Ser Arg Gly Asp Tyr Trp Gly Gln Gly Thr
115 120 125
Thr Leu Thr Val Ser Ser
130
[0004]
<210>4
<211>131
<212>PRT
<213>mice (mus musculus)
<223>2-189-H-1 variable region of light chain
<400>4
Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala
1 5 10 15
Ser Ser Ser Asp Val Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Ile
35 40 45
Val His Ser Asn Gly Ile Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro
50 55 60
Gly Gln Ser Pro Lys Ala Leu Ile Ser Lys Val Ser Asn Arg Leu Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys
100 105 110
Phe Gln Gly Ser His Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys
130
[0005]
<210>5
<211>8
<212>PRT
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<223>2-189-H-1 heavy chain CDR1
<400>5
Gly Phe Ser Leu Thr Thr Tyr Gly
1 5
[0006]
<210>6
<211>11
<212>PRT
<213>mice (mus musculus)
<223>2-189-H-1 light chain CDR1
<400>6
Gln Thr Ile Val His Ser Asn Gly Ile Thr Tyr
1 5 10
[0007]
<210>7
<211>7
<212>PRT
<213>mice (mus musculus)
<223>2-189-H-1 heavy chain CDR2
<400>7
Ile Trp Gly Asp Gly Ser Thr
1 5
[0008]
<210>8
<211>3
<212>PRT
<213>mice (mus musculus)
<223>2-189-H-1 light chain CDR2
<400>8
Lys Val Ser
[0009]
<210>9
<211>9
<212>PRT
<213>mice (mus musculus)
<223>2-189-H-1 heavy chain CDR3
<400>9
Ala Gln Gln Gly Ser Arg Gly Asp Tyr
1 5
[0010]
<210>10
<211>9
<212>PRT
<213>mice (mus musculus)
<223>2-189-H-1 light chain CDR3
<400>10
Phe Gln Gly Ser His Ile Pro Tyr Thr
1 5
Claims (11)
1. the anti-human PD-1 monoclonal antibody separated or its antigen-binding portion thereof, it comprises:
A) the variable region of heavy chain CDR1 selected from SEQ ID NO:5 aminoacid sequence is comprised;
B) the variable region of heavy chain CDR2 selected from SEQ ID NO:7 aminoacid sequence is comprised;
C) the variable region of heavy chain CDR3 selected from SEQ ID NO:9 aminoacid sequence is comprised;
D) the variable region of light chain CDR1 selected from SEQ ID NO:6 aminoacid sequence is comprised;
E) the variable region of light chain CDR2 selected from SEQ ID NO:8 aminoacid sequence is comprised;
F) the variable region of light chain CDR3 selected from SEQ ID NO:10 aminoacid sequence is comprised.
2. the anti-human PD-1 monoclonal antibody separated or its antigen-binding portion thereof, it comprises:
A) variable region of heavy chain of the aminoacid sequence selected from the 20th to 134 of SEQ ID NO:3 is comprised;
B) variable region of light chain of the aminoacid sequence selected from the 20th to 131 of SEQ ID NO:4 is comprised.
3. the coding anti-human PD-1 monoclonal antibody gene described in claim 2, it is characterised in that:
A) nucleotide sequence of encoding heavy chain is if the 58th of SEQ ID NO.1 is to shown in 402;
B) nucleotide sequence of coding light chain is if the 58th of SEQ ID NO.2 is to shown in 393.
Anti-human PD-1 variable region of mab the most according to claim 2 sequence, changes at least one variable region antibody
At least one amino acid residue in sequence, described sequence is selected from variable fragments of heavy chain sequence and light chain variable domain antibodies sequence
Row, to create at least one altered antibody sequence and by altered antibody
Sequence table reaches protein.
Anti-human PD-1 variable region of mab the most according to claim 3 sequence, replaces through one or several bases
Still there is after changing, lack or adding the aminoacid sequence produced with described nucleotide sequence there is the nucleotides sequence of identical activity
Row.
6. an expression vector, comprises the nucleotide sequence described in claim 1 to 5 any one.
7. an expressive host, comprises the expression vector described in claim 6.
8. the application prepared on albumen and antibody, above-mentioned albumen and antibody it is listed according to the nucleotides sequence described in claim 1 to 5
Including recombiant protein, recombinant antibodies, ScFv antibody, humanized antibody, chimeric antibody, bi-specific antibody, single domain antibody and
ADC coupled antibody and albumen.
9. the antibody any one of claim 1-8 or antibody fragment purposes in preparation is used for the medicine of following aspect:
A). improve activated immune cell;
B). treatment cancer;
C). treatment is infected or infectious disease.
10. antibody any one of claim 1-11 or antibody fragment are for the purposes of diagnostic application.
11. 1 kinds of pharmaceutical compositions, including the described anti-PD-1 antibody of therapeutically effective amount.
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Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
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CN106749666A (en) * | 2016-12-22 | 2017-05-31 | 福州大学 | A kind of monoclonal antibodies of people's source program death receptor hPD 1 |
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CN108948203B (en) * | 2018-08-09 | 2019-06-04 | 南京鼓楼医院 | Anti- PD-1 monoclonal antibody and its preparation method and application |
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CN109593135B (en) * | 2018-12-29 | 2021-01-15 | 百奥赛图江苏基因生物技术有限公司 | Anti-human PD-L1 monoclonal antibody and application thereof |
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