CN108948202A - A kind of anti-human PD1 monoclonal antibody and application thereof - Google Patents
A kind of anti-human PD1 monoclonal antibody and application thereof Download PDFInfo
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Abstract
The present invention relates to a kind of anti-human PD1 monoclonal antibodies and application thereof, while providing coding nucleic acid molecule, expression vector, host cell and the method for expressing the antibody of the antibody.Additionally provide antibody mediated immunity coupling conjugate, bispecific molecule, embedding and antigen receptor, pharmaceutical composition and purposes including antibody of the present invention.Compared with the existing technology, the invention has the following advantages that the monoclonal antibody of energy specific recognition people PD1 of the invention, affinity of the antibody in conjunction with people PD1 are better than existing anti-human PD1 monoclonal antibody, sequence is novel.
Description
Technical field
The present invention relates to a kind of high-affinity and with the monoclonal antibody or antibody fragment of functional anti-human PD1.
Invention also provides the coding nucleic acid molecule of the antibody, expression vector, host cell and sides for expressing the antibody
Method.Additionally provide antibody mediated immunity coupling conjugate, bispecific molecule, embedding and antigen receptor, drug including antibody of the present invention
Composition and diagnostic and therapeutic method.
Background technique
Programmed death receptor 1, also known as PD-1 or CD279 belong to CD28 superfamily member.PD-1, which is mainly expressed, to be activated
B cell, (Agata et al., supra in T cell and bone marrow cell;Okazaki et al.(2002)
Curr.Opin.Immunol.14:391779-82;Bennett et al.(2003)J Immunol 170:711-8).With
Other members of CD28 family are different, due to being free of the cysteine of non-matching in PD-1 structure, prompt PD1 mainly with monomer shape
Formula exists.
PD-1 is the I type transmembrane protein of 55kDa a kind of, extracellular to have immune globulin variable region spline structure, is contained in intracellular region
One nearly film end immunity receptor Tyrosine Inhibitory Motifs (immunoreceptor tyrosine inhibitory motif,
ITIM) and the immunity receptor tyrosine at remote film end converts motif (immunoreceptor tyrosine based switch
Motif, ITSM) (Thomas, M.L. (1995) J Exp Med181:1953-6;Vivier,E and Daeron,M(1997)
Immunol Today 18:286-91)。
Although PD1 cannot combine B7-1 and B7-2 similar in construction to CTLA4, due to lacking MYPPY motif.PD-
L1 and PD-L2 is two ligand moleculars for the PD-1 being proved.
A large amount of evidences show that PD1 and its ligand interaction negative regulator are in immune response.PD-L1 is in most cancer cell tables
Face height expresses (Dong et al. (2002) Nat.Med.8:787-9).PD-1/PD-L1 interaction can lead to tumor infiltrating
Lymphocyte reduce, TCR mediate cell inhibitory effect and immunologic escape (Dong et al. (2003) J.Mol.Med.81:
281-7;Blank et al.(2005)Cancer Immunol.Immunother.54:307-314;Konishi et al.
(2004)Clin.Cancer Res.10:5094-100).Immunosupress can be reversed by inhibiting PD-L1/PD-1 to interact,
If blocking PD-L2/PD-1 that can further enhance effect (Iwai et al. (2002) Proc.Nat' of reverse simultaneously
l.Acad.Sci.USA 99:12293-7;Brown et al.(2003)J.Immunol.170:1257-66).
PD-1 depleted mice shows various autoimmune phenotype, concurrent including certainly immune cardiomyopathy, arthritis/ephritis
Lupoid acne syndrome (Nishimura et al. (1999) Immunity 11:141-51;Nishimura et al.(2001)
Science 291:319-22).Moreover, PD-1 is in itself property encephalomyelitis, systemic loupus erythematosus, graft versus host disease(GVH disease), I
Play a significant role in the diseases such as patients with type Ⅰ DM, rheumatoid key inflammation (Salama et al. (2003) J Exp Med 198:
71-78;Prokunina and Alarcon-Riquelme(2004)Hum Mol Genet 13:R143;Nielsen et
al.(2004)Lupus 13:510).In the B cell tumour cell of a mouse, what the ITSM motif of PD-1 mediated BCR
The tyrosine phosphorylation of Ca fluctuation and downstream molecules rise crucial blocking part with (Okazaki et al. (2001) PNAS98:
13866-71)。
The cancer immunotherapy drug of a large amount of targeting PD-1 molecules has been succeeded in developing.Such as one cried Nivolumab's
Antibody (i.e. BMS company), to facing for 296 cancer patients of non-small cell lung cancer, melanoma, clear-cell carcinoma
Complete or partial inhibitory effect (Topalian SL et al. (2012) The New England is shown in bed test
Journal of Medicine.366(26):2443–54).Treatment transfer is approved in Japan and the U.S. within the medicine 2014
The melanoma of property.The PD-1 antibody drug of another targeting, (trade name KEYTRUDA, Merck are public by Pembrolizumab
Department) it is also the same year to be approved by the fda in the United States for treating metastatic melanoma.It faces lung cancer, lymthoma, celiothelioma
Bed test is also just carrying out clinical experimental stage in the U.S..
Although targeting PD-1 antibody medicine have been developed that with it is granted, for PD-1 target spot there is still a need for exploitation more high-affinity
It is also very necessary and with important medical value with the monoclonal antibody of other pharmacological properties.
Summary of the invention
In order to overcome drawbacks described above, the object of the present invention is to provide a kind of anti-human PD1 monoclonal antibody and purposes, the antibody
With high-affinity and functionality, it is better than existing anti-human PD1 monoclonal antibody.
In order to achieve the above object, the present invention provides a kind of anti-human PD1 monoclonal antibody, which is characterized in that the antibody packet
Contain: heavy chain and light chain;
The heavy chain and light chain includes variable region, which includes complementary determining region;
Complementary determining region CDR1, CDR2 and CDR3 of the heavy chain are indicated with CDR-H1, CDR-H2 and CDR-H3 respectively;
Complementary determining region CDR1, CDR2 and CDR3 of the light chain are indicated with CDR-L1, CDR-L2 and CDR-L3 respectively;
The amino acid sequence of the CDR-H1 is shown in SEQ ID NO:3;
The amino acid sequence of the CDR-H2 is shown in SEQ ID NO:4;
The amino acid sequence of the CDR-H3 is shown in SEQ ID NO:5;
The amino acid sequence of the CDR-L1 is shown in SEQ ID NO:6;
The amino acid sequence of the CDR-L2 is shown in SEQ ID NO:7;
The amino acid sequence of the CDR-L3 is shown in SEQ ID NO:8.
Heavy chain variable amino acid sequence is shown in SEQ ID NO:1;Chain variable region amino acid sequence is SEQ ID
Shown in NO:2.
A kind of nucleic acid molecule, the nucleic acid molecule coding anti-human PD1 monoclonal antibody;
The sequence of the nucleic acid molecule is selected from SEQ ID NO:9 and/or SEQ ID NO:10;
The heavy chain variable region of the sequence SEQ ID NO:9 coding antibody;
The light chain variable region of the sequence SEQ ID NO:10 coding antibody.
A kind of expression vector, the expression vector contain the nucleic acid molecule.
A kind of host cell, the host cell contain the expression vector.
A kind of preparation method of anti-human PD1 monoclonal antibody, the preparation method comprise the following steps:
Step 1: the expression of nucleic acid molecule of the preparation containing the expression anti-human PD1 monoclonal antibody
Carrier;
Step 2: transfecting eukaryotic host cell with the expression vector of step 1;
Step 3: the eukaryotic host cell that incubation step 2 transfects;
Step 4: isolating and purifying, obtain the antibody.
The invention further relates to the antibody mediated immunities for including anti-human PD1 monoclonal antibody above-mentioned to be coupled conjugate, bispecific
Molecule, embedding and antigen receptor or pharmaceutical composition.
The invention further relates to anti-human PD1 monoclonal antibodies to prepare anti-tumor drug, communicable disease or other be situated between by PD1
Application in the drug for the pathologic conditions led.
The heavy chain and light chain all further includes constant region, which is the constant region of human IgG, it is therefore preferable to IgG1's
Constant region.
Compared with the existing technology, the invention has the following advantages that the monoclonal of energy specific recognition people PD1 of the invention is anti-
Body, affinity of the antibody in conjunction with people PD1 are better than existing anti-human PD1 monoclonal antibody, and sequence is novel.
Specific embodiment
The following further describes the technical solution of the present invention with reference to embodiments.
One, the acquisition of antibody
Embodiment 1 obtains the mouse monoclonal antibody of the anti-PD1 of specificity by fusion hybridoma technology
1.1 animal immune
According to (E Harlow, D.Lane, Antibody:A Laboratory Manual, Cold Spring in document
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) mouse is immunized in general method.It is immune
It originally was recombined human PD1 (Amino Acid Range Leu 25-Gln 167) albumen (Acro that C-terminal contains human IgG1's Fc label
biosystems,Cat#PD1-H5257).Using recombined human PD1 with hi s label albumen (Sino biological inc.,
Cat#10377-H08H) the detection antigen as measurement serum titer and hybridoma screening.Briefly, suitable Fu Shi assistant is taken out
Into 1.5ml EP pipe, oscillator is mixed for agent.Antigen protein solution is prepared with PBS.Amount mixes adjuvant as required and albumen is anti-
Original solution mutually pushes away the solution that fully emulsified antigen forms stable Water-In-Oil by syringe, then carries out animal injection.According to blood
Clear titration after first immunisation as a result, usually require to can be only achieved good immune effect after carrying out 2 to 3 booster immunizations.
The high immune mouse row intraperitoneal injection of selection serum titer carries out cell fusion after exempting from eventually.
1.2 hybridoma fusions and screening
Before cell fusion, culture murine myeloma cell (SP2/0-Ag14, ATCC#CRL-1581) is in logarithmic growth
Phase.It puts to death immune mouse gnotobasis and takes spleen, PEGylated fusion spleen B cell and SP2/0 myeloma are used according to method in document
Cell (E Harlow, D.Lane, Antibody:A Laboratory Manual, (Cold Spring Harbor
Laboratory Press,Cold Spring Harbor,N.Y.,1998);Kohler G,and Milstein C,"
Continuous cultures of fused cell secreting antibody of predefined
specificity,"Nature,256:495-497(1975)).Fused 96 porocyte culture plates of plating cells, usual 7 arrive
The Growth of Hybridoma Cell that survival can be observed after 10 days under microscope comes out.Plating cells after two weeks, are collected in each hole culture
Clearly, hybridoma screening is carried out with ELISA method employment PD1-his proteantigen.It is summarized as follows, with the people of 60ul 2ug/ml
The PBS solution coated elisa plate of PD1-his proteantigen, 4 degree (" degree " in the present invention refers to degree Celsius) overnight.Then PBST is washed
After plate 4 times, the PBST solution for 5% skimmed milk power that 200 holes μ l/ are added is placed in 37 degree and closes 2 hours.60 μ are added in board-washing again
The hybridoma supematant in the hole l/, 37 degree are incubated for 40 minutes, then board-washing 4 times.The diluted horseradish mistake of 1:5000 is added with 100 holes μ l/
The sheep anti-Mouse secondary antibody (JacksonImmuno research, cat#115-036-071) of oxide enzyme label, 37 degree are incubated for 40
Minute, then board-washing 4 times, pat dry.Add the TMB chromogenic substrate (Huzhou English creation object, TMB-S-002) in 100 holes μ l/, room temperature
Colour developing 5 to 15 minutes is then terminated with the sulfuric acid solution of 1M, measures the light absorption value in each hole of 450 nanometers.Choose ELISA combination
Positive hybridoma holes cell, is transferred in 24 orifice plates, continues to cultivate.And the second wheel secondary screening, screening are carried out by ELISA method
Out specific recognition PD1 and can block PD1/PDL1 combine hybridoma (the results are shown in Table 1, wherein D2G3 be the present invention obtain it is miscellaneous
Hand over tumor, anti-human PD1 monoclonal antibody obtained by the hybridoma expression present invention), it is subcloned by limiting dilution assay, obtains mesh
Mark monoclonal cell strain.Then these monoclonal antibodies of a small amount of Expression products, antibody carry out the Function Appraising analysis of next step.
The ELISA of table 1PD1 Mouse Hybridoma Cells supernatant tests and assesses
Two, ira vitro analytical methods:
Embodiment 2 is used to measure the ira vitro analytical methods of PD1 monoclonal antibody functional activity
2.1 binding abilities based on capture ELSIA measurement antibody
Special secondary antibody (Jackson the Immuno Research, #115- of goat anti-mouse igg Fcr is prepared with 1xPBS
006-071), make its final concentration of 2 μ g/ml, with 100 μ l/ hole liquid feedings in 96 hole elisa Plates, 4 degree of coatings are overnight.Next day, with containing
After PBS solution (i.e. the 1xPBST) board-washing 4 times of 0.05% polysorbas20, the PBST that 5% skimmed milk power in 200 holes μ l/ is added is molten
Liquid is placed in 37 degree and closes 2 hours.Board-washing again, after the dilution antibody-solutions or hybridoma supematant in 100 holes μ l/ are added, 37 degree are incubated
It educates 40 minutes, then board-washing 4 times.With 100 holes μ l/ be added prepare 60nM biotin labeling people PD1-Fc protein solution (
In the PBST of 2.5% skimmed milk power), 37 degree are incubated for 40 minutes, then board-washing 4 times.It is diluted that 1:10000 is added with 100 holes μ l/
The Streptavidin (Jackson Immuno Research, #016-030-084) of horseradish peroxidase-labeled, 37 degree of incubations
40 minutes, then board-washing 4 times, patted dry.Add the ELISA chromogenic substrate TMB in 100 holes μ l/, color development at room temperature 5 to 15 minutes, then
With the sulfuric acid solution color development stopping of 1M, the light absorption value in each hole of 450 nanometers is measured.
2.2 competitive ELISA
2.2.1 ligand receptor, which combines, blocks ELISA
The blocking ability that PD1/PDL1 is combined by competitive ELISA method assessment PD1 antibody.It is summarized as follows, uses 1xPBS
The PDL1-Fc albumen (Biosion inc., cat#BS-TA1601103-90) for preparing people, makes its final concentration of 2 μ g/ml, with
100 μ l/ hole liquid feedings are in 96 hole elisa Plates, and 4 degree of coatings are overnight.Next day, with containing 0.05% polysorbas20 PBS solution (i.e.
1xPBST) after board-washing 4 times, the PBST solution for 5% skimmed milk power that 200 holes μ l/ are added is placed in 37 degree and closes 2 hours.It washes again
Plate 4 times.
PD1 antibody or reference antibody gradient dilution in people's PD1Fc protein solution containing biotin labeling, after preparing
Room temperature is incubated 40 minutes in advance.The solution of the antibody and PD1Fc biotin that have been incubated for is added to 100 holes μ l/ then and is coated with
On the plate of PDL1-Fc, 37 degree are incubated for board-washing 4 times again after forty minutes.Horseradish peroxidase mark then is added with 100 holes μ l/
The Streptavidin of note, 37 degree of incubation board-washing 4 times after forty minutes, pats dry.Add the colour developing of TMB solution and the 50 μ l/ in 100 holes μ l/
The 1M sulfuric acid in hole terminates reaction, measures the absorption value of 450 nanometers.Data processing is carried out using Graphpad prism software,
Obtain the IC50 concentration value that antibody blocking PD1/PDL1 is combined.
2.2.2 reference antibody blocks ELISA
Pass through competitive ELISA method assessment PD1 antibody blocking reference antibodyThe blocking of/people PD1 antigen binding
Ability.It is summarized as follows, prepares reference antibody with 1xPBSMake its final concentration of 2 μ g/ml, with 100 hole μ l/ liquid feedings in 96
Hole elisa Plates, 4 degree of coatings are overnight.Next day is added after PBS solution (i.e. 1xPBST) board-washing 4 times containing 0.05% polysorbas20
The PBST solution of 5% skimmed milk power in 200 holes μ l/ is placed in 37 degree and closes 2 hours, board-washing 4 times again.
PD1 antibody or reference antibody gradient dilution in the people PD1-Fc protein solution (10nM) containing biotin labeling
In, it prepares rear room temperature and incubates in advance 40 minutes.Then the solution of the antibody and PD1Fc biotin that have been incubated for is added to 100 holes μ l/
It has been coated with reference antibodyPlate on, 37 degree be incubated for after forty minutes, board-washing 4 times again.It is then added with 100 holes μ l/ peppery
The Streptavidin of root peroxidase labelling, 37 degree of incubation board-washing 4 times after forty minutes, pats dry.Add TMB colour developing and 1M sulfuric acid
Reaction is terminated, the absorption value of 450 nanometers is measured.Data processing is carried out using Graphpad prism software, show that antibody hinders
The IC50 concentration value that disconnected reference antibody/people PD1 is combined.
The 2.3 antibody functional analyses based on cell
The present invention further uses the Reporter System based on cell, to analyze the blocking of assessment PD1 monoclonal antibody
Sexual function activity.The experimental system by two kinds be engineered cell line form, one is PD1 effector cell system (Genscript,
), GS-J2/PD1 the PD1 of cytotostatic expression people and the luciferase reporter gene driven by NFAT response element;It is another
For PDL1 cell line (Genscript, GS-C2/PD-L1 are a kind of antigen presenting cell), PDL1 cell line stablizes expression people's
PDL1 and the cell surface antigen of engineering peptide/major histocompatibility complex (MHC).When both cells co-culture,
Since combination negative regulation of the PDL1 to PD1 acts on, the activation to NFAT reporter gene for causing MHC/TCR to mediate is made at blocking
With luciferase gene is not expressed.After introducing the ligand binding blocking antibody of PD1 in system, PD1 albumen is enclosed, is made
The combination negative regulation effect for obtaining PDL1 is prevented from, and luciferase expression is gradually recovered.Experiment is summarized as follows: culture is raw in logarithm
Long-term PDL1 cell line is with 5*105The density of a/ml is inoculated in 384 orifice plates.Next day, according to experimental design gradient dilution PD1
Antibody.Suck in 384 orifice plates after the culture supernatant of PDL1 cell, sequence add the diluted PD1 test antibodies of 20ul/ gradient pores or
Reference antibody and density are 6.25*105The PD1 cell (hole 20ul/) of a/ml.Then cell plates are placed in 37 degree of incubators and continue
After culture 6 hours, cell plates are taken out, the uciferase activity (One-Glo in every hole is tested according to the operating instruction for producing house
Luciferase Assay system,Promega inc.#E6120).Using Graphpad prism software according to measurement
Uciferase activity corresponds to antibody concentration mapping, obtains the EC50 concentration of antibody in PD1 antibody/luciferase signal response curve
Value.
The combination activity of the anti-PD1 mouse monoclonal antibody of embodiment 3
It is as follows according to the combination Activity Summary of analysis method described in embodiment 2.1, PD1 mouse monoclonal antibody of testing and assessing
Table 2.Wherein with reference antibodyIt is the existing anti-human PD1 monoclonal antibody having been commercialized as control.It can from table 2
Know, the combination activity of anti-human PD1 monoclonal antibody of the invention and people's PD1 antigen is equal to reference antibody.
The combination activity of table 2PD1 antibody
4 functional experiment of embodiment (analysis method referring to 2.2 and 2.3) is obtained anti-based on ELISA and cellular level
Body function experimental evaluation data
Such as the following table 3, the functional evaluating result of anti-PD1 mouse monoclonal antibody is summarized.
The functional evaluating result of the anti-PD1 mouse monoclonal antibody of table 3
The competitive ELISA experiment of antibody on human PD1/PDL1 shows that D2G3 antibody of the invention can specific blockage in table 3
PD1 combines its ligand PDL1, and blocking ability is close to reference antibody;Antibody is to reference antibody/people PD1 competition simultaneously
ELISA experiment shows D2G3 antibody blocks reference antibody combination people's PD1 antigen, shows that the epitope that D2G3 is combined is approximate
But it is different from reference antibody;The reporter gene assays of cell are additionally based on, show the function assessment activity of D2G3 close to referring to anti-
Body.
Embodiment 5DNA clone and sequencing, the variable region protein sequencing of anti-PD1 mouse monoclonal antibody
Using Trizol reagent (Invitrogen, catalog#15596-018) from the mouse monoclonal cell strain of culture
Middle extraction total serum IgE.Process is summarized as follows, the cell of 5x106 is collected by centrifugation into 1.5ml centrifuge tube, blots supernatant.Add 1ml
Trizol reagent and blow and beat repeatedly and be placed at room temperature for 5 minutes afterwards for several times for lytic cell.And then, every pipe is added 0.2ml's
Chloroformic solution, acutely concussion was placed at room temperature for 3 minutes after 15 seconds.Then, 4 degree of 12000g of centrifuge tube are centrifuged 10 minutes, take out centrifugation
Pipe draws upper strata aqueous phase solution into new 1.5ml centrifuge tube, adds the isopropanol of 0.4ml for precipitating from water phase
RNA.After mixing EP is managed and placed room temperature 10min manually, 4 degree of 12000g are centrifuged 10 minutes, abandon supernatant.The second of 1ml 75% is added
Alcohol, 4 degree of 7500rpm are centrifuged 5min again, abandon supernatant.Tube bottom RNA precipitate after ten minutes, is added 30 and arrives 50ul's in drying at room temperature
Sterile DEPC processing water dissolves RNA sample.
And then, selecting the reverse transcription cDNA kit (catalog#6110A) of Taraka becomes CDNA total serum IgE.It is real
The system of testing is formulated as follows, and+8.5 μ l RNase-free water (total 14ul) of+0.5 μ l Oligo (dT) of total serum IgE of 5 μ l is first placed in 65
5min initial denaturation is spent, is then put 2 minutes on ice.Further it is added+0.5 μ l's of dNTP mixture of+1 μ l of 5x buffer of 4 μ l
+ 1 μ l reverse transcriptase (20.5ul system in total) of RNase inhibitor, mixes after preparing, and runs 40 degree 50 minutes using PCR instrument, and 70
10min is spent, cDNA synthesis is completed.
CDNA is further formulated as follows at 3 ' ends plus Poly G, reaction system: the ddH2O of+33.5 μ l of cDNA sample of 5 μ l
Terminal deoxynucleotidyl transferase (the total volume of the dGTP+0.5 μ l of the CoCl2+1 μ l of+5 μ l of 10xTdT buffer of+5 μ l
50ul), it is mixed after preparing, runs 37 degree 30 minutes, 70 degree of 10min using PCR instrument, complete Poly G tailing.
Further, the gene magnification of antibody variable region is carried out using the cDNA of tailing as template.It can for amplification heavy chain of antibody
Become region sequence, prepare PCR reaction system: the general 0.5 μ l+ of poly C primer (forward primer) of 5 μ l+ of 10x Taq enzyme buffer is small
0.5 μ l+dNTP of mouse IgG1 reverse primer, 11 μ l+ddH2O of μ l+Taq 1 μ l+cDNA of polymerase, 41 μ l.It is light for amplification antibody
Chain variable region sequence prepares PCR reaction system: the general 0.5 μ l of poly C primer (forward primer) of 5 μ l+ of 10x Taq enzyme buffer
0.5 μ l+dNTP of+mouse IgG kappa reverse primer, 11 μ l+ddH2O of μ l+Taq polymerase 1ul+cDNA, 41 μ l.Antibody
The temperature cycles of heavy chain and light chain variable region PCR amplification are following (wherein step 2 to 4 repeats 25 circulations):
95 DEG C of .5min. of 1- initial denaturation
2- is denaturalized 95 DEG C of .20sec.
56 DEG C of .20sec. of 3- annealing
4- extends 72 DEG C of .30sec.
5- saves 25 DEG C of .60min.
PCR product is analyzed through 1% agarose gel electrophoresis, cut out corresponding size DNA section band (the about 600bp of VH,
Vkappa light chain about 500bp), the extracting of DNA is carried out with the gel DNA QIAquick Gel Extraction Kit (catalog#28704) of Qiagen.Letter
State as follows: gel weighing adds the QG buffer of 3 times of gel volumes, is then incubated for 10 minutes until gel is completely dissolved for 50 degree.Add
After the isopropanol mixing for entering 1 times of colloid product, sample moves to QIA purification column, is centrifuged 13000rpm 1 minute.The PE of 750ul is added
Buffer is into column, and then 13000rpm is centrifuged 1 minute.And 13000rpm is centrifuged liquid residual in 1 minute removal column again.Add
The water elution column for entering 30ul is centrifuged 1 minute, obtains the DNA sample of preparation, and the PCR product of purifying obtains the variable of antibody through sequencing
Region sequence is as shown in table 4, and the amino acid full length sequence of PD1 mouse monoclonal antibody is as shown in table 5, PD1 mouse monoclonal antibody
DNA sequences encoding it is as shown in table 6.
The variable region amino acid sequence and CDR region of table 4PD1 mouse monoclonal antibody
The amino acid full length sequence of table 5PD1 mouse monoclonal antibody
The DNA sequences encoding of table 6PD1 mouse monoclonal antibody
In conclusion the monoclonal antibody of energy specific recognition people PD1 of the invention, the antibody is affine in conjunction with people PD1
Power is strong, and sequence is novel.
It is discussed in detail although the contents of the present invention have passed through above preferred embodiment, but it should be appreciated that above-mentioned
Description is not considered as limitation of the present invention.After those skilled in the art have read above content, for of the invention
A variety of modifications and substitutions all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Sequence table
<110>letter biotechnology (Nanjing) Co., Ltd difficult to understand is won
<120>a kind of anti-human PD1 monoclonal antibody and application thereof
<130> xhx2018072402
<141> 2018-07-24
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 114
<212> PRT
<213> Mus musculus
<400> 1
Gln Glu Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Glu Ser Tyr
20 25 30
Gly Val Asp Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Gly Ser Thr Ser Tyr Asn Ser Gly Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Lys Gln Val Phe Leu
65 70 75 80
Glu Met Lys Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ile Asp Gly Gly Phe Ala Ser Trp Gly Gln Gly Thr Leu Val Thr Val
100 105 110
Ser Ala
<210> 2
<211> 112
<212> PRT
<213> Mus musculus
<400> 2
Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly
1 5 10 15
Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Gln Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Arg Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser
85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 3
<211> 8
<212> PRT
<213> Mus musculus
<400> 3
Gly Phe Ser Leu Glu Ser Tyr Gly
1 5
<210> 4
<211> 7
<212> PRT
<213> Mus musculus
<400> 4
Ile Trp Gly Ser Gly Ser Thr
1 5
<210> 5
<211> 8
<212> PRT
<213> Mus musculus
<400> 5
Ala Ile Asp Gly Gly Phe Ala Ser
1 5
<210> 6
<211> 16
<212> PRT
<213> Mus musculus
<400> 6
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
1 5 10 15
<210> 7
<211> 7
<212> PRT
<213> Mus musculus
<400> 7
Tyr Met Ser Asn Leu Ala Ser
1 5
<210> 8
<211> 9
<212> PRT
<213> Mus musculus
<400> 8
Met Gln Ser Leu Glu Tyr Pro Tyr Thr
1 5
<210> 9
<211> 342
<212> DNA
<213> Mus musculus
<400> 9
caggagcagc tgaagcagtc aggacctggc ctggtggcgc cctcacagac cctgtccatc 60
acatgtactg tctctggatt ctcattagag agctatggtg tagactgggt tcgccagtct 120
ccaggaaagg gtctggagtg gctgggagtg atatggggta gtggaagcac aagttataat 180
tcaggtctca aatccagact gagcatcagc aaggacaact ccaagaagca agttttctta 240
gaaatgaaaa gtctgcaaac tgatgacaca gccatgtact actgcgccat cgacgggggg 300
tttgcttcct ggggccaagg gactctggtc actgtctctg ca 342
<210> 10
<211> 336
<212> DNA
<213> Mus musculus
<400> 10
gatattgtga tgactcaggc tgcaccctct gtacctgtca ctcctggaga gtcagtatcc 60
atctcctgca ggtctagtaa gagtcttcta catagtaatg gcaacactta tttatattgg 120
ttcctgcaga ggccaggcca gtctcctcag cgcctgatat actatatgtc caaccttgcc 180
tcaggagtcc cagacaggtt cagtggcaga gggtcaggaa ctgatttcac attgagaatc 240
agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaaagtct agaatatccg 300
tacacgttcg gaggggggac caagctggaa ataaaa 336
<210> 11
<211> 972
<212> DNA
<213> Mus musculus
<400> 11
gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 60
tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 120
tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 180
ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 240
acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 300
gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 360
cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 420
gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 480
gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 540
agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 600
aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660
aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 720
agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780
aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 840
tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 900
acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 960
tctcctggta aa 972
<210> 12
<211> 321
<212> DNA
<213> Mus musculus
<400> 12
cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60
ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120
tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180
agcaaagaca gcacctacag catgagcagc accctcacgt tgactaagga cgagtatgaa 240
cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300
agcttcaaca ggggagagtg t 321
<210> 13
<211> 324
<212> PRT
<213> Mus musculus
<400> 13
Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
1 5 10 15
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
100 105 110
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
115 120 125
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
130 135 140
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
145 150 155 160
Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
165 170 175
Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
180 185 190
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
195 200 205
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
210 215 220
Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
225 230 235 240
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
260 265 270
Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
275 280 285
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
290 295 300
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
305 310 315 320
Ser Pro Gly Lys
<210> 14
<211> 107
<212> PRT
<213> Mus musculus
<400> 14
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
1 5 10 15
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
20 25 30
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
65 70 75 80
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
85 90 95
Pro Ile Val Lys Ser Phe Asn Arg Gly Glu Cys
100 105
Claims (9)
1. a kind of anti-human PD1 monoclonal antibody, which is characterized in that the antibody includes: heavy chain and light chain;
The heavy chain and light chain includes variable region, which includes complementary determining region;
Complementary determining region CDR1, CDR2 and CDR3 of the heavy chain are indicated with CDR-H1, CDR-H2 and CDR-H3 respectively;
Complementary determining region CDR1, CDR2 and CDR3 of the light chain are indicated with CDR-L1, CDR-L2 and CDR-L3 respectively;
The amino acid sequence of the CDR-H1 is shown in SEQ ID NO:3;
The amino acid sequence of the CDR-H2 is shown in SEQ ID NO:4;
The amino acid sequence of the CDR-H3 is shown in SEQ ID NO:5;
The amino acid sequence of the CDR-L1 is shown in SEQ ID NO:6;
The amino acid sequence of the CDR-L2 is shown in SEQ ID NO:7;
The amino acid sequence of the CDR-L3 is shown in SEQ ID NO:8.
2. a kind of anti-human PD1 monoclonal antibody according to claim 1, which is characterized in that heavy chain variable amino acid sequence
It is classified as shown in SEQ ID NO:1;Chain variable region amino acid sequence is shown in SEQ ID NO:2.
3. a kind of nucleic acid molecule, which is characterized in that it is mono- that the nucleic acid molecule encodes anti-human PD1 as claimed in claim 1 or 2
Clonal antibody;
The sequence of the nucleic acid molecule is selected from SEQ ID NO:9 and/or SEQ ID NO:10;
The heavy chain variable region of the sequence SEQ ID NO:9 coding antibody;
The light chain variable region of the sequence SEQ ID NO:10 coding antibody.
4. a kind of expression vector, which is characterized in that the expression vector contains nucleic acid molecule as claimed in claim 3.
5. a kind of host cell, which is characterized in that the host cell contains expression vector as claimed in claim 4.
6. a kind of preparation method of anti-human PD1 monoclonal antibody of any of claims 1 or 2, which is characterized in that the preparation method
It comprises the following steps:
Step 1: the expression vector of nucleic acid molecule of the preparation containing the expression anti-human PD1 monoclonal antibody;
Step 2: transfecting eukaryotic host cell with the expression vector of step 1;
Step 3: the eukaryotic host cell that incubation step 2 transfects;
Step 4: isolating and purifying, obtain the antibody.
7. the preparation method of anti-human PD1 monoclonal antibody according to claim 6, which is characterized in that the expression carries
Body contains the nucleotide sequence selected from SEQ ID NO:9 and/or SEQ ID NO:10.
8. including antibody mediated immunity coupling conjugate, the bispecific point of anti-human PD1 monoclonal antibody of any of claims 1 or 2
Sub, embedding and antigen receptor or pharmaceutical composition.
9. anti-human PD1 monoclonal antibody of any of claims 1 or 2 prepare anti-tumor drug, communicable disease or it is other by
Application in the drug for the pathologic conditions that PD1 is mediated.
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CN201810824078.3A CN108948202A (en) | 2018-07-25 | 2018-07-25 | A kind of anti-human PD1 monoclonal antibody and application thereof |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110156895A (en) * | 2019-05-30 | 2019-08-23 | 上海甲贝生物医药技术股份有限公司 | A kind of anti-PD-L1 antibody or its functional fragment and application thereof |
CN113150154A (en) * | 2021-04-15 | 2021-07-23 | 博奥信生物技术(南京)有限公司 | Anti-human PDL1 monoclonal antibody and application thereof |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102131828A (en) * | 2007-06-18 | 2011-07-20 | 奥根农股份公司 | Antibodies to human programmed death receptor pd-1 |
CN105683217A (en) * | 2013-05-31 | 2016-06-15 | 索伦托治疗有限公司 | Antigen binding proteins that bind PD-1 |
WO2017011580A2 (en) * | 2015-07-13 | 2017-01-19 | Cytomx Therapeutics, Inc. | Anti-pd-1 antibodies, activatable anti-pd-1 antibodies, and methods of use thereof |
-
2018
- 2018-07-25 CN CN201810824078.3A patent/CN108948202A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102131828A (en) * | 2007-06-18 | 2011-07-20 | 奥根农股份公司 | Antibodies to human programmed death receptor pd-1 |
CN105683217A (en) * | 2013-05-31 | 2016-06-15 | 索伦托治疗有限公司 | Antigen binding proteins that bind PD-1 |
WO2017011580A2 (en) * | 2015-07-13 | 2017-01-19 | Cytomx Therapeutics, Inc. | Anti-pd-1 antibodies, activatable anti-pd-1 antibodies, and methods of use thereof |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110156895A (en) * | 2019-05-30 | 2019-08-23 | 上海甲贝生物医药技术股份有限公司 | A kind of anti-PD-L1 antibody or its functional fragment and application thereof |
CN113150154A (en) * | 2021-04-15 | 2021-07-23 | 博奥信生物技术(南京)有限公司 | Anti-human PDL1 monoclonal antibody and application thereof |
CN113150154B (en) * | 2021-04-15 | 2022-05-10 | 博奥信生物技术(南京)有限公司 | Anti-human PDL1 monoclonal antibody and application thereof |
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