CN106046162A - Preparation and application of anti-human programmed death factor 1 (PD-1) monoclonal antibody - Google Patents

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

Anti-human programmed death factor 1(PD-1) preparation of monoclonal antibody and application

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]

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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.