CN114539406B

CN114539406B - Fully human anti-PD-1 monoclonal antibody No.8-3 and application thereof

Info

Publication number: CN114539406B
Application number: CN202111662735.7A
Authority: CN
Inventors: 詹金彪; 梁可莹; 梅圣圣; 彭珊珊; 陈洁; 高向征
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2023-04-14
Anticipated expiration: 2041-12-31
Also published as: CN114539406A

Abstract

The invention provides a full-length antibody No.8-3 of fully human anti-PD-1 and application thereof, and provides amino acid sequences of light chain and heavy chain variable regions of the antibody. The invention screens out single-chain antibody scFv-8 of anti-PD-1 from a human phage single-chain antibody library, splices the variable region and constant region sequence of the antibody by a genetic engineering technology, establishes an engineered cell strain expressing the full-length antibody, and finally screens and purifies to obtain the full-length antibody No.8-3. The structure of the polypeptide comprises an antibody heavy chain variable region, a light chain variable region and a constant region; having high affinity, K _D Value of 10 ^‑12 M; can recognize PD-1 on the surface of cells and effectively block the combination of PD-1 and PD-L1; has better effect of reversing T cell immunosuppression in a co-culture system with tumor cells. The antibody has low immunogenicity and high stability, and can be used for tumor immunotherapy.

Description

Fully human anti-PD-1 monoclonal antibody No.8-3 and application thereof

Technical Field

The invention belongs to the technical field of antibody engineering, and particularly relates to a fully human anti-PD-1 monoclonal antibody No.8-3 and application thereof.

Background

Programmed cell death receptor 1 (PD-1) is a type I transmembrane glycoprotein encoded by the pdcd1 gene, a 288 amino acid protein. The structure is divided into an extracellular immunoglobulin variable region-like domain, a transmembrane region and an intracellular region. Wherein, the intracellular domain of PD-1 comprises two important tyrosine motifs, namely an immunoreceptor tyrosine base inhibition motif (ITIM) and an immunoreceptor tyrosine base transfer motif (ITSM). PD-1 belongs to a member of the CD28 superfamily, and is mainly expressed on the surface of activated T cells, NK cells, B cells, tumor-associated macrophages and the like. In 1992, professor Tasuku Honjo first isolated and identified the PD-1 gene in mice and thought that activation of the PD-1 molecule would lead to apoptosis of T cells. However, until 1999, researchers observed the development of autoimmune diseases in mice through studies on PD-1 deficient mice, and gradually elucidated the immunosuppressive function of PD-1. The ligand of PD-1 is PD-L1 (also called B7-H1 or CD 274) and PD-L2 (also called B7-DC or CD 273). PD-L1 may be expressed on different types of cells, including immune cells, epithelial cells, endothelial cells, and especially tumor cells. PD-L2 is expressed only in Antigen Presenting Cells (APCs). Expression of pro-inflammatory cytokines, such as type I and type II interferons, tumor necrosis factor alpha (TNF- α), and Vascular Endothelial Growth Factor (VEGF) induces the expression of cellular PD-L1 and PD-L2. During the immune response in the body, activation of T cells requires, in addition to a first signal that the T cell receptor recognizes an antigenic peptide presented by the histocompatibility complex (MHC) on the APCs, a co-stimulatory signal that the co-stimulatory receptor CD28 binds to the B7 receptor of the APCs. However, if the co-inhibitory receptor PD-1 binds to the ligand PD-L1 or PD-L2, the proliferation of T cells is inhibited. Therefore, the PD-1/PD-L1 antibody can block the PD-1/PD-L1 signal of an immune check point and recover the proliferation and killing capacity of T cells, thereby achieving the aim of killing tumor cells.

More than 100 monoclonal antibody drugs have been approved for sale to the market since the first FDA approved monoclonal antibody drug in 1986. At present, the emergence of antibody engineering technology accelerates the preparation and screening of human antibodies, and recombinant antibodies prepared by genetic engineering technology mainly include human-mouse chimeric antibodies, humanized antibodies, fully humanized antibodies, and the like. The phage display technology has the advantages of short screening period, high in-vitro affinity maturation, no need of antigen immunization and the like, and becomes a main technical platform for preparing fully human antibodies.

The phage display technology is characterized in that a foreign coding polypeptide or protein gene is inserted into a proper position of a structural gene of a coat protein of a phage through a gene engineering technology, so that the foreign coding polypeptide or protein gene can form a fusion protein with a capsid protein of the phage, and the fusion protein is displayed on the surface of the phage along with the reassembly of a progeny phage and can keep a relative spatial structure and biological activity. The antibody fragment and the phage capsid protein are displayed on the surface of the phage in a fusion expression way to obtain the phageThere are three main types of displayed antibody libraries: scFv library (composed of VH and VL, and consisting of a small peptide (Gly 4 Ser) ₃ Linked as a single chain polypeptide), fab libraries (containing VH-CH and VL-CL, linked by disulfide bonds) and VHH libraries (heavy chain variable region only). Screening of phage antibody libraries mimics the process of antibody affinity maturation. The non-specifically bound phage are washed away using the target antigen using a suitable panning procedure. Then the phage combined under the elution of acid or competitive molecules is used, the neutralized phage infects escherichia coli for amplification, the proportion of the phage capable of specifically recognizing the target antigen is gradually improved through 3-5 rounds of enrichment, and finally the high-affinity phage antibody capable of recognizing the target antigen is obtained.

It is reported in the literature that GC-rich sites on DNA strands can regulate the transcription process by trans-acting factors (Cys 2His 2). Since the opening of eukaryotic chromatin plays a crucial role in gene expression, the GC-rich structure near the target gene can promote the opening of chromatin by giving better rigidity to DNA duplexes. The pMH3 expression vector is a mammalian expression vector based on a 'GC rich' mechanism, and contains 3 GC-rich DNA fragments with the length of about 1000bp and derived from chicken beta actin gene, so that the pMH3 expression vector has ultrahigh exogenous protein expression capacity. In addition, the pMH3 expression vector not only contains prokaryotic DNA sequences (such as replicons, antibiotic resistance genes, single restriction enzyme sites and the like) which are convenient for amplification and replication in prokaryotes, but also has expression elements (including promoters, enhancers, 5 'non-coding sequences, 3' non-coding sequences, S/MARs elements, introns, polyA tails and the like) which can only be applied to mammalian cells, and is suitable for construction of high-expression stable cell strains of mammalian cell systems.

Chinese hamster ovary Cells (CHO) can continuously proliferate, stably integrate exogenous genes and maintain high-efficiency exogenous protein expression level; as a mammalian cell, the function of exogenous gene post-transcriptional modification can be accurately carried out, so that the expressed protein is close to a natural protein molecule in function and structure; the recombinant protein can be produced in a large scale by high-density culture in a serum-free culture environment in a suspension culture mode. Therefore, the CHO mammalian cell expression system is one of the most important expression systems for the development and production of genetic engineering drugs at present.

Disclosure of Invention

One of the purposes of the invention is to provide a fully human anti-PD-1 monoclonal antibody No.8-3, which is a novel fully human anti-PD-1 full-length antibody No.8-3 obtained by gene recombination.

The monoclonal antibody No.8-3 comprises a heavy chain variable region and a light chain variable region, as well as an antibody constant region. The polynucleotide sequence of the heavy chain variable region VH is shown in SEQ ID No.1, the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID No.2, the polynucleotide sequence of the light chain variable region VL is shown in SEQ ID No.3, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID No. 4.

The monoclonal antibodies No.8-3 contain the complete complementarity determining regions CDR1, CDR2 and CDR3. The amino acid sequence of the VH CDR1 of the heavy chain variable region is QSISSY, and the amino acid sequence of the VH CDR2 of the heavy chain variable region is: AAP, the amino acid sequence of VH CDR3 of the heavy chain variable region is as follows: QQYQTLPLT; the amino acid sequence of the light chain variable region VL CDR1 is as follows: EDTFGNHA, the amino acid sequence of VL CDR2 in the light chain variable region is as follows: IIPMFSKV; the amino acid sequence of light chain variable region VL CDR3 is: AKEGEPPRDDAFDI.

Another objective of the invention is to provide a recombinant antibody expression vector, comprising the heavy chain polynucleotide sequence SEQ ID No.1 or the light chain polynucleotide sequence SEQ ID No.3 of the antibody.

The invention also provides a host cell transfected with the recombinant antibody expression vector, wherein the host cell comprises prokaryotic cells E.coil DH5 alpha and mammalian cells CHO-K1.

The preparation method of the monoclonal antibody No.8-3 is realized by the following steps:

respectively constructing recombinant plasmids of pMH3-VH (containing heavy chain variable region genes and heavy chain constant region genes) and pMH3-VL (containing light chain variable region genes and light chain constant region genes), then co-transfecting the recombinant plasmids into CHO-K1 cells, and obtaining stably transfected cell strains through G418 screening. And (3) screening subclones with high antibody yield by a limiting dilution method, and domesticating to obtain the suspension cells cultured in a serum-free manner. Collecting cell culture supernatant, and purifying by Protein A affinity chromatography to obtain No.8-3 anti-PD-1 full-length antibody.

Still another object of the present invention is to provide the use of said fully human PD-1 but cloned antibody No.8-3 in the preparation of a medicament for the immunotherapy of antibodies against tumors.The application refers to the antibody itself or the light chain variable region sequence and the heavy chain variable region sequence thereof Application of medicine in preparing medicine for treating triple negative breast cancer。

The No.8-3 anti-PD-1 full-length antibody obtained by the above method was compared with the biological functions such as affinity, antigen recognition ability, ability to block PD-1/PD-L1, and the like of the Keytruda monoclonal antibody approved by FDA to be marketed. The antibody is shown to be capable of effectively blocking the binding of PD-1 and a ligand PD-L1 thereof and inhibiting the biological activity of PD-1.

The invention relates to a method for preparing a full-length antibody No.8-3 of anti-PD-1, which is characterized in that a single-chain antibody scFv-8 which is screened from a fully human leukemia phage single-chain antibody library and can be specifically combined with extracellular region PD-1 protein is transformed by a genetic engineering technology, the antibody variable region of the scFv-8 is spliced with an antibody constant region (Fc end) sequence by the genetic engineering technology to establish an engineered cell strain for expressing the full-length antibody, and finally, the full-length antibody No.8-3 of the anti-PD-1 is obtained by screening and purifying.

The invention has the beneficial effects that: (1) The antibody of the targeted human PD-1 is screened by adopting a fully human phage antibody library, and the obtained antibody is fully humanized, so that the immunogenicity can be reduced to the greatest extent, and the immune reaction of a human mouse can be eliminated; (2) The anti-PD-1 antibody has high affinity, can effectively recognize PD-1 on the surface of a cell, and can effectively block the combination of the PD-1 and a ligand PD-L1 thereof; can reverse the immune suppression of T lymphocyte in cell co-culture system and raise the secretion level of T cell to tumor killing cell factor IFN-gamma.

Drawings

FIG. 1 is a scheme showing the experimental design of construction of pMH3-VH and pMH3-VL recombinant plasmids.

FIG. 2 shows that monoclonal cell lines secreting high expression levels of anti-PD-1 monoclonal antibodies were screened by ELISA.

FIG. 3, purification and detection of antibody Nos. 8-3, wherein A is Protein A purification antibody Nos. 8-3; b is No.8-3 antibody detected and purified by reducing SDS-PAGE; m: marker;1: non-reducing property; 2: and (4) reducing property.

FIG. 4, binding dissociation curves of anti-PD-1 monoclonal antibodies Keytruda and Nos. 8-3.

FIG. 5 is a flow chart of the detection of the binding ability of the anti-PD-1 monoclonal antibody to the cell surface PD-1 antigen.

FIG. 6 flow cytometry detection of the blocking effect of anti-PD-1 monoclonal antibodies on PD-1/PD-L1.

FIG. 7, ELISA test for IFN-. Gamma.release levels from T cells.

Detailed Description

The detailed implementation method of the invention refers to the examples, experimental methods and reagents described in the examples, and the experimental methods and reagents are all conventional experimental methods and reagents unless otherwise specified. The following examples are intended only to illustrate and explain the invention, and are not intended to limit the invention in any way.

EXAMPLE 1 recombinant pMH3-VL and pMH3-VH eukaryotic expression vector construction

Obtaining scFv-8 through screening of a phage antibody library, respectively carrying out PCR amplification on an Ig kappa signal peptide sequence and VH and VL fragments of the scFv-8, then respectively connecting a signal peptide (S) to the N ends of the VH and VL fragments through an overlapping PCR method to obtain S + VH and S + VL fragments, and respectively inserting the amplified S + VH and S + VL fragments into a pMH3-KL recombinant plasmid containing a human IgG heavy chain constant region pMH3-KH and a human kappa light chain constant region to obtain a pMH3-VH and pMH3-VL recombinant plasmid. The experimental design route for constructing pMH3-VH, pMH3-VL recombinant plasmids is shown in FIG. 1.

Example 2 construction and screening of cell line expressing human anti-PD-1 full-Length antibody No.8-3

2.1pMH3-VH and pMH3-VL plasmids co-transfect CHO-K1 cells

CHO-K1 cells in log phase of growth were seeded into six well plates of cell culture, 1X 10 cells per well ⁵ The pMH3-VH and pMH3-VL plasmids obtained in example 1 were co-transfected by the lipofection method until the confluency of the cells reached 90%. After 24h of cell transfection, a small amount of cells are taken for culture and the full-length antibody is detected by ELISA methodAnd (4) expressing.

2.2 screening of stably transfected monoclonal cell lines with high expression level of anti-PD-1 antibody

At 48h after cell transfection, positive cell lines were selected using 700. Mu.g/ml G418 antibiotic, since the pMH3 plasmid has the neomycin resistance gene. The culture medium is replaced every 2 to 3 days, and the drug concentration is maintained for screening for a period of one month to obtain the stably transfected positive cell strain. Then, the stably transfected positive cell strain is subjected to monoclonality by a limiting dilution method so as to screen and obtain a single cell strain with high antibody expression amount. The stably transfected CHO-K1 cells were digested with pancreatin to obtain a single cell suspension. Cell counts were performed and diluted in multiples to a cell density of 100 cells/ml. The diluted cell suspension was inoculated into a 96-well cell culture plate at 100. Mu.l/well and placed in a cell incubator overnight. Wells containing only one monoclonal cell were labeled. The confluency can be 90% in about 2 weeks of cell culture, and the cell culture supernatants from each well can be collected for ELISA detection by comparing OD ₄₅₀ And screening out monoclonal cell strains with high antibody expression quantity, and then carrying out expanded culture.

2.3ELISA method for detecting expression of anti-PD-1 full-length antibody of stably transfected monoclonal cell strain

PD-1ECD protein was added to the microplate and coated overnight at 4 ℃. The next day, the microplate was washed 3 times with PBST, and the washing solution was removed by inverting the microplate. Add 200. Mu.l of blocking solution to each well and block for 1h at 37 ℃. The blocking solution was discarded and then washed 3 times with the washing solution. Adding 200 μ l of cell culture supernatant into each well, incubating at 37 deg.C for 2h, and using PBS as blank control; the negative control was control cell culture supernatant. The cell culture supernatant was discarded and washed 3 times with washing solution. The sealing liquid is prepared by mixing 1: goat anti-human IgG-HRP secondary antibody was diluted at a rate of 1000, 200. Mu.l of secondary antibody was added to each well, and incubated at 37 ℃ for 1h. The secondary antibody was discarded and washed 5 times with washing solution. Mu.l of freshly prepared TMB solution was added to each well and developed for 30min at 37 ℃. 50 mul of stop solution (2M H) was added to each well ₂ SO ₄ ) And measuring the OD value under the wavelength of 450nm by using a microplate reader. The results showed that most of the selected monoclonal cell lines were able to bind to the PD-1ECD protein, while Nos. 8-3 had higher affinity than the other monoclonal (FIG. 2). Subsequent selectionExperiments were performed with monoclonal cell lines expressing antibodies No.8-3.

EXAMPLE 3 expression and purification of antibodies No.8-3

3.1 suspension acclimatization of monoclonal cells stably expressing No.8-3 antibody

In order to improve the antibody expression of the CHO-K1 cells after the monoclonal reaction, the cells in an adherent state need to be subjected to suspension domestication. When the confluency of the CELLs reaches 90%, the CELLs are passaged, and the original DMEM medium is replaced by EX-CELL 302 medium. The serum concentration of the cell culture medium was reduced to half of the original concentration at each passage, i.e., from 10%,5%, and then halved until the serum concentration was reduced to 1%. When the serum concentration of the cell culture medium is reduced to 1%, the cells need to continue to passage for 2-3 generations to maintain and adapt to the low-concentration serum. The serum concentration of the subsequent cell culture is continuously and sequentially reduced to 0.5 percent and 0 percent, and each serum concentration needs to maintain the stable passage of the cells for 2-3 generations. When the CELLs have grown in suspension, the suspension CELLs are transferred to a 250mL CELL shake flask, 10mL EX-CELL 302 medium is added, and supplemented with 4 mM/L-glutamine, 37 ℃,5% CO ₂ The culture was performed in an incubator at 120rpm with shaking. When the CELL density increased, EX-CELL 302 medium was gradually added to a total volume of 100ml, and glutamine was maintained at 4 mM/L. Cell viability was checked daily by trypan blue staining until cell viability decreased to 50%, cell culture supernatants were harvested by centrifugation at 3000rpm and frozen at-20 ℃. The suspended and domesticated cells can be directly frozen by using a serum-free freezing medium.

3.2 Affinity chromatography purification of No.8-3 antibody

Cell culture supernatants were purified using the Bio-Rad NGC Protein purification chromatography system and Protein A purification columns using the principle that Protein A binds specifically to the Fc domain of the constant region of IgG molecules. The liquid on the column was filtered through a 0.22 μ M filter. The Protein A purification column was equilibrated with 3-5 Column Volumes (CV) of column wash buffer at a flow rate of 1 mL/min. 100mL of cell culture supernatant can be loaded each time, and after loading is finished, the deproteinized protein is continuously washed by 10CV of washing column buffer until the UV 280 curve returns to the baseline level. The antibody was eluted with 5CV of elution buffer and the eluate was collected in a 1.5mL EP tube, 1 mL/tube volume. The eluted product was rapidly neutralized with 1M Tris-HCl. FIG. 3A shows the purification scheme of antibodies No.8-3. After the elution was completed, the column was washed with 10CV of column washing buffer, and finally the Protein A purification column was stored in 20% ethanol. The antibodies in the collection tubes located within the elution peak range were concentrated. The antibody was concentrated using a 30kDa molecular cut-off ultrafiltration tube and centrifuged at 10,000xg for 5min. Finally, PBS was added, and the mixture was centrifuged 3 times to replace the buffer with PBS. After the BCA method to determine protein concentration, SDS-PAGE electrophoresis was performed to determine the structural integrity and purity of the antibody (FIG. 3B).

EXAMPLE 4 determination of the affinity of antibodies No.8-3

The binding kinetics of No.8-3 antibody and PD-1 antigen were analyzed and determined by Fortibio Octet biomacromolecule interaction analyzer. The results of the analysis are shown in FIG. 4. As a result, it was found that the affinity of the antibody No.8-3 was higher than that of Keytruda (Table 1).

TABLE 1 determination of affinity constant of anti-PD-1 antibody

Sample	Ka(1/Ms)	Kd(1/s)	K _D (M)
				Keytruda	9.97×10 ⁵	2.13×10 ^-3	2.14×10 ^-9
No.8-3	2.16×10 ⁴	4.49×10 ^-7	2.08×10 ^-11

Example 5 flow cytometry to detect the binding Capacity of an anti-PD-1 antibody to PD-1 on the surface of a cell

Selecting PD-1 positive human acute leukemia mononuclear cells THP-1, 000rpm, centrifuging for 5min, and collecting cell precipitates. Add 500. Mu.L of FACS buffer to resuspend the cells and wash 2 times. Each group of cell treatment was set at 2-3 replicates and the blank control was not treated at all. FACS buffer diluted anti-PD-1 antibody was added and incubated on ice for 1h. Add 500. Mu.L of FACS buffer to resuspend the cells and wash 2 times. FACS buffer diluted FITC labeled anti-human fluorescent secondary antibody (1: 500 dilution ratio) was added, incubated on ice for 1h in the dark and washed 2 times by FACS buffer. Finally, the cells were resuspended in 500. Mu.L PBS and filtered through a 300 mesh cell screen, and the mean fluorescence intensity of the cells and the percentage of positive cells were measured on a flow cytometer. As a result, it was found that both antibodies No.8-3 and Keytruda bind to cell surface PD-1, as shown in FIG. 5.

Example 6 detection of blocking Effect of anti-PD-1 antibody on PD-1/PD-L1 by flow cytometry

Selecting positive PD-1 THP-1 and U937 cells, centrifuging at 1,000rpm for 5min, and collecting cell precipitate; add 500. Mu.L of FACS buffer to resuspend the cells and wash 2 times. Adding 2 mu g of PD-L1-his recombinant protein and anti-PD-1 antibody mixed solution with different concentrations into each group of cells, fully mixing uniformly, and incubating for 2h at 4 ℃. Each group was set to 2 replicates, one of which was incubated with only PD-L1-his recombinant protein, and the blank control was not treated. FACS buffer diluted anti-mouse His monoclonal antibody (1: 400 dilution) was added and incubated at 4 ℃ for 1h. Add 500. Mu.L of FACS buffer to resuspend the cells and wash 2 times. FACS buffer diluted Alexa Flior 647 labeled anti-mouse fluorescent secondary antibody (1 dilution ratio 500) was added, incubated at 4 ℃ in the dark for 1h and FACS buffer washed 2 times. Finally, the cells were resuspended in 500. Mu.L PBS and filtered through a 300 mesh cell sieve, and the mean fluorescence intensity of the cells was measured on a flow cytometer. The flow-through results were processed by FlowJo and Excel software to calculate the competitive inhibition rate of the anti-PD-1 antibody, and as a result, as shown in FIG. 6, it was found that both antibodies No.8-3 and Keytruda inhibited the binding of PD-L1 to PD-1, and that the inhibition effect of antibody No.8-3 was superior to that of Keytruda at the same antibody concentration.

Example 7 ELISA detection of the level of IFN-. Gamma.Release from T cells

Resuscitated human peripheral blood lymphocytes (PBMCs) were stimulated with 300U/mL IL-2 and 1. Mu.g/mL anti-CD 28 antibody to increase the proportion of activated T lymphocytes after 48h of stimulation. Triple negative breast cancer MDA-MB 231 cells and stimulated PBMCs were mixed according to 1:2 in a 96-well plate and 100. Mu.L of anti-PD-1 antibody (final concentration of 1. Mu.g/mL or 10. Mu.g/mL) was added for a total incubation of 48h. The cell supernatant was collected by centrifugation, and the concentration of IFN-. Gamma.released into the cell supernatant was measured by ELISA kit. Adding 100 mu L of IFN-gamma standard substances or samples to be detected with different concentrations into each hole of the antibody pre-coated enzyme label plate, and incubating for 2h at 37 ℃. After washing the plate 3 times with wash solution, 100. Mu.L of biotinylated antibody (100X) working solution was added and incubated for 1h at 37 ℃. After washing the plate 3 times, 100. Mu.L of streptomycin-HRP (100X) working solution was added and incubated at 37 ℃ for 30min. After washing the plate 3 times, 100. Mu.L of TMB substrate was added, incubated at 37 ℃ for 15min, followed by 50. Mu.L of stop buffer, and OD at 450nm was immediately detected using a microplate reader. As shown in FIG. 7, both the No.8-3 antibody and Keytruda were effective in stimulating IFN-. Gamma.secretion from T lymphocytes in the cell co-culture system, i.e., promoting the activation of T cells, thereby killing tumor cells. Also, at a concentration of 10. Mu.g/mL, antibody No.8-3 had a stronger effect of restoring T cell activation than Keytruda.

It is obvious to those skilled in the art that the present invention is not limited to the above embodiments, and the invention is not limited to the above embodiments, and it is within the scope of the present invention to use the method concept and technical solution of the present invention directly in other fields without any substantial modification or improvement.

Sequence listing

<110> Zhejiang university

<120> monoclonal antibody No.8-3 of fully human anti-PD-1 and application thereof

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gggaaagccc ctaagctcct gatctatgct gcacccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gaagtcgatc tgggacacaa tatactttat ccatcgacag cctgcaacct 240

gaagattttg caaattatta ttgtcaacaa tatcagactc tcccactcac tttcggcgga 300

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Claims

1. The monoclonal antibody No.8-3 of the fully human anti-PD-1 is characterized in that the monoclonal antibody No.8-3 comprises a heavy chain variable region and a light chain variable region as well as an antibody constant region, wherein a polynucleotide sequence for coding the VH of the heavy chain variable region is shown in SEQ ID No.1, an amino acid sequence of the VH of the heavy chain variable region is shown in SEQ ID No.2, a polynucleotide sequence for coding the VL of the light chain variable region is shown in SEQ ID No.3, and an amino acid sequence of the VL of the light chain variable region is shown in SEQ ID No. 4.

2. The fully human anti-PD-1 monoclonal antibody No.8-3 of claim 1, which comprises the complete complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of VH CDR1 of the heavy chain variable region is QSISSY, and the amino acid sequence of VH CDR2 of the heavy chain variable region is: AAP, the amino acid sequence of VH CDR3 of the heavy chain variable region is as follows: QQYQTLPLT; the amino acid sequence of the light chain variable region VL CDR1 is as follows: the amino acid sequence of the VL CDR2 of the light chain variable region of EDTFGNHA is as follows: IIPMFSKV; the amino acid sequence of light chain variable region VL CDR3 is: AKEGEPPRDDAFDI.

3. A recombinant DNA expression vector comprising the polynucleotide sequence of claim 1 encoding the heavy chain variable region VH and the polynucleotide sequence of claim 3 encoding the light chain variable region VL SEQ ID No. 1.

4. Use of the fully human PD-1 monoclonal antibody No.8-3 according to claim 1 for the preparation of a tumor immunotherapy antibody medicament, characterized in that the tumor is triple negative breast cancer.