CN108840932B

CN108840932B - PD-1 specific antibody and anti-tumor application thereof

Info

Publication number: CN108840932B
Application number: CN201810822534.0A
Authority: CN
Inventors: 严景华; 陈丹青; 谭曙光; 仝舟; 高福
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2018-04-28
Filing date: 2018-07-24
Publication date: 2022-03-29
Anticipated expiration: 2038-07-24
Also published as: CN108840932A

Abstract

The invention provides an anti-PD-1 antibody or an antibody fragment thereof, which can specifically bind to a PD-1 molecule, can block the binding of PD-1 and PD-L1, PD-L2 or a combination thereof after the binding, and can generate biological effects such as T cell activation and anti-tumor.

Description

PD-1 specific antibody and anti-tumor application thereof

Technical Field

The invention belongs to the field of medicine, and particularly relates to an antibody or an antigen binding fragment thereof, wherein the antibody or the antibody fragment thereof specifically recognizes programmed cell death molecules (PD-1) and can be used as an immune activator to stimulate the immune reaction of an organism so as to generate the effect of resisting diseases such as tumors and the like.

Background

In 2011, cancer surpasses heart disease, and becomes the first leading cause of death worldwide. WHO published 12 months in 2013, the number of newly added cancer patients worldwide has exceeded 1400 million every year, which is greatly increased compared to 1270 million patients as a statistical result in 2008. At the same time, the number of deaths among cancer patients has increased, from 760 to 820 million in the past. The immune anticancer therapy in 2013 is judged as the first breakthrough of 10 years of Science journal, and since then, the research of the tumor immunotherapy continuously gets breakthrough progress, the clinical application of the immune anticancer therapy also gets huge success, and the immune anticancer therapy is the most promising treatment means in the current tumor treatment research field and is expected to become a new conventional treatment method following the operation and the chemoradiotherapy method.

Early in the 80's of the 20 th century, Allison and other researchers determined the genetic structure of the α β T Cell Receptor (TCR) responsible for antigen recognition on the surface of T cells. In the later 80 s, Boone, Rosenberg, Old and other researches respectively find that some tumor specific antigens exist in different tumor patients, can be recognized by T cells and specifically kill the tumor cells, so that the hope of tumor immunotherapy is reignited, and a great deal of research is dedicated to the research and development of tumor therapeutic vaccines. However, Schwartz et al found that TCR signaling alone was not sufficient to activate antigen-specific T cells, and that T cell activation also required the involvement of other molecules, namely the synergistic effect of a so-called second signal, a "co-stimulatory molecule". It has also been found that only certain Antigen Presenting Cells (APCs) are capable of expressing the costimulatory molecule, whereas most cells, including tumor cells, are not capable of providing a costimulatory molecule signal. In the early 90 s of the 20 th century, Allison et al discovered the molecule CD28, which provided a secondary signal required for T cell activation. Later, Linsley et al researches find that B7 molecules expressed on the cell surface of APCs are ligands of CD28 molecules, while Allison et al researches through a mouse model, and tumor cells which can express B7 molecules after being modified can be rapidly eliminated by a mouse immune system. Therefore, the lack of expression of the tumor cell B7 molecule may be an important factor for the body's inability to efficiently stimulate T cell immunity.

In the 20 th century, studies in the 90 s indicated that cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) performed a completely opposite function to that of CD28 in vivo, and that if the CD28 molecule was compared to the throttle of an automobile, the CTLA-4 molecule performed a "brake" function. Such molecules are also referred to as "immune checkpoint" molecules because, upon activation of T cells in the body, they "check" the degree of immune cell activation, up-regulate expression in activated cells and exert immunosuppressive functions, so that T cells in the body are not so hyperproliferative and activated as to damage normal cells. Cancer cells use the immunosuppressive mechanism of these molecules to evade the killing of the body's immune system. Researches show that the CTLA-4 specific monoclonal antibody is used for blocking CTLA-4 signals, the activity of T cells can be obviously improved, and the monoclonal antibody can greatly improve the tumor inhibition capability of mice after blocking CTLA-4 in mouse model researches of various tumors. In addition to CTLA-4, immune checkpoint molecules include PD-1, PD-L1, TIM-3, LAG-3, TIGIT, etc., B7 superfamily and CD28 superfamily molecules. By blocking these "inhibitory" signals by specific monoclonal antibodies, the activity of the T cells can be re-released, thus enabling these T cells to exert an anti-tumor effect. The contribution of tumor immune checkpoint therapy to anti-tumor strategies lies in: on the one hand, immune checkpoint therapies do not directly target tumor cells, but act on the patient's immune system, releasing T cell activity by releasing signals that limit the function of T cells; on the other hand, the activation of T cells is not antigen-specific, but rather reactivates the entire immune system, and thus can be applied to the treatment of a variety of different tumors, as a universal therapy for tumors. Moreover, the success of CTLA-4 antibody blocking therapy has opened up the development and application of immunosuppressive related molecular block in tumor therapy, and blocking antibodies developed based on immunosuppressive molecules represented by PD-1 and PD-L1 have also made a major breakthrough, and in 2014, the FDA has approved two PD-1 blocking antibodies, nivolumab and pembrolizumab, for clinical treatment of melanoma.

At present, there is still a need to develop further anti-PD-1 antibodies.

Disclosure of Invention

One aspect of the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof capable of specifically binding to a PD-1 molecule, said anti-PD-1 antibody or antigen-binding fragment thereof comprising an amino acid sequence as set forth in SEQ ID NO: 3. SEQ ID NO:4 and SEQ ID NO:5, the heavy chain CDRs; and as shown in SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8, and light chain CDRs as shown in figure 8. Wherein the fragment is selected from the group consisting of Fab, Fab '-SH, Fv, scFv, F (ab')₂An anti-PD-1 antibody or an antigen-binding fragment thereof that blocks the binding of PD-1 to a substance selected from the group consisting of PD-L1, PD-L2, or a combination thereof.

In a specific embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises SEQ ID NO:9 and SEQ ID NO:10, or the anti-PD-1 antibody or antigen-binding fragment thereof comprises the light chain sequence set forth in SEQ ID NO:1 and SEQ ID NO:2, or a light chain sequence set forth in seq id no.

In a specific embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises SEQ ID NO:11 and SEQ ID NO:12, or the anti-PD-1 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:13 and SEQ ID NO:14, or a light chain variable region as shown in fig. 14.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a murine or humanized anti-PD-1 monoclonal antibody, preferably the humanized PD-1 monoclonal antibody comprises a human Fc region, more preferably the Fc region of human IgG 4.

In a specific embodiment, the invention relates to a polypeptide having a sequence as set forth in SEQ ID NO:11, wherein the polypeptide is part of an antibody that specifically binds to a PD-1 molecule, and wherein the antibody further comprises the amino acid sequence of SEQ ID NO: 12.

In a specific embodiment, the invention relates to a polypeptide having a sequence as set forth in SEQ ID NO:13, wherein the polypeptide is part of an antibody that specifically binds to a PD-1 molecule, and wherein the antibody further comprises the amino acid sequence of SEQ ID NO: 14.

In a specific embodiment, the invention relates to a polypeptide having a sequence as set forth in SEQ ID NO:12, wherein the polypeptide is part of an antibody that specifically binds to a PD-1 molecule, and wherein the antibody further comprises the amino acid sequence of SEQ ID NO: 11.

In a specific embodiment, the invention relates to a polypeptide having a sequence as set forth in SEQ ID NO:14, wherein the polypeptide is part of an antibody that specifically binds to a PD-1 molecule, and wherein the antibody further comprises the amino acid sequence of SEQ ID NO: 13.

A second aspect of the invention provides an isolated polynucleotide encoding the anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ id no:9 and SEQ ID NO:10, or the anti-PD-1 antibody or antigen-binding fragment thereof comprises the light chain sequence set forth in SEQ ID NO:1 and SEQ ID NO:2, or a light chain sequence set forth in seq id no.

In a specific embodiment, the invention relates to a polynucleotide encoding the anti-PD-1 antibody or antigen-binding fragment thereof.

In a specific embodiment, the present invention relates to a polynucleotide encoding the polypeptide of SEQ ID NO:11, wherein the polypeptide is part of an antibody that specifically binds to a PD-1 molecule, and wherein the antibody further comprises the amino acid sequence set forth in SEQ ID NO:12, preferably the polynucleotide sequence is SEQ ID NO: shown at 15.

In a specific embodiment, the present invention relates to a polynucleotide encoding the polypeptide of SEQ ID NO:12, wherein the polypeptide is part of an antibody that specifically binds to a PD-1 molecule, and wherein the antibody further comprises the amino acid sequence set forth in SEQ ID NO:11, preferably the polynucleotide sequence is SEQ ID NO: shown at 16.

In a specific embodiment, the present invention relates to a polynucleotide encoding the polypeptide of SEQ ID NO:13, wherein the polypeptide is part of an antibody that specifically binds to a PD-1 molecule, and wherein the antibody further comprises the amino acid sequence set forth in SEQ ID NO:14, preferably the polynucleotide sequence is SEQ ID NO: shown at 17.

In a specific embodiment, the present invention relates to a polynucleotide encoding the polypeptide of SEQ ID NO:14, wherein the polypeptide is part of an antibody that specifically binds to a PD-1 molecule, and wherein the antibody further comprises the amino acid sequence set forth in SEQ ID NO:13, preferably the polynucleotide sequence is SEQ ID NO: 18, respectively.

A third aspect of the invention is to provide an expression vector comprising the polynucleotide.

A fourth aspect of the present invention is to provide a host cell comprising the above-described expression vector.

A fifth aspect of the present invention provides a method of preparing the anti-PD-1 antibody or an antigen-binding fragment thereof, the method comprising: 1) culturing the host cell; 2) recovering the anti-PD-1 antibody or antigen-binding fragment thereof from the host cell or culture medium.

The sixth aspect of the present invention provides a pharmaceutical composition comprising the anti-PD-1 antibody or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

The seventh aspect of the invention provides the use of the anti-PD-1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for increasing the level of IFN- γ secretion by T cells.

The eighth aspect of the invention is to provide the use of the anti-PD-1 antibody or an antigen-binding fragment thereof in the preparation of an anti-tumor medicament for treating non-small cell lung cancer and the like.

The anti-PD-1 antibody or the fragment thereof provided by the invention can specifically bind to a PD-1 molecule, can block the binding of PD-1 to a compound selected from PD-L1, PD-L2 or a combination thereof after being bound, and can generate a series of biological effects. These biological effects include, for example: can improve the level of IFN-gamma secretion of tumor-specific T cells in tumor cases, and particularly can inhibit the growth of tumors in mice.

In the present invention, the expression "PD-1 antibody" or "murine PD-1 antibody" is a murine monoclonal antibody directed against PD-1, in a particular embodiment a PD-1.C antibody. The expression "humanized PD-1 antibody" is made by humanization on the basis of murine PD-1 antibody, and in a particular embodiment, by humanization of PD-1.C antibody.

PD-1 molecules are important members of the CD28 family, which also includes CD28, CTLA-4, ICOS, etc. PD-1 molecule is expressed in activated B cells, T cells, bone marrow cells and the like, and is a type I membrane protein with the molecular weight of about 55kDa, and PD-1 is found to have two ligands, namely PD-L1 and PD-L2, and the PD-1 inhibits the activity of T cells through the interaction with (PD-L1 and/or PD-L2). PD-L1 can be highly expressed on the surfaces of various tumor cells, and through the interaction between PD-1 and PD-L1, the functional activity of tumor infiltrating lymphocytes, including TCR-mediated T cell proliferation capacity, cytokine secretion level and the like, can be inhibited, so that the PD-L1 is utilized by various tumors and is used as an important mechanism for monitoring the escape of tumor cells from an immune system. And the antibody is used for specifically blocking the interaction between PD-1 and (PD-L1 and/or PD-L2), so that the T cells in an inhibition state can be activated, the functions of the T cells are released, and the functions of the T cells are recovered, thereby achieving the effect of killing tumor cells by using an immune system of an organism to treat tumors.

The invention is based on the principle that the anti-PD-1 antibody or the antigen binding fragment thereof specifically binds to a PD-1 molecule to block the binding of PD-1 to (PD-L1 and/or PD-L2), so that T cells are activated, and the level of IFN-gamma secretion of the T cells is increased.

The present application includes antibodies or derivatives that specifically bind to PD-1, as well as antibody fragments that exhibit substantially the same antigen specificity as the original antibody. "fragments of an antibody" or "antigen-binding fragments" refers to antigen-binding fragments and antibody analogs of an antibody, which typically include at least a portion of the antigen-binding or variable region of the parent antibody, e.g., one or more CDRs. Fragments of an antibody retain at least some of the binding specificity of the parent antibody. Antigen binding fragments include those selected from the group consisting of Fab, Fab '-SH, Fv, scFv, F (ab')₂Diabodies, peptides containing CDRs, and the like.

A "Fab fragment" consists of one light and one heavy chain of CH1 and the variable domains.

The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains.

A "Fab ' fragment" contains a light chain and a portion of a heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, with an interchain disulfide bond between the two heavy chains of the two Fab ' fragments to form the F (ab ')₂A molecule.

“F(ab′)₂A fragment "comprises two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, F (ab')₂The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks the constant region.

"Single chain Fv antibody (scFv antibody)" refers to an antibody fragment comprising the VH and VL domains of an antibody, which domains are present in a single polypeptide chain. Generally, Fv polypeptides additionally comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain. By using linkers that are so short that they cannot pair between two domains of the same chain, the domains pair with complementary domains of another chain and form two antigen binding sites.

"humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. Humanized antibodies are largely human immunoglobulins in which residues from a hypervariable region of the recipient antibody are replaced by residues from a hypervariable region of a non-human species, such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are substituted for corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not present in the recipient antibody or the donor antibody. These modifications were made to further improve antibody performance.

"specific" binding, when referring to a ligand/receptor, antibody/antigen or other binding pair, refers to determining the presence or absence of a binding reaction for a protein, such as PD-1, in a heterogeneous population of proteins and/or other biological agents. Thus, under the conditions specified, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The invention also provides pharmaceutical compositions comprising the PD-1 antibodies of the invention, or antigen-binding fragments thereof. To prepare a pharmaceutical composition, the antibody or antigen-binding fragment thereof can be prepared into various desired dosage forms by mixing with a pharmaceutically acceptable carrier or excipient. Examples of the dosage form of the pharmaceutical composition of the present invention include tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated preparations, pellets, sublingual tablets, and ointments, which are oral preparations, and examples of non-oral preparations include injections, suppositories, transdermal preparations, ointments, plasters, and external liquid preparations, and those skilled in the art can select an appropriate dosage form according to the administration route and the administration target.

The dose of the active ingredient of the pharmaceutical composition of the present invention varies depending on the subject, the target organ, the symptom, the administration method, and the like, and can be determined by the judgment of the doctor in consideration of the type of the formulation, the administration method, the age and weight of the patient, the symptom of the patient, and the like.

The pharmaceutical compositions of the present invention may also contain other agents, including but not limited to cytotoxic, cytostatic, antiangiogenic or antimetabolic agents, tumor-targeting agents, immunostimulants or immunomodulators or antibodies that bind to cytotoxic, cytostatic or other toxic agents.

Drawings

FIG. 1 is an SDS-PAGE identification of PD-1 protein prepared from three different expression systems.

FIG. 2 is a graph showing Protein A elution and SDS-PAGE identification of the ability of PD-1.C antibody to block the binding of PD-1 to PD-L1.

FIG. 3 is a graph showing that PD-1.C antibody is capable of blocking the binding of PD-1 to PD-L1.

FIG. 4 is a graph showing that PD-1.C antibody can activate tumor antigen-specific T cell responses in tumor cases.

FIG. 5 is a graph showing SPR measurement of the affinity of PD-1.C antibody binding to PD-1 protein prepared in different expression systems.

FIG. 6 shows the results of molecular sieve chromatography and SDS-PAGE purity detection of the humanized PD-1.C antibody protein.

FIG. 7 is a graph showing SPR detection of the affinity of binding of the humanized PD-1.C antibody to PD-1.

FIG. 8 is a graph showing the results of a tumor model inhibition experiment of humanized PD-1.C antibody NCG mouse HCC-827.

Detailed Description

The present invention will be described more specifically with reference to examples. However, it will be understood by those skilled in the art that the following examples are for illustrative purposes only and are not intended to limit the present invention.

Example 1 preparation of PD-1.C antibody

Construction of PD-1 recombinant expression plasmid

And (2) taking the cDNA (NM-005018.2) of the PD-1 as a reference, synthesizing a DNA sequence of the PD-1 (amino acids 1-170), respectively introducing restriction sites EcoRI and BglII, and introducing 6 His amino acid tags into the C end, wherein the EcoRI restriction site is positioned at the 5 'end of the sequence, and the 6 His amino acid tags and the restriction sites BglII are sequentially positioned at the 3' end of the sequence. The synthesized DNA sequence of PD-1 is cloned into an expression vector pCAGGS (Addgene company) by utilizing restriction enzyme cutting sites EcoRI and BglII, and a recombinant eukaryotic expression plasmid of the PD-1 full-length protein is established.

Expression and purification of PD-1 recombinant proteins

1. HEK293T cells: HEK293T cells (ATCC) were transferred to a petri dish at a ratio of 1: 3 for further culture; add 7.5mL DMEM (serum free and antibiotic) (GIBCO) to a 50mL tube and add 300. mu.L Polyetherimide (PEI))1.0, mixing evenly; adding 75 mu g of PD-1 recombinant plasmid DNA into the mixed solution, mixing uniformly and standing for 30 minutes; mu.L of each solution was added to each plate at 37 ℃ with 5% CO₂Culturing in an incubator.

2) Collecting a supernatant: after transfection for 48 hours, the cells were centrifuged at 4 ℃ to collect the supernatant.

3) Purifying by using a nickel affinity chromatography column: filtering the supernatant with 0.45 μ M, 33mm PVDF membrane filter, centrifuging, and binding with nickel affinity chromatography column (GE Health) at 4 deg.C for more than 4 hr; then, elution was carried out with an eluent containing imidazole at various concentrations such as 10mM, 20mM, 50mM, 100mM, 200mM, 300mM, 500mM, etc., and then the elution was changed to 20mM Tirs-HCl, 150mM NaCl buffer for further use, and the purity was confirmed by SDS-PAGE (first lane of FIG. 1).

Expression and purification of PD-1-mFc protein

Taking cDNA (NM-005018.2) of PD-1 as reference, synthesizing DNA sequence of PD-1 (amino acid 1-170), and respectively introducing restriction sites EcoRI and BglII, and introducing a label of mouse Fc amino acid at C end, wherein the EcoRI restriction site is positioned at 5 'end of the sequence, and the Fc label and the restriction site BglII are sequentially positioned at 3' end of the sequence. The synthesized DNA sequence of PD-1 is cloned into an expression vector pCAGGS (Addgene company) by utilizing restriction enzyme sites EcoRI and BglII, and a recombinant eukaryotic expression plasmid of PD-1-mFc protein is established.

PD-1-mFc protein expression:

1) transfection of HEK293T cells: HEK293T cells (ATCC) were transferred to a petri dish at a ratio of 1: 3 for further culture; adding 7.5mL DMEM (serum-free and antibiotic) into a 50mL tube, adding 300 mu L Polyetherimide (PEI)1.0, and mixing uniformly; adding 75 mu g of PD-1-mFc recombinant plasmid DNA into the mixed solution, mixing uniformly and standing for 30 minutes; mu.L of each solution was added to each plate at 37 ℃ with 5% CO₂Culturing in an incubator.

3) Protein A affinity chromatography column purification: the centrifuged supernatant was filtered through a 0.45. mu.M, 33mM PVDF membrane filter, and after removing the precipitate by centrifugation, an equal volume of 20mM Na was added thereto₃PO₄(pH 7.0), filtering with 0.22 μm filter membrane at 4 deg.C, and subjecting to Protein A affinity chromatography (GE Heal)th) binding for more than 4 hours; the PD-1-mFc protein was then eluted from the column with 0.1M Gly pH3.0, 1M Tris pH9.0 about 0.8mL (3.2 mL in collection volume) was added, and the solution was changed to 20mM Tirs-HCl, 150mM NaCl buffer for further use.

Preparation and screening of PD-1 murine monoclonal antibody

The recombinantly produced purified full-length PD-1 recombinant protein (hereinafter abbreviated as PD-1 antigen) was used for immunization of B6/C57 mice (experimental animal technology ltd, viton, beijing) according to the french complete adjuvant intraperitoneal immunization method. The specific method comprises the following steps:

1) animal immunization: the purified PD-1 antigen is emulsified by complete Freund's adjuvant (Sigma), B6/C57 mice of 6-8 weeks old are immunized by an intraperitoneal injection method, the immunization dose is 50 mu g/mouse, the second immunization is carried out after two weeks, and the immunization dose is 50 mu g/mouse by the incomplete Freund's adjuvant. After twice immunization, tail blood is taken and subjected to gradient dilution by an ELISA method to determine the serum titer; and determining whether to strengthen the immunity according to the result, and selecting the mouse with the highest antibody titer for cell fusion.

2) Cell fusion: the myeloma cells adopt BALB/c-derived sp2/0, and are in logarithmic growth phase when fused; taking the spleen of an immunized mouse, and preparing a lymphocyte single cell suspension; mixing mouse spleen lymphocyte and myeloma cell at 1: 5-1: 10, adding 1mL of 50% PEG (pH 8.0) at 37 deg.C, adding incomplete culture medium and rest stop solution, centrifuging, removing supernatant, adding HAT culture medium, suspending, mixing, metering MC to 50mL, subpackaging in 3.5cm culture dish, placing in wet box, placing at 37 deg.C and 5% CO₂Culturing in a constant temperature incubator.

3) Screening and cloning: cell clones were selected within 7-10 days of fusion and hybridoma cell supernatants were tested for binding to PD-1 protein by ELISA using purified PD-1 recombinant protein. Coating the PD-1 recombinant protein on an ELISA plate according to the concentration of 300 ng/hole, incubating overnight at 4 ℃, washing 3 times by using PBS buffer solution, adding a blocking solution of 5% skimmed milk powder (Yili), and incubating for 2 hours at 25 ℃; then washing with PBS buffer solution for 3 times, adding culture supernatant of cell clone, and incubating for 1 hour at room temperature of 25 ℃; then washing with PBS buffer solution for 3 times, adding horseradish peroxidase-labeled goat anti-mouse IgG antibody (three arrows), and incubating for 1 hour at room temperature of 25 ℃; and then washing the substrate for 3 times by using PBS buffer solution, adding TMB developing solution, adding stop solution after 15min, and detecting the absorption value of OD450 by using a spectrophotometer. Screening of cell clones with OD450 > 0.5 for subsequent screening. The cell line number was labeled. And (3) performing limited dilution on the positive hole cells, measuring the ELISA value 5-6 days after each limited dilution, and selecting the monoclonal hole with the higher OD450 positive value in the ELISA detection to perform limited dilution until the whole plate result of the 96-well plate in the ELISA detection is positive. And (4) selecting a monoclonal fixed strain with a high positive value. The corresponding fused plate cell strain is PD-1. C.

4) Preparing and purifying cell supernatant monoclonal antibody: culturing cell line PD-1.C in DMEM medium containing 15% serum in 10cm culture dish, expanding to about 4 × 10⁷At this time, the cells were centrifuged at 800rpm for 5min, the supernatant was discarded and the cells were transferred to a 2L spinner flask and serum-free medium was added to give a cell density of about 3X 10⁵One per ml. After further culturing for 1-2 weeks, when the cell death rate reaches 60% -70% (at this time, the cell density is about 1-2 × 10⁶And each ml), collecting cell suspension, centrifuging at 6000rpm for 20min, collecting supernatant, purifying the supernatant by affinity chromatography, selecting corresponding column material according to antibody profiling, wherein the monoclonal antibody PD-1.C subtype is IgG1, and purifying by protein G. Measuring the concentration of purified monoclonal antibody, subpackaging (100 uL/tube, concentration of 1mg/ml), and storing at 4-8 deg.C.

5. Expression purification of murine PD-1.C antibody

2 x 10 to⁶The cell strain PD-1.C is prepared by injecting 6-8 weeks old BALB/C mouse (purchased from Wintolite Corp.) into abdominal cavity, collecting ascites after 2-3 weeks, centrifuging the obtained ascites to remove precipitate, adding equal volume of 20mM Na₃PO₄(pH 7.0), and then filtering the ascites by using a filter membrane of 0.22 mu m, mainly preventing other impurities in the ascites from damaging the column; after filtration is complete, the sample is ready for purification.

Protein G (5mL) HP affinity column (GE Co.) was attached to AKTAPurifier/Explorer/FPLC/START (GE Co.) and the following procedure was run on the machine: the column was flushed with 20% ethanol and then with 20mM ethanol Na₃PO₄Balancing the column with a buffer solution with pH 7.0, and injecting the ascites into the device to be combined with Protein G in a 5mL loop ring loading mode at a flow rate of 1mL/min after the conductivity is 4.5% on the device; after UV is stabilized, 1M Tris pH9.0 about 0.8mL (collection volume 3.2mL) is added to the subsequent collection tube, then the procedure is changed to 100% 0.1M Gly pH3.0 to elute the antibody hung on the column, the eluted sample is collected, the sample is prepared, gel electrophoresis identification is carried out, the size of the gel image strip is judged to be correct, and the purity of the gel image strip is identified by SDS-PAGE (figure 2); if the identification is correct, the concentrated antibody is continuously diluted by PBS by adopting a method of concentrating and changing liquid, and the sample is subpackaged after repeatedly concentrating and diluting by more than 100 times and is directly used or stored in a refrigerator at the temperature of minus 80 ℃.

Example 2 PD-1 blocking antibody screening and affinity analysis

Mouse immunization is carried out on the PD-1 protein expressed in vitro by 293T cells, and the obtained monoclonal antibody is subjected to blocking experiment of PD-1 and PD-L1 to screen an antibody capable of specifically blocking the interaction of PD-1 and PD-L1.

Preparation of PD-L1 full-Length expression 293T cells

In this example, 293T cells (ATCC) expressing the full length PD-L1 were obtained by transfecting 293T cells (ATCC) with a PD-L1-GFP-p plasmid (Clontech) containing the full length PD-L1 (pEGFP-N1 vector-GFP tag plasmid). 1 day before transfection, the ratio of the total amount of the active components to the total amount of the active components is 0.5-2 × 10⁵Cells were seeded in 24-well culture plates per well, and 500. mu.L of antibiotic-free DMEM complete medium (GIBCO Co.) was added to ensure that the cells were confluent at 70-80% at the time of transfection. Mu.g of the PD-L1-GFP-p plasmid was diluted in 50. mu.L of medium without serum and antibiotics and mixed gently. Mu.l PEI (Sigma) (4mg/mL) was diluted in 50. mu.L medium without serum and antibiotics and mixed gently. After 5 minutes, 50. mu.L of the LPEI dilution was added dropwise to 50. mu.L of the PD-L1-GFP-p plasmid dilution, gently mixed and incubated at room temperature for 20 minutes. Mu.l PEI/PD-L1-GFP-p plasmid complex was added dropwise to each well and mixed well with fresh medium with gentle shaking. And (3) putting the cells into an incubator for incubation for 4-6h, and then replacing a serum-containing culture solution to remove the compound. The cells were placed at 37 ℃ CO₂After incubation for 24 hours, the incubator is passed through flow cytometryThe analyzer (BD ARIA II) measures GFP expression levels and evaluates the expression levels of the full-length PD-L1 expressing 293T cells.

2. Antibody blocking assay

The PD-1.C antibody prepared in example 1 and the PD-1-mFc protein (obtained in example 1) were mixed at a molar ratio of 2: 1 and incubated on ice for 1 hour, after which they were added to a mixture containing 2X 10⁵The full-length expression 293T cells of PD-L1 were incubated on ice for 30 minutes. Setting Ebola virus GP protein specific antibody 4G7(Mapp Biopharmaceutical) as a negative control; after that, PBS was washed twice, APC-labeled anti-mouse IgG secondary antibody (BD) was added, and after incubation for 30 minutes, washing was performed twice with PBS buffer, and finally flow cytometry analysis was performed after resuspension with 300mL of PBS solution. The results are shown in fig. 3, and indicate that PD-1-mFc was able to significantly bind to 293T cells expressed in full length of PD-L1, while the addition of PD-1.C antibody completely inhibited the binding of PD-1 to PD-L1, thereby rendering PD-1-mFc unable to bind to PD-L1 protein on the surface of 293T cells (fig. 3). Thus, the PD-1.C antibody was able to significantly inhibit the binding of PD-1 and PD-L1 at the cellular level.

Example 3 in vitro activation of tumor-specific T cells in non-Small cell Lung cancer cases by PD-1 blocking antibodies

One of the important applications of the PD-1 blocking antibody is the anti-tumor effect, in the embodiment, 15 cases of non-small cell lung cancer peripheral blood are collected, tumor specific polypeptide is used for in vitro culture, then the activation capability of the PD-1.C antibody on tumor polypeptide library specific T cells is evaluated for detection, and the anti-tumor potential of the PD-1 blocking antibody screened by the invention at the in vitro cell level is evaluated.

1. Case grouping screening

The included cases in this example are non-small cell lung cancer cases positive for tumor cells from needle biopsies.

Sorting of PBMCs

The lymphocytes used in the present invention are derived from the venous peripheral blood of an individual. After the screened individuals are qualified by the physical examination of a clinician, the testers inform the specific project flow and the required blood quantity, the volunteers agree and sign an informed consent, and the clinical medical staff take blood from the volunteers. MiningThe blood product contains EDTA-K₂Anticoagulated 9mL disposable vacuum blood collection tubes (VACUETTE, Graina, Austria) collected about 20-25mL of blood from each volunteer, and immediately after collection, they were inverted to prevent clotting.

1) Diluting freshly collected peripheral blood by one time with phosphate buffer solution (PBS, pH7.4) which is sterilized and cooled at 121 ℃ under high pressure, carefully adding the diluted blood sample into 15mL of prepared lymphocyte separation solution (purchased from Tianjin third ocean biotechnology Co., Ltd.) with great care during addition, and slowly adding to avoid interface disorder;

2) centrifuging at 25 deg.C for 20min with 700g horizontal centrifuge (SOLVAL Stratos, Thermo Co., USA) and slowing down when stopping;

dividing the centrifuged sample into four layers, sucking the uppermost layer of plasma by a Pasteur pipette, and carefully sucking the second layer of lymphocyte layer into a new sterile centrifuge tube to obtain crude pure PBMCs cells;

3) diluting the crude PBMCs cells with an equal volume of phosphate buffer solution (PBS, pH7.4), and centrifuging at 25 deg.C with 800g centrifugal force for 10 min;

the supernatant was discarded, resuspended in 7mL serum-free RPMI1640 (Hyclone brand, GE USA), and centrifuged at 500g for 5min at 25 ℃;

the supernatant was discarded, resuspended, washed with 7mL of RPMI-1640 medium containing 10% fetal bovine serum (FBS, Australian origin, Thermo Fisher company, USA), and centrifuged at 500g for 5 minutes at 25 ℃;

4) discarding supernatant, resuspending with 3mL RPMI-1640 medium containing 10% FBS, taking appropriate amount of resuspension solution, counting cells on blood count plate, and finally adjusting to 2.5 × 10 with RPMI-1640 medium containing 10% serum⁶Obtaining PBMCs cells for later use according to the cell/mL density;

5) adding a tumor antigen polypeptide library (the tumor antigen polypeptide library is shown in table 1) with the final concentration of each peptide of 5 mu g/mL into the cell suspension for stimulation, adding rIL-2 (double Lut pharmacy) with the final concentration of 20U/mL on the third day after culture for continuous culture, changing the liquid half according to the state of a culture medium on the third day and the seventh day in the culture process, supplementing 20U/mL rIL-2 (double Lut pharmacy), and harvesting cells on the tenth day for relevant detection.

6) ELISPOT detects T cell activation levels of antibodies

ELISPOT detection cell stimulation culture

The ELISPOT plate (Merck Michibo Co.) was coated with anti-human gamma interferon monoclonal antibody (BD) diluted with phosphate buffer (pH7.4) more than 12 hours in advance, and placed horizontally at 4 ℃. Blocking was performed with RPIM-1640 medium containing 10% serum (HyClone) for 1 hour at room temperature before addition of stimulating antigen and cells.

The tumor specific polypeptide library (see Table 1 for the tumor antigen polypeptide library) was diluted to 5. mu.g/ml per polypeptide in RPMI-1640 medium with 10% serum (HyClone), 100. mu.L of each peptide was added to each well of the ELISPOT plate wells, two replicate wells per peptide library, a blank control well without polypeptide stimulation and a lectin (PHA) (Sigma) -stimulated positive control well were provided. Controls for this experimental setup included:

no polypeptide stimulation, no antibody, negative control;

adding antibody without polypeptide stimulation;

polypeptide stimulation, no antibody;

polypeptide stimulation, adding antibody;

wherein the added antibodies include: PD-1.C antibody and irrelevant antibody (EBOLA virus GP protein antibody 4G7(Mapp Biopharmaceutical)). The antibody was added to 96 wells at a final concentration of 10. mu.g/mL for stimulation culture.

The diluted PBMCs cells were added to 100. mu.l/well, and after the addition, the ELISPOT plate containing 100. mu.L of the polypeptide diluent and 100. mu.L of the PBMCs diluent was placed at 37 ℃ in a 5% CO plate₂Incubate for 18 hours under (carbon dioxide) conditions.

ELISPOT plate washing and result acquisition

a) After incubation, the cell sap in the wells was discarded, 200. mu.L of normal temperature deionized water was rapidly added to each well for washing 2 times, and then PBS (PBST) containing 5% Tween-20 was washed 3 times.

b) The wash solution was removed, blotted onto absorbent paper, 100. mu.L of diluted detection antibody (BD) was added to each well, and incubated at room temperature for 2 h.

c) The detection antibody solution was removed, PBST washed 3 times, and then 100 μ L of diluted streptavidin-HRP conjugate (BD) was added to each well and incubated at room temperature for 1 h.

d) Color development: the streptavidin-HRP conjugate solution was removed, PBST washed 3 times, followed by PBS washing 2 times. After the wells were vigorously blotted on absorbent paper, 100. mu.L of AEC substrate (BD) solution was added to each well, and the reaction was stopped by washing with distilled water when a clear spot was observed after incubating at room temperature for 15 to 30 minutes.

e) And (3) airing at 37 ℃ or room temperature, then counting reaction spots in the ELISPOT holes by using an automatic plate reader (C.T.L), then adjusting parameters and performing quality control to give a final reaction result.

3. Analysis of results

By counting 10⁵The number of specific spots produced in the cells after stimulation was analyzed to evaluate the effect of the PD-1 blocking antibody on T cell activation.

In 15 cases of non-small cell lung cancer, 4 cases of non-small cell lung cancer were cultured in vitro to amplify tumor-specific T cells, and the response of the 4 cases of tumor PBMCs to PD-1 antibody was evaluated. The results show (fig. 4) that the stimulated wells with PD-1.C antibody were able to produce a stronger T cell immune response and a comparable level of T cell immune response to the negative control wells without polypeptide stimulation, whereas the 4G7 negative control antibody was not significantly different from the negative control wells. Through comparison of the activation levels of T cells after the antibodies are added under the condition of tumor polypeptide stimulation, the stimulation wells added with the PD-1.C antibodies can generate stronger T cell immune response compared with negative control wells, and the 4G7 negative control antibodies have no significant difference compared with the negative control wells.

Therefore, the PD-1.C antibody can effectively activate the activity of T cells in tumor cases under in vitro culture conditions, particularly tumor-specific T cells, promote the generation of IFN-gamma, and further enhance the T cell immune function.

Table 1: tumor antigen polypeptide library (synthesized by Zhongke Suguang Co., Ltd.)

Example 4 affinity validation of PD-1.C antibodies with PD-1 protein produced by different expression systems

In this example, the affinity of the PD-1.C antibody was identified by Surface Plasmon Resonance (SPR).

1. Preparation of PD-1 protein of insect cell expression system

Taking cDNA (NM-005018.2) of PD-1 as reference, synthesizing DNA sequence of PD-1 (amino acid 1-170), respectively introducing enzyme cutting sites EcoR I and Xho I, introducing 6 His tags at C end, wherein EcoRI enzyme cutting site is positioned at 5 'end of the sequence, and the tags of 6 His amino acids and the enzyme cutting site Xho I are sequentially positioned at 3' end of the sequence. The synthesized DNA sequence of PD-1 was cloned into an expression vector pFastBac (Invitrogen corporation) using restriction sites EcoRI and Xho I to construct an insect cell recombinant expression plasmid for PD-1-His protein.

The specific operation process is as follows: transferring the expression plasmid into DH10 competent cells, obtaining recombinant Bacmid through blue-white screening, transfecting Bacmid into insect cells SF-9 cells (Invitrogen) to obtain recombinant baculovirus carrying target genes, carrying out 2-3 rounds of amplification, adding the amplified P3 virus into cells with the cell density of 2 multiplied by 10 according to a certain proportion (according to the virulence of P3 generation virus)⁶Expanding P4 toxin substitute in one/mL SF-9 cells; after 3 days, the cell density was 2X 10 at a ratio of about 1: 4⁶Adding P4 poison into the cell suspension, culturing for 2 days at 27 ℃ in a shaking table at 120rpm/min, collecting cell supernatant, and purifying the target protein by a Histrap pre-packed column and a molecular sieve.

Combining the filtered supernatant with 20mM Tris, 150mM NaCl and pH8.0-well-balanced pre-packed column at 4 ℃ freezer at flow rate of less than 2.5mL/min by using peristaltic pump, connecting the nickel column combined with the target protein to AKTA after combination, eluting by using Buffer A (20mM Tris, 150mM NaCl, pH8.0) for more than 20mL (mainly removing non-specific combination), adjusting Buffer B (20mM Tris, 150mM NaCl, 1M imidazole and pH8.0) by using AKATA GE (Health) to form target protein on the imidazole elution nickel column with different concentration gradients, collecting protein eluted by imidazole Buffer with different concentration gradients, performing gel electrophoresis on samples eluted by different imidazole gradients, and judging the target band by using gel map (a second lane of figure 1).

2. Preparation of PD-1 protein of prokaryotic cell expression system

Taking cDNA (NM-005018.2) of PD-1 as reference, synthesizing DNA sequence of PD-1 (amino acids 25-170), and respectively introducing enzyme cutting sites Nde I and Xho I, wherein, the EcoRI enzyme cutting site is positioned at 5 'end of the sequence, and the enzyme cutting site Xho I is positioned at 3' end of the sequence. The synthesized DNA sequence of PD-1 was cloned into an expression vector pET21 a (Invitrogen corporation) using the restriction sites Nde I and Xho I, and a prokaryotic recombinant expression plasmid for PD-1 protein was constructed.

Transferring the expression plasmid into E.coli.BL21(DE3) competent cells, adding IPTG to perform induction expression, and obtaining the PD-1 protein in an inclusion body state. The inclusion bodies of PD-1 were dropped into 1L of prepared renaturation solution (20mM Tris-HCl, 400mM L-arginine, EDTA 2mM, GSH/GSSG 5mM/1mM), and 3mL of each drop was added in two drops with a minimum interval of 8h, and then concentrated in a concentration cup, PD-1 was exchanged with a buffer of 20mM Tris-HCl, 150mM NaCl, pH8.0, and then purified with a Hiload 16/60superdex200pg molecular sieve column, and the target protein was detected by SDS-PAGE (third lane of FIG. 1).

SPR detection of affinity of PD-1 protein and PD-1.C antibody produced by different expression systems

PD-1 protein and PD-1.C antibody from 293T mammalian cells, insect cells and prokaryotic cells, respectively, produced by three expression systems were exchanged into SPR buffer (10mM HEPES-HCl, 150mM Na-Cl, 0.005% Tween-20, pH 7.4). The PD-1.C antibody was diluted to 20. mu.g/ml and immobilized on a CM5 chip (GE Health), after which the gradient diluted PD-1 protein was flowed through each channel of the CM5 chip, the binding kinetics parameters were analyzed using BIA evaluation software, and the affinity constant (kD) was calculated.

This example identifies the PD-1.C antibody and PD-1 protein produced by different expression systems to evaluate the binding characteristics of the PD-1.C antibody to PD-1 protein (fig. 5). PD-1 protein has four glycosylation sites of N-link, namely aspartic acid (Asn, N) at positions 49, 58, 74 and 116, different expression systems produce proteins with different glycosylation modifications, and the difference of glycosylation level may influence the binding of the antibody and the PD-1 protein. More importantly, the glycosylation modification of the human functional protein is different under the conditions of different cell types, different tissues, different organs, ages, diseases and the like, so that the detection of the affinity of the antibody to the PD-1 protein produced by different expression systems has certain guiding significance for guiding the administration of the PD-1 antibody.

The result shows that the PD-1.C antibody can be combined with PD-1 protein obtained by insect cell production and renaturation after escherichia coli expression, and the affinity is 5.23 multiplied by 10 respectively^-10M and 3.41X 10^-10M, 1.33X 10 affinity to PD-1 expressed by eukaryotic 293T cells^-9M, no obvious difference from each other. The results of this example show that the binding of PD-1.C antibody to PD-1 is not affected by the expression system, suggesting that it may have a broader spectrum of applicability to the population and disease state (see fig. 5).

Example 5 humanization of murine PD-1.C antibody and affinity detection of humanized antibody to PD-1

According to the sequence homology of the PD-1.C antibody, the humanized PD-1.C antibody (hu-PD-1.C) is obtained by replacing the framework of the humanized antibody on the basis of reserving CDR regions of a light chain and a heavy chain of the antibody.

SEQ ID No.: 1: heavy chain of PD-1.C murine PD-1.C antibody

SEQ ID No.: 2: PD-1.C murine PD-1.C antibody light chain

SEQ ID No.: 3: PD-1.C heavy chain CDR1

SEQ ID No.: 4: PD-1.C heavy chain CDR2

SEQ ID No.: 5: PD-1.C heavy chain CDR3

SEQ ID No.: 6: PD-1.C light chain CDR1

SEQ ID No.: 7: PD-1.C light chain CDR2

SEQ ID No.: 8: PD-1.C light chain CDR3

SEQ ID No.: 9: humanized PD-1.C heavy chain

SEQ ID No.: 10: humanized PD-1.C light chain SEQ ID NO: 11: heavy chain variable region of murine PD-1.C antibody

SEQ ID NO: 12: light chain variable region of murine PD-1.C antibody

SEQ ID NO: 13: heavy chain variable region of humanized PD-1.C antibody

SEQ ID NO: 14: light chain variable region of humanized PD-1.C antibody

SEQ ID NO: 15: coding sequence of heavy chain variable region of murine PD-1.C antibody

SEQ ID NO: 16: coding sequence of light chain variable region of murine PD-1.C antibody

SEQ ID NO: 17: coding sequence of heavy chain variable region of humanized PD-1.C antibody

SEQ ID NO: 18: coding sequence of light chain variable region of humanized PD-1.C antibody

SEQ ID NO: 55: coding sequence of heavy chain of murine PD-1.C antibody

SEQ ID NO: 56: coding sequence of light chain of murine PD-1.C antibody

SEQ ID NO: 57: coding sequence of heavy chain of humanized PD-1.C antibody

SEQ ID NO: 58: coding sequence of light chain of humanized PD-1.C antibody

1) Expression purification of humanized PD-1.C antibody:

humanized PD-1.C antibodies were generated by separately constructing a humanized antibody comprising a heavy chain variable region encoding SEQ ID No.: 9 and SEQ ID No.: 10 (SEQ ID NO: 57 and SEQ ID NO: 58) and introducing cleavage sites EcoRI and BglII, respectively, wherein the EcoRI cleavage site is located at the 5 'end of the sequence and the BglII is located at the 3' end of the sequence. The synthesized DNA sequence of the hu-PD-1.C light chain or heavy chain is cloned into an expression vector pCAGGS (Addgene company) by utilizing restriction enzyme cutting sites EcoRI and BglII, and the recombinant eukaryotic expression plasmid of the hu-PD-1.C antibody light chain and heavy chain is established. The expression plasmids of the light chain and the heavy chain of the hu-PD-1.C antibody are transfected into 293T cells together according to the proportion of 2: 1, and the expressed antibody is purified by Protein G affinity column chromatography. The method specifically comprises the following steps:

a. cells with higher cell density are divided into dishes (for example, a dish of 10cm culture dish with 100% of confluent cells is used for passage at a ratio of 1: 3) 14-16h before transfection, and transfection can be carried out when the cell density reaches 70% 14-16h later.

b. For example, adherent 293T cells were transfected in 10cm dishes: the amount of plasmid required for transfection was 20 μ g/disc (light chain: heavy chain ═ 1: 1, mass ratio), diluted into 100 μ L/disc of HBS solution, mixed well and left to stand; the amount of PEI (1mg/mL) was determined at a ratio of PEI (μ L) to plasmid mass (μ g) of 1: 4, diluted into 100 μ L/disc of HBS solution, mixed well and left to stand. Standing and mixing the two solutions for 5min, mixing the two solutions, standing for 20min, and adding into cell culture solution to be transfected.

c. After 4-6h of transfection, the transfected cells were changed to fluid, rinsed twice with 2-3mL PBS and then changed to fresh serum-free DMEM medium (with streptomycin added at 1: 1000), maintained at 37 deg.C with 5% CO₂Culturing and expressing in the incubator.

And (3) collecting supernatant after the transfected cell culture solution is cultured for 3 days, replacing the supernatant with a DMEM medium, and receiving the supernatant again by the seventh day. The supernatants collected 2 times were mixed and the objective Protein was purified by the method of purifying the Protein G murine PD-1.C antibody in step 5 of this example. The purity of the antibody after affinity chromatography on Protein G column was more than 95% (see FIG. 6).

2) Affinity validation of humanized PD-1.C antibody:

in this example, affinity identification was performed for the PD-1.C antibody and the humanized PD-1.C antibody by Surface Plasmon Resonance (SPR).

PD-1 protein prepared in example 1 and humanized PD-1.C antibody were exchanged into SPR buffer (10mM HEPES-HCl, 150mM Na-Cl, 0.005% Tween-20, pH 7.4). The PD-1 protein was diluted to 20. mu.g/ml and immobilized on a CM5 chip (GE Health), after which the gradient diluted antibodies were flowed through each channel of the CM5 chip, the binding kinetics parameters were analyzed using BIA evaluation software, and the affinity constants were calculated.

By humanizing the PD-1.C antibody with PD-1 affinity detection shows that the affinity of the humanized PD-1.C antibody and PD-1 is 1.88 multiplied by 10^-9And M. Thus, it can be seen from the SPR results that the affinity of the humanized PD-1.C antibody is comparable to that of the murine PD-1.C antibody, and still remains 10^-9Affinity of the order of M (see figure 7).

Example 6 NCG immunodeficient mouse HCC827 tumor suppression assay for hu-PD-1.C antibody

This example uses the NCG immunodeficient mouse HCC827 tumor model to evaluate the tumor-inhibiting effect of hu-PD-1.C antibody.

NCG mouse tumor suppression assay procedure for hu-PD-1.C antibody included:

reconstruction of human immune System in NCG mice

NCG mice were derived from the university of south kyo-south kyo biomedical research institute, and first, PBMCs cells derived from healthy individuals were inoculated into each NCG mouse, and mice having an immune system derived from human were established in the NCG mice:

i. number of seeded human PBMCs cells: 1X 10⁷Cells/200. mu.l/cell;

ii site of inoculation: the tail vein;

tumorigenesis of HCC-827 cell line

Each NCG mouse was inoculated with HCC-827 non-small cell lung cancer cell line (ATCC) 3 days after PBMCs were inoculated:

a) number of HCC-827 cells seeded: 5X 10⁷Cells/200. mu.L/cell;

b) inoculation part: subcutaneous on the back;

3. grouping and processing:

mice with more uniform tumor formation were selected for grouping about 1 week after tumor cell injection, followed by intraperitoneal injection of antibody. In this example, a parallel experiment was performed using an Ebola virus-specific antibody 13C6(Mapp Biopharmalogical) injection group as a negative control and a humanized PD-1.C antibody as a treatment group, each of which contained 10 mice.

Grouping of mice	Antibody injection and dosage	Number of mice
			Negative antibody control group	10mg/kg，100μL	10
hu-PD-1.C antibody group	10mg/kg，100μL	10

Antibody injection: after the mice had developed significant tumors (7 days), the injection of the antibody was divided into 7 injections, the first 200. mu.g/mouse, two days later, i.e., the second injection of 200. mu.g/mouse on the third day, and every three or four days later (all 200. mu.g/mouse).

Tumor size was measured every three to four days after tumor formation and continued for two weeks after the last injection.

4. Observation of treatment effect:

1) tumor size detection:

a) diameter measurements were made with calipers after antibody injection in mm and were calculated as: v is 1/2 × a × b × b (a is the long diameter, b is the short diameter);

b) terminating the experiment after the last observation, and separating tumor tissues for direct photographing and observation;

the results showed that the control mice injected with 13C6 antibody all grew rapidly after the injection of 13C6 antibody. The hu-PD-1.C antibody injection group rapidly entered a plateau in tumor growth after antibody injection, with two

mice accelerating growth

10 and 14 days after antibody injection, respectively, but still significantly less than the 13C6 antibody control group. The results of this example show that hu-PD-1.C antibody can effectively inhibit tumor growth, and has potential tumor therapeutic value (see FIG. 8).

Sequence information

1) SEQ ID No.: 1: PD-1.C murine PD-1.C antibody heavy chain:

2) SEQ ID No.: 2: PD-1.C murine PD-1.C antibody light chain

3) SEQ ID No.: 3: PD-1.C heavy chain CDR1

GDTFTDYE

4) SEQ ID No.: 4: PD-1.C heavy chain CDR2

IHPGSGGT

5) SEQ ID No.: 5: PD-1.C heavy chain CDR3

TREGMNTDWYFDV

6) SEQ ID No.: 6: PD-1.C light chain CDR1

QTIVHTNGNTY

7) SEQ ID No.: 7: PD-1.C light chain CDR2

KVS

8) SEQ ID No.: 8: PD-1.C light chain CDR3

FQGSHVPYT

9) SEQ ID No.: 9: humanized PD-1.C heavy chain

10) SEQ ID No.: 10: humanized PD-1.C light chain

11) SEQ ID NO: 11: heavy chain variable region of murine PD-1.C antibody

12) SEQ ID NO: 12: light chain variable region of murine PD-1.C antibody

13) SEQ ID NO: 13: heavy chain variable region of humanized PD-1.C antibody

14) SEQ ID NO: 14: light chain variable region of humanized PD-1.C antibody

15) SEQ ID NO: 15: coding sequence of heavy chain variable region of murine PD-1.C antibody

16) SEQ ID NO: 16: coding sequence of light chain variable region of murine PD-1.C antibody

17) SEQ ID NO: 17: coding sequence of heavy chain variable region of humanized PD-1.C antibody

18) SEQ ID NO: 18: coding sequence of light chain variable region of humanized PD-1.C antibody

19) SEQ ID NO: 55: coding sequence of heavy chain of murine PD-1.C antibody

20) SEQ ID NO: 56: light chain coding sequence of murine PD-1.C antibody

21) SEQ ID NO: 57: heavy chain coding sequence of humanized PD-1.C antibody

22) SEQ ID NO: 58: light chain coding sequence of humanized PD-1.C antibody

Claims

1. An anti-PD-1 antibody or an antigen-binding fragment thereof that specifically binds to a PD-1 molecule, comprising the heavy chain CDRs shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and as SEQ ID NO: 5 The light chain CDRs shown in ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

2. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab') ₂ , bi- Antibodies and peptides comprising CDRs, the anti-PD-1 antibody or antigen-binding fragment thereof blocks the binding of PD-1 to a substance selected from PD-L1, PD-L2 or a combination thereof, the "diabody" is a Small antibody fragments of two antigen-binding sites comprising a variable heavy domain (VH) linked to a variable light domain (VL) in the same polypeptide chain, i.e. VH-VL or VL- VH.

3. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1 or 2, comprising the heavy chain sequence shown in SEQ ID NO:9 and the light chain sequence shown in SEQ ID NO:10.

4. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1 or 2, comprising the heavy chain sequence shown in SEQ ID NO:1 and the light chain sequence shown in SEQ ID NO:2.

5. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1 or 2, comprising the heavy chain variable region sequence shown in SEQ ID NO: 11 and the light chain variable region sequence shown in SEQ ID NO: 12 The variable region sequence, alternatively, comprises the heavy chain variable region sequence set forth in SEQ ID NO:13 and the light chain variable region sequence set forth in SEQ ID NO:14.

6. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1 or 2, which is a murine or humanized anti-PD-1 monoclonal antibody.

7. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 6, wherein the humanized anti-PD-1 monoclonal antibody comprises a human Fc region.

8. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 7, wherein the human Fc region is the Fc region of human IgG4.

9. A polynucleotide encoding an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises the heavy chain sequence shown in SEQ ID NO:9 and SEQ ID NO:10 The light chain sequence shown, or the anti-PD-1 antibody or antigen-binding fragment thereof comprises the heavy chain sequence shown in SEQ ID NO:1 and the light chain sequence shown in SEQ ID NO:2.

10. An expression vector comprising the polynucleotide of claim 9.

11. A host cell comprising the expression vector of claim 10.

12. A method of making the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-6, the method comprising:

1) culturing the host cell as claimed in claim 11;

2) recovering the anti-PD-1 antibody or its antigen-binding fragment from the host cell or culture medium.

13. A pharmaceutical composition comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-6 and a pharmaceutically acceptable carrier.

14. Use of the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-6 in the manufacture of a medicament for increasing the level of IFN-γ secreted by T cells.

15. Use of the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-6 in the preparation of an anti-tumor drug for treating non-small cell lung cancer.