Novel Antibody of Small Molecule Immune Agonist Conjugated with PD-1 Antibody and Its Application in Anti-Tumor

Abstract

The present invention provides a novel antibody obtained by conjugating a small molecule immune agonist with a PD-1 antibody and its application. The novel antibody is obtained by a conjugation reaction of a small molecule immune agonist and a PD-1 antibody. The novel antibody comprises a compound of the following general formula [Ⅰ]: The novel antibody in the present invention can be used for the immunotherapy and immunomodulation of malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of immunochemistry, and more particularly, to a novel antibody formed by conjugating a PD-1 antibody having an inhibitory effect on the immune system with a class of small molecule immune agonists, and the preparation and application of the novel antibody in anti-tumor treatment. Background Art

[0002] In recent years, an important progress in the field of immune anti-tumor is the application of the immune anti-tumor effect of PD-1 antibody. As a breakthrough antibody drug for anti-tumor, its target is the PD-1 protein expressed on immune cells (such as T cells). PD-1 is an immunosuppressive protein that plays an inhibitory and immune balance role in the over-immune response of the normal human body. However, in tumor patients, PD-1 also has an inhibitory effect on the normal anti-tumor immune response; therefore, PD-1 antibody can relieve this immune inhibitory effect on tumors (Chinese Journal of Biologicals, Vol. 27, No. 6, June 2014, pp. 856-860), and this effect has produced many long-term therapeutic effects in the clinical treatment of tumors.

[0003] Currently, PD-1 antibodies have been clinically applied in the United States, Japan and other countries (for details, please refer to the following website: http: / / www.fiercebiotech.com / biotech / anti-pd-1-cancer-star-nivolumab-wins -world-s-first-regulatory-approval; http: / / www.pharmatimes.com / news / japan _approves_worlds_first_pd-1_drug%2C_nivolumab_1002153).

[0004] However, the effect of PD-1 antibody is only to relieve the inhibitory "brake" effect of the immune system. Therefore, although its curative effect is significant, it is only effective in about 20% to 30% of patients. In order to improve the curative effect ratio, a product is needed that can simultaneously enhance the autoimmune ability of tumor patients to produce a synergistic effect and a higher therapeutic effect. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a novel antibody of a small molecule immune agonist conjugated with a PD-1 antibody and its application in anti-tumor treatment in view of the low curative effect ratio of the above-mentioned PD-1 antibody.

[0006] The technical solution for the present invention to solve the above technical problem is to provide a novel antibody of a small molecule immune agonist conjugated with a PD-1 antibody, which is obtained by a conjugation reaction of a small molecule immune agonist and a PD-1 antibody, and the novel antibody includes a compound of the following general formula [Ⅰ]:

[0007]

[0008] In formula [Ⅰ]: X 1 represents OH or SH; R 1 represents an alkoxy group or an alkylamino group, and X 2 represents a linking group; wherein the antibody part and the small molecule immune agonist part are covalently combined and linked by the small molecule immune agonist and the said antibody to form, m is the number of small molecule agonists (m is defined as the coupling degree), which is a number from 1 to 10; the antibody part refers to a monoclonal antibody against PD-1 (IgG1, IgG2 or IgG4).

[0009] When the small molecule immune agonist is a compound of formula 1:

[0010]

[0011] The said X 2 represents a thiocarbonyl group:

[0012] When the small molecule immune agonist is a compound of formula 2-1, 2-2, 2-3:

[0013]

[0014] The said X 2 represents the following group:

[0015]

[0016] When the small molecule immune agonist is a compound of formula 3:

[0017] In formula 3, u is an integer from 0 to 12;

[0018] X 2 represents the following group:

[0019] wherein u is an integer from 0 to 12;

[0020] When the small molecule immune agonist is a compound of formula 4:

[0021]

[0022] X 2 represents the following group:

[0023]

[0024] wherein PEG is a polyethylene glycol group such as the diethylene glycol group is the triethylene glycol group is the tetraethylene glycol group is

[0025] The present invention provides a novel bifunctional antibody 10, 12, 14, 16, 18, which has both immunosuppression release function (represented by a significantly improved anti-tumor effect) and immune activation function at the same time.

[0026] The innovation of the present invention lies in providing a novel antibody having both immunosuppression release function (represented by a significantly improved anti-tumor effect) and immune activation function at the same time, and a preparation method thereof.

[0027] The innovation of the present invention lies in providing a novel antibody having the above bifunction and capable of promoting the proliferation of activated T cells, and a preparation method thereof.

[0028] The object of the present invention is to provide a conjugate precursor small molecule compound for preparing the above novel antibody, and a compound or a salt thereof for synthesizing these conjugate precursor small molecules.

[0029] Another object of the present invention is to provide the application of the prepared novel antibody in immunosuppression release and activation, anti-virus, tumor immune regulation and tumor biological immunotherapy.

[0030] The novel conjugate antibody in the present invention can be used for the immunotherapy and immune regulation of malignant tumors, and the administration method can be intraperitoneal injection, subcutaneous injection, intramuscular injection and intravenous injection; or a method of separating and reinfusing immune cells in vivo after co-culturing the novel antibody in the present invention and immune cells (such as dendritic cells, natural killer cells NK, lymphocytes, monocytes / macrophages, granulocytes, etc.) can be adopted.

[0031] The above novel antibody and the corresponding small molecule immune agonist compound or a salt thereof for preparing them can be made into various therapeutic drugs suitable for the above, can be made into a compound drug, a synergistic drug with other drugs, or a complex or conjugate of a pharmaceutically acceptable carrier.

[0032] The novel bifunctional antibody or a salt thereof of the present invention can be used for preparing therapeutic drugs in various proportions. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the interleukin-6 (IL-6) excitation activity of the novel antibody 10, 12, 14, 16, 18.

[0034] Figure 2 is the interleukin-12 (IL-12) excitation activity of the novel antibody 10, 12, 14, 16, 18.

[0035] Figure 3 is the IFN-γ excitation activity of the novel antibody 10, 12, 14, 16, 18.

[0036] Figure 4 is the anti-tumor activity of novel antibodies 10, 12, 14, 16, 18.

[0037] Figure 5 is the activity of promoting T cell proliferation of novel antibodies 10, 12, 14, 16, 18.

[0038] Figure 6 is the IFN-γ stimulating activity of small molecule immune agonists 19, 20, 21, 22, 20-2.

[0039] Figure 7 is the interleukin-6 (IL-6) stimulating activity of small molecule immune agonists 19, 20, 21, 22.

[0040] Figure 8 is the functional characteristic of the PD-1 antibody.

[0041] Figure 9 , 10 , 11 is the PD-1 antibody functional characteristic of the novel antibody of the present invention. Detailed implementation manners

[0042] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0043] The present invention provides a novel antibody in which a small molecule immune agonist is conjugated to a PD-1 antibody, having the following general structural formula [Ⅰ]:

[0044]

[0045] In formula [Ⅰ]: X 1 represents OH or SH; R 1 represents an alkoxy group or an alkylamino group, and X 2 represents a linking group; wherein the antibody and the small molecule immune agonist component are covalently conjugated and linked by the small molecule immune agonist and the antibody, m is the number of small molecule agonists (m is defined as the conjugation degree), which is a number from 1 to 10; the antibody part refers to a monoclonal antibody against PD-1 (IgG1, IgG2 or IgG4).

[0046] When the small molecule immune agonist is a compound of formula 1:

[0047]

[0048] The X 2 represents a thiocarbonyl group:

[0049] When the small molecule immune agonist is a compound of Formula 2-1, 2-2, or 2-3:

[0050]

[0051]

[0052] said X 2 represents the following group:

[0053]

[0054] When the small molecule immune agonist is a compound of Formula 3:

[0055] In Formula 3, u is an integer from 0 to 12;

[0056] X 2 represents the following group:

[0057] wherein u is an integer from 0 to 12;

[0058] When the small molecule immune agonist is a compound of Formula 4:

[0059]

[0060] X 2 represents the following group:

[0061]

[0062] wherein PEG is a polyethylene glycol group such as the diethylene glycol group is the triethylene glycol group is the tetraethylene glycol group is and so on.

[0063] When the small molecule immune agonist is a compound represented by Formula 1, 2-1, 2-2, 2-3, or 4, taking the PD-1 antibody and other antibodies as examples, the typical representative structural formula of the novel antibody formed as the antibody part is as follows:

[0064]

[0065] wherein m is a number between 1 and 10.

[0066] When the precursor small molecule conjugated to the novel antibody is a compound represented by Formula 1, 2-1, 2-2, 2-3, or 4, taking the PD-1 antibody as an example, the typical representative synthesis and structural formula of the novel antibody formed as the representative antibody are as follows:

[0067]

[0068]

[0069]

[0070] Among the novel antibodies formed by the above coupling, the antibody is selected from PD-1 monoclonal antibodies; it can also be applied to other similar immunosuppressive antibody, such as PD-L1 antibody, CTLA-4 antibody, TIM-3 antibody, LAG3 antibody, TIGIT antibody, etc.; the PD-1 monoclonal antibody includes various monoclonal antibodies against human PD-1 protein (IgG1, IgG2 and IgG4), and the sequence of the PD-1 protein is as follows:

[0071] 1 MQIPQAPWPWWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTS 61 ESFVLNWYR 70 MSPSNQTDKLAAFPEDR 86 SQPGQDCRFRVTQLPNGRDFHMSWRARRND 118 SGTYLCGAISLAPKAQIKE 136 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGS 181 LVLLVWVLAVICSRAARGTIGARRTGQPLKEDP5AVPVFSVDYGELDFQWREKTPEPPVP 241 CVPEQTEYATIVFP5GMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0072] The PD-1 antibody targets the epitope sequence with this sequence as the antigen.

[0073] Example of the synthesis of coupling precursor compound 11:

[0074]

[0075] Dissolve 20-1 in anhydrous DMSO, cool to 10 °C and add an equivalent amount of succinic anhydride. Stir the mixture at room temperature for 24 hours. Pour the mixed reactants into 20 times the volume of water to precipitate a large amount of white solid compound SZU-101; yield 88%; MS (ESI) 445.27 (M+1);

[0076] Dissolve SZU-101 (1 eq), NHS (1.2 eq) and EDC (1.3 eq) in anhydrous DMF, stir at room temperature for 4 h to end the reaction. Pour the reaction solution into dichloromethane, filter by suction, dry to obtain the white solid of active ester 23, which is directly used for the next reaction. Dissolve the active ester and NH2-PEG2-COOH (Compound 24) in an equivalent ratio in DMF, stir at room temperature until the reaction ends (monitored by TLC). Add water, a large amount of solid precipitates, filter by suction, dry, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain the white solid Compound 19, with a yield of 86%; melting point 221 °C; high-resolution mass spectrometry molecular weight HRMS (ESI) theoretical value m / z 603.2746, found 604.2757 (M+H).

[0077] Dissolve 19 (1 eq), NHS (1.2 eq) and EDC (1.3 eq) in anhydrous DMF, stir at room temperature for 12 h to end the reaction. Pour the reaction solution into dichloromethane, filter by suction, dry to obtain the white solid of active ester Compound 11, with a yield of 82%; melting point 187 °C; high-resolution mass spectrometry molecular weight HRMS (ESI) theoretical value m / z 700.2839, found 701.2842 (M+1).

[0078] Example of the synthesis of coupling precursor Compound 13:

[0079]

[0080] Dissolve Compound 20-1 (refer to Chinese Patent CN201210382202.8) (1 eq) and mercaptoacetic acid (1 eq) in anhydrous DMF, add 1.2 eq HBTU, 3 eq triethylamine and 0.1 eq DMAP, stir at room temperature. After the reaction ends, add water, filter by suction, dry to obtain 20-2 light yellow solid. Then stir with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid (1 eq) in DMF at room temperature, monitor by TLC until the raw materials disappear, pour the reaction mixture into cold water, filter the product solid, dry to obtain Compound 20, MS (ESI) 656.27 (M+1). Add Compound 20 (1 eq) to the DMF solution of NHS (1.2 eq) and EDC (1.3 eq), stir at room temperature overnight. After the reaction is completed, separate by preparative liquid phase to obtain the active ester Compound 13, melting point 201 °C, yield 35% (calculated from the starting material 20-1); high-resolution mass spectrometry molecular weight HRMS (ESI) theoretical value m / z 752.2630, found 753.2632 (M+1).

[0081] The synthesis preparation methods, reaction conditions and feeding ratios of coupling precursors Compound 15 and Compound 17 are the same as those for preparing Compound 13, except that is used to replace;

[0082] Synthetic route of coupling precursor compound 15:

[0083]

[0084] Compound 21 was obtained as a white solid. The molecular weight was identified by mass spectrometry: HRMS(ESI) 630.2356 (M+1). Coupling precursor compound 15 was obtained as a white solid. The molecular weight was identified by mass spectrometry: HRMS(ESI) 727.2431 (M+1).

[0085] Synthetic route of coupling precursor compound 17:

[0086]

[0087] Compound 22 was obtained as a white solid. The molecular weight was identified by mass spectrometry: HRMS(ESI) 588.1819 (M+1). Coupling precursor compound 17 was obtained as a white solid. The molecular weight was identified by mass spectrometry: HRMS(ESI) 685.2022 (M+1).

[0088] Synthesis examples of the novel bifunctional antibody formed by PD-1 antibody in the present invention:

[0089] Model of the mass spectrometer for substance identification: LDI-1700 laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), manufacturer: Linear Scientific, USA

[0090] The method for determining the ratio (coupling degree) of the small molecule immune agonist by mass spectrometry is as follows: Subtract the molecular weight of the original PD-1 antibody from the molecular weight of the obtained coupled novel antibody product identified by mass spectrometry to obtain the increased value. Calculate: Increased value of molecular weight / (molecular weight of coupling precursor small molecule - 18) = coupling degree; (for each coupling of a small molecule compound, the molecular weight of one water molecule is subtracted).

[0091] The PD-1 antibody is preferably Nivolumab (BMS-936558), Pembrolizumab (MK-3475), AMP-514, AMP-224, Pidilizumab; however, it is also applicable to PD-1 antibodies from other sources.

[0092] The preparation of Nivolumab is carried out according to the methods for preparing 5C4, 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 in patent WO2006121168; and the methods disclosed in CN201380062005.0.

[0093] The preparation of pembrolizumab is carried out according to the methods disclosed in Patent WO2008156712(A1) for the preparation of h409A11, and in US Patents 8354509 and 8900587.

[0094] The preparation of AMP-514 and AMP-224 is carried out according to the methods disclosed in Patent WO201214549.

[0095] The preparation of pidilizumab is carried out according to the methods disclosed in Patents WO2009014708 and WO2009114335.

[0096] The preparation method of the PD-1 antibody can also adopt other publicly known methods in the art, such as the methods disclosed in Chinese Patents CN201410838610.9; CN201310199947.5; CN201380079581.6; CN201480011008.6; CN201310258289.2.

[0097] The PD-1 antibody can also be sourced from market purchases, such as the PD-1 antibody purchased from BioXCell, USA, named anti h PD-1(h CD279)IgG1, with a content of 95%; and the anti-PD-1 fully human monoclonal antibody from Beijing Kexin Biotechnology Co., Ltd. The source of the PD-1 antibody in the novel antibody synthesis method of the present invention is not limited to this.

[0098] The reaction concentration of the following PD-1 antibodies is 1 equivalent concentration (1eq, relative to other reactants).

[0099] Preparation of novel antibody 10:

[0100] For the synthesis and preparation method of the coupling precursor small molecule compound 1, please refer to Chinese Patent (CN201210382202.8).

[0101] Mix compound 1 (10eq equivalent concentration) and the PD-1 antibody (Nivolumab) in 1 mL of DMSO solvent, add 0.1 mL of triethylamine to the mixture, and stir and react at 5°C to 20°C for 12 hours. Mix the reaction mixture with 10 mL of pure water at 0°C, shake well, filter through a 10kD biological filter membrane to remove small molecules, wash the product with pure water, and freeze-dry the eluate to obtain novel antibody 10 (yield 60%). The molecular weight increase was identified by mass spectrometry as 1167; the calculated coupling degree was 3. (1167 / 386 = 3.02).

[0102] Preparation of novel antibody 12:

[0103] Compound 11 (at a concentration of 30 eq) and the PD-1 antibody (Nivolumab) were mixed in an appropriate amount of DMSO solvent. Triethylamine was added to the mixture to adjust the pH to 8, and the reaction was stirred at 5 °C to 20 °C for 12 hours. The reaction mixture was mixed with 10 volumes of pure water at 0 °C, shaken well, filtered through a 10 kD biologic filter membrane to remove small molecules, the product was washed with pure water, and the eluate was freeze-dried to obtain the novel antibody 12 (yield 72%). Mass spectrometry identified an increase in molecular weight to 2931; the calculated coupling degree was 5. (2931 / 585.6 = 5).

[0104] (In the preparation method of the novel antibody 12, Nivolumab was replaced with the PD-1 antibody from BioXCell: antih PD-1(h CD279)IgG1 to obtain the novel antibody 12-2. The preparation process and the results of the obtained novel antibody 12-2 were the same as those of the novel antibody 12).

[0105] Preparation of the novel antibody 14:

[0106] Compound 13 (at a concentration of 40 eq) and the PD-1 antibody (AMP-514) were mixed in an appropriate amount of DMSO solvent. Triethylamine was added to the mixture to adjust the pH to 8, and the reaction was stirred at 5 °C to 20 °C for 12 hours. The reaction mixture was mixed with 10 volumes of pure water at 0 °C, shaken well, filtered through a 10 kD biologic filter membrane to remove small molecules, the product was washed with pure water, and the eluate was freeze-dried to obtain the novel antibody 14 (yield 66%). Mass spectrometry identified an increase in molecular weight to 1917; the calculated coupling degree was 3.

[0107] Preparation of the novel antibody 16:

[0108] Compound 15 (at a concentration of 40 eq) and the PD-1 antibody (Pembrolizumab) were mixed in an appropriate amount of DMSO solvent. Triethylamine was added to the mixture to adjust the pH to 8, and the reaction was stirred at 5 °C to 20 °C for 12 hours. The reaction mixture was mixed with 10 volumes of pure water at 0 °C, shaken well, filtered through a 10 kD biologic filter membrane to remove small molecules, the product was washed with pure water, and the eluate was freeze-dried to obtain the novel antibody 16 (yield 73%). Mass spectrometry identified an increase in molecular weight to 2377; the calculated coupling degree was approximately 4.

[0109] Preparation of the novel antibody 18:

[0110] Compound 17 (40 eq) and PD-1 antibody (AMP-224) were mixed in an appropriate amount of DMSO solvent. Triethylamine was added to the mixture to adjust the pH value to 8, and the reaction was stirred at 5 °C to 20 °C for 12 hours. The reaction mixture was mixed with 10 volumes of pure water at 0 °C, shaken well, and filtered through a 10 kD biofilter membrane to remove small molecules. The product was washed with pure water, and the eluate was freeze-dried to obtain the novel antibody 18 (yield 57%). The molecular weight was identified by mass spectrometry to increase to 1143; the calculated coupling degree was about 2.

[0111] Immunoactivation experimental method of the novel antibody of the present invention (mice):

[0112] 1. Take Balb / C mouse splenic lymphocytes and plate them at 1x10 6 / ml / well

[0113] 2. Preferred antibody concentration: Add the drug at 0.1 μM and stimulate for 24 h.

[0114] 3. Collect the supernatant and detect IL-6 / IL-12 by ELISA method (the results are as Figure 1 、 2 shown).

[0115] Immunoactivation experimental method of the small molecule immunoagonist in the present invention (mice):

[0116] 1. Take Balb / C mouse splenic lymphocytes and plate them at 1x10 6 / ml / well

[0117] 2. Select the concentration of the small molecule immunoagonist: Add the drug at 0.1, 10, 20 μM and stimulate for 24 h.

[0118] 3. Collect the supernatant and detect IFN-γ / IL-6 by ELISA method (the results are as Figure 6 、 7 shown).

[0119] Immunoactivation experimental method of the novel antibody of the present invention (humans):

[0120] 1. Take peripheral monocytes from volunteers, isolate T cells (suspension cells), culture them in a serum-containing medium at 37 °C and 5% CO2 for 7 days, and plate them at 1x10 6 / ml / well

[0121] 2. Preferred antibody concentration: Add the drug at 0.1 μM and stimulate for 24 h.

[0122] 3. Collect the supernatant and detect IFN-γ by ELISA method (as Figure 3 shown).

[0123] Antitumor effect experimental method of the novel antibody of the present invention:

[0124] Balb / C mice at 6 - 8 weeks of age were randomly divided into 7 groups. 2.5x10 5 4T1 tumor cells were subcutaneously implanted on the back of the mice. PBS was used as a blank control, and a PD - 1 antibody at a dose of 10 mg / kg was used as a control drug; the dosing doses of the control drug and the novel antibodies 10, 12, 14, 16, and 18 were 10 mg / kg with a volume of 100 μL; the administration method for each group was intraperitoneal injection. On the 1st day after tumor implantation, each group was administered on days 7, 15, 22, and 29. The mice were euthanized when the tumor reached 1500 mm 3 or was greater than 15% of the body weight. The tumor inhibition results are as Figure 4 shown. It can be seen that the novel antibody treatment group in the present invention has a significantly enhanced anti - tumor effect.

[0125] Experimental method for promoting T - cell proliferation of the novel antibody of the present invention:

[0126] Peripheral monocytes of volunteers were taken, and T cells (suspension cells) were isolated and cultured in a serum - containing medium at 37°C and 5% CO2 for 7 days. Then, the serum - free medium was changed, and the novel antibody of the present invention and PD - 1 (control) (both at a concentration of 1 μM) were added. After 3 days, the CCK - 8 cell staining method was used for detection. It can be seen that the novel antibody of the present invention can promote T - cell proliferation by strengthening the blockade of PD - 1 and activating innate immunity. The results are as Figure 5 shown.

[0127] Experimental method for the functional characteristics of the PD - 1 antibody of the novel antibody of the present invention (detecting the PD - 1 antibody with a specific secondary antibody against PD - 1):

[0128] 1. Draw peripheral blood from healthy volunteers

[0129] 2. Place the peripheral blood in a centrifuge tube and centrifuge at 800 g for 30 min. Separate the serum and blood cells.

[0130] 3. After obtaining the blood cells, resuspend them in physiological saline and gently add them to the upper layer of the peripheral blood lymphocyte separation solution along the wall.

[0131] 4. Centrifuge at 800 g for 30 min. Aspirate the buffy coat (peripheral lymphocyte layer).

[0132] 5. Resuspend the separated peripheral blood lymphocytes in physiological saline and centrifuge at 900 g for 10 min. Wash twice.

[0133] 6. Count the separated peripheral blood lymphocytes.

[0134] 7. Take 1x106 cells, resuspend them in 100 ul of normal saline, and incubate them with PD-1 antibody, the novel antibody 10, 12, 16 of the present invention, and IgG1 (control) at 37 °C for 30 min.

[0135] 8. Wash with normal saline three times, resuspend with 100 ul of normal saline, add the secondary antibody Anti-PD-1-FITC, and incubate at room temperature for 30 min.

[0136] 9. Wash with normal saline and detect by flow cytometry.

[0137] The results are shown in Figure 8 、 9 、10, 11 as follows: It can be found that compared with the control group and the uncoupled group,

[0138] the PD-1 antibody functional characteristics of the novel antibody of the present invention are well maintained.

[0139] As described above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. An antibody conjugating a small molecule immune agonist with a PD-1 antibody, wherein the structural formula of the antibody is as follows: , wherein the PD-1 antibody is AMP-514 , wherein the PD-1 antibody is Pembrolizumab , wherein the PD-1 antibody is AMP-224.

2. A pharmaceutical composition comprising the antibody according to claim 1.

3. Use of the antibody according to claim 1 in the preparation of a drug for immunosuppression relief, activation and cancer immunotherapy.

4. Use according to claim 3, wherein the drug is administered by intraperitoneal injection, subcutaneous injection, intramuscular injection, intravenous injection or oral administration; or by the method of separating immune cells after co-culture with immune cells and then reinfusing the immune cells in vivo.

5. Use according to claim 4, wherein the immune cells are dendritic cells, natural killer cells NK, lymphocytes, monocytes, macrophages or granulocytes.

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