WO2018032793A1 - Novel antibody obtained by coupling small molecule immuno-agonist and pd-1 antibody, and application thereof in tumor resistance - Google Patents

Abstract

A coupled antibody obtained by coupling a small molecule immuno-agonist and PD-1 antibody, and application thereof. The coupled antibody is obtained by means of a coupling reaction between a small molecule immuno-agonist and PD-1 antibody, comprises a compound of formula [I], and can be used in immunotherapy and immunomodulation of malignant tumors.

Description

Novel antibody conjugated to PD-1 antibody by small molecule immunoagonist and its application in antitumor Technical field

The present invention relates to the field of immunochemistry, and more particularly to a PD-1 antibody having an inhibitory effect on the immune system and a novel type of small molecule immunoagonist to form a novel antibody, and the preparation and resistance of the novel antibody Application in tumors.

Background technique

In recent years, an important advance in the field of immunological anti-tumor is the application of the anti-tumor effect of PD-1 antibody as an anti-tumor breakthrough antibody drug whose target is expressed on immune cells (such as T cells). PD-1 protein. PD-1 is an immunosuppressive protein that inhibits immune balance in normal human immunity. However, in tumor patients, PD-1 also inhibits the normal anti-tumor immune response; therefore, PD-1 antibodies can relieve this tumor's immunosuppressive effect (Chinese Journal of Biologicals, June 2014, Volume 27, Issue 6) 856-860), this effect has produced many long-term treatment effects in clinical treatment of tumors.

At present, PD-1 antibodies have been marketed in the United States, Japan and other countries (see 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).

However, the role of the PD-1 antibody is only to relieve the "brake" of the immune system, so its efficacy is significant, but it is only effective in about 20% to 30% of patients. In order to increase the proportion of its efficacy, a product is needed to simultaneously improve the autoimmune ability of tumor patients to produce synergistic effects and higher therapeutic effects.

Summary of the invention

The technical problem to be solved by the present invention is that the above-mentioned PD-1 antibody has a low therapeutic effect ratio, A novel antibody that conjugates a PD-1 antibody to a small molecule immunoagonist and provides its antitumor application.

The technical solution of the present invention is to provide a novel antibody of a small molecule immunoagonist coupled with a PD-1 antibody, which is obtained by coupling a small molecule immunoagonist and a PD-1 antibody. The novel antibodies include the following compounds of the general formula [I]:

In the 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 moiety and the small molecule immunoagonist component are composed of a small molecule immunoagonist and The antibody is covalently linked to form, m is the number of small molecule agonists (m is defined as the degree of coupling), is a number from 1 to 10; antibody portion is referred to as a monoclonal antibody against PD-1 (IgG1, IgG2 or IgG4) ).

When the small molecule immunoagonist is a compound of formula 1:

The X ² represents a thiocarbonyl group:

When the small molecule immunoagonist is a compound of formula 2-1, 2-2, 2-3:

The X ² represents the following group:

When the small molecule immunoagonist is a compound of formula 3:

式3中，u为0～12的整数；

In Formula 3, u is an integer of 0 to 12;

X ² represents the following groups:

其中u为0～12的整数；

Where u is an integer from 0 to 12;

When the small molecule immunoagonist is a compound of formula 4:

X ² represents the following groups:

如二聚乙二醇基团为

三聚乙二醇基团为

四聚乙二醇基团为

Wherein PEG is a polyethylene glycol group

Such as diethylene glycol groups

Triethylene glycol group is

Tetraethylene glycol group is

The present invention provides a novel bifunctional antibody 10, 12, 14, 16, 18 which has both an immunosuppressive release function (represented by a significantly enhanced antitumor effect) and an activated immune function.

The invention is created by providing both an immunosuppressive release function (with a significantly improved anti-tumor) The tumor effect is representative), and at the same time, it has a novel antibody that activates an immune function and a preparation method thereof.

The invention is created by providing a novel antibody having the above dual functions and promoting the proliferation of active T cells and a preparation method thereof.

It is an object of the present invention to provide a coupled precursor small molecule compound for preparing the above novel antibody and a compound or a salt thereof for synthesizing these coupled precursor small molecules.

Another object of the present invention is to provide a novel antibody prepared for use in the inhibition and stimulation of immunosuppression, antiviral, tumor immunomodulation and tumor bioimmunotherapy.

The novel conjugated antibody of the present invention can be used for immunotherapy and immunomodulation of malignant tumors, and the administration method can be intraperitoneal injection, subcutaneous injection, intramuscular injection, and intravenous injection; or the novel antibody and immune cells of the present invention can be used ( For example, dendritic cells, natural killer cells NK, lymphocytes, monocytes/macrophages, granulocytes, etc. are co-cultured, and methods for isolating immune cells in vivo are isolated.

The above novel antibodies and the corresponding small molecule immunoagonist compounds or salts thereof for preparing the same can be formulated into various therapeutic drugs, can be combined with other drugs, synergistic drugs, or made pharmaceutically acceptable. A complex or combination of carriers.

The novel bifunctional antibodies of the invention or their salts can be used in the preparation of various ratios of therapeutic agents.

DRAWINGS

Figure 1 shows the interleukin-6 (IL-6) eliciting activity of novel antibodies 10, 12, 14, 16, 18.

Figure 2 is an interleukin-12 (IL-12)-stimulating activity of novel antibodies 10, 12, 14, 16, 18.

Figure 3 is the IFN-γ eliciting activity of novel antibodies 10, 12, 14, 16, 18.

Figure 4 is the antitumor activity of novel antibodies 10, 12, 14, 16, 18.

Figure 5 is a graph showing the promotion of T cell proliferation activity of novel antibodies 10, 12, 14, 16, 18.

Figure 6 is the IFN-γ-stimulating activity of small molecule immunoagonists 19, 20, 21, 22, 20-2.

Figure 7 shows interleukin-6 (IL-6) eliciting activity of small molecule immunoagonists 19, 20, 21, 22.

Figure 8 is a functional feature of the PD-1 antibody.

Figures 9, 10 and 11 are functional features of the PD-1 antibody of the novel antibodies of the present invention.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The present invention provides a novel antibody which conjugates a PD-1 antibody to a small molecule immunoagonist, and has the following structural formula [I]:

In the 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 immunoagonist component are composed of a small molecule immunoagonist and The antibody is covalently linked to form, m is the number of small molecule agonists (m is defined as the degree of coupling), a number from 1 to 10; the antibody portion is referred to as a monoclonal antibody against IgG-1 (IgG1, IgG2 or IgG4) .

When the small molecule immunoagonist is a compound of formula 1:

The X ² represents a thiocarbonyl group:

When the small molecule immunoagonist is a compound of formula 2-1, 2-2, 2-3:

The X ² represents the following group:

When the small molecule immunoagonist is a compound of formula 3:

式3中，u为0～12的整数；

In Formula 3, u is an integer of 0 to 12;

X ² represents the following groups:

其中u为0～12的整数；

Where u is an integer from 0 to 12;

When the small molecule immunoagonist is a compound of formula 4:

X ² represents the following groups:

如二聚乙二醇基团为

三聚乙二醇基团为

四聚乙二醇基团为

等。Wherein PEG is a polyethylene glycol group

Such as diethylene glycol groups

Triethylene glycol group is

Tetraethylene glycol group is

Wait.

When a small molecule immunoagonist is a compound represented by Formula 1, 2-1, 2-2, 2-3, and 4, a typical representative structural formula of a novel antibody formed by using a PD-1 antibody and other antibodies as an antibody moiety is as follows :

Where m is a number between 1 and 10.

When the small molecule of the novel antibody-conjugated precursor is a compound represented by Formula 1, 2-1, 2-2, 2-3, and 4, a typical representative of a novel antibody formed by using a PD-1 antibody as a representative antibody is exemplified. The synthesis and its structural formula are as follows:

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

The PD-1 antibody is directed against the sequence as an epitope sequence of an antigen.

Synthesis of coupled precursor compound 11:

20-1 was dissolved in anhydrous DMSO, and an equivalent amount of succinic anhydride was added thereto by cooling at 10 ° C, and the mixture was stirred at room temperature for 24 hours. The mixed reactant was poured into 20 volumes of water to precipitate a large amount of white solid compound SZU-101; yield 88%; MS (ESI) 445.27 (M+1);

SZU-101 (1 eq), NHS (1.2 eq) and EDC (1.3 eq) were dissolved in anhydrous DMF at room temperature After stirring for 4 h, the reaction was completed, and the reaction mixture was poured into methylene chloride, filtered, and dried to give a white solid. The active ester and NH2-PEG2-COOH (compound 24) were dissolved in DMF in an equivalent ratio and stirred at room temperature until the end of the reaction (TLC monitoring). Water was added, a large amount of solid was precipitated, suction filtration, dried, and purified by column chromatography (dichloromethane:methanol = 10:1) to afford white solid compound 19, yield 86%; melting point 221 ° C; high-resolution mass spectrometry molecular weight HRMS (ESI) Theoretical value m/z 603.2746, found 604.2757 (M+H).

19 (1 eq), NHS (1.2 eq) and EDC (1.3 eq) were dissolved in anhydrous DMF, stirred at room temperature for 12 h, the reaction was completed, the reaction mixture was poured into dichloromethane, filtered and dried to give an active ester compound. 11 white solid, yield 82%; mp 187 ° C; mp.

Examples of the synthesis of the coupled precursor compound 13:

Compound 20-1 (refer to Chinese patent CN201210382202.8) (1 eq) and thioglycolic acid (1 eq) were dissolved in anhydrous DMF, 1.2 eq HBTU, 3 eq triethylamine and 0.1 eq DMAP were added, stirred at room temperature, and water was added after the reaction was completed. Filter by suction and dry to give a 20-2 pale yellow solid. Stirring with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid (1 eq) in DMF at room temperature, the disappearance of the starting material by TLC, reaction mixture The product was poured into cold water, and the product was filtered and evaporated tolulujjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjs After completion of the reaction, the active ester compound 13 is obtained by preparative liquid phase, melting point 201 ° C, yield 35% (from starting material 20-1); high resolution mass spectrometry molecular weight HRMS (ESI) theoretical value m / z 752.2630 , found 753.2632 (M+1).

用

替换；The preparation method, reaction conditions and feed ratio of the coupled precursor compound 15 and 17 are the same as those of the preparation of the compound 13, except that

use

replace;

Coupling of precursor compound 15 synthesis route:

Compound 21 was obtained as a white solid. Mass molecular weight: HRMS (ESI) 630.2356 (M+1). The precursor compound 15, a white solid, was identified by mass spectrometry to determine the molecular weight: HRMS (ESI) 727.2431 (M + 1).

Synthetic route of coupling precursor compound 17:

Compound 22 was obtained as a white solid. Mass spectrum was identified by mass spectrum: HRMS (ESI) 588.1819 (M+1). The precursor compound 17, a white solid, was identified by mass spectrometry to determine the molecular weight: HRMS (ESI) 685.2022 (M + 1).

A synthetic example of the preparation of a novel bifunctional antibody of the present invention formed by a PD-1 antibody:

Material Identification Mass Spectrometer Model: LDI-1700 Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS), manufacturer: Linear Scientific, USA.

The method for determining the ratio of the small molecule immunoagonist (coupling degree) by mass spectrometry is as follows: mass spectrometry identifies the molecular weight of the coupled novel antibody product minus the molecular weight of the original PD-1 antibody, and calculates: molecular weight increase value / (before coupling) The molecular weight of the small molecule is -18) = the degree of coupling; (the molecular weight of the small molecule compound is reduced by one minus one water molecule).

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

Preparation of Nivolumab According to the method of preparing 5C4, 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 in the patent WO2006121168; and the method disclosed in CN201380062005.0.

The preparation of Pembrolizumab is in accordance with the method disclosed in the patent WO2008156712 (A1), the preparation of h409A11, and its US Pat. No. 8,354,509 and US Pat. No. 8,900,587.

The preparation of AMP-514 and AMP-224 is in accordance with the method disclosed in the patent WO201214549.

The preparation of Pidilizumab is in accordance with the methods disclosed in the patents WO2009014708 and WO2009114335.

The method of preparing the PD-1 antibody can also employ other disclosed methods known in the art, such as those disclosed in Chinese Patent No. CN201410838610.9; CN201310199947.5; CN201380079581.6; CN201480011008.6; CN201310258289.2.

PD-1 antibody can also be purchased from the market, such as the PD-1 antibody from BioXCell, USA, name: anti h PD-1 (h CD279) IgG1, content 95%; Beijing Kehao Biotechnology Co., Ltd. anti-PD-1 Fully monoclonal antibody. The source of the PD-1 antibody in the novel antibody synthesis method of the present invention is not limited thereto.

The reaction concentrations of the following PD-1 antibodies were all 1 equivalent (1 eq, relative to other reactants).

Preparation of novel antibody 10:

For the synthetic preparation method of the coupled precursor small molecule compound 1, please refer to the Chinese patent (CN201210382202.8).

Compound 1 (10 eq equivalent concentration) and PD-1 antibody (Nivolumab) were mixed in 1 mL of DMSO solvent, and the mixture was added with 0.1 mL of triethylamine, and stirred at 5 ° C to 20 ° C for 12 hours. The reaction mixture was mixed with 10 mL of pure water at 0 ° C, shaken, and filtered through a 10 kD biofilter to remove small molecules, and the product was eluted with pure water, and the eluate was freeze-dried to obtain a novel antibody 10 (yield 60%). Mass spectrometry identified a molecular weight increase of 1167; a coupling degree of 3 was calculated. (1167/386=3.02).

Preparation of novel antibody 12:

Compound 11 (30 eq equivalent concentration) and PD-1 antibody (Nivolumab) were mixed in an appropriate amount of DMSO solvent, the mixture was added with triethylamine 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 0 ° C pure water, shaken, and filtered to remove small molecules by a 10 kD biofilter, and the product was washed with pure water, and the eluate was freeze-dried to obtain a novel antibody 12 (yield 72%). Mass spectrometry identified an increase in molecular weight of 2931; a coupling degree of 5 was calculated. (2931/585.6=5).

(In the preparation method of novel antibody 12, Nivolumab was replaced with BioXCell PD-1 antibody: anti h PD-1 (h CD279) IgG1 to obtain novel antibody 12-2, the preparation process thereof and the obtained novel antibody 12-2 The result is the same as the novel antibody 12).

Preparation of novel antibody 14:

Compound 13 (40 eq equivalent concentration) and PD-1 antibody (AMP-514) were mixed in an appropriate amount of DMSO solvent, the mixture was added with triethylamine 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 0 ° C pure water, shaken, and filtered to remove small molecules by a 10 kD biological filter, and the product was washed with pure water, and the eluate was freeze-dried to obtain a novel antibody 14 (yield 66%). Mass spectrometry identified an increase in molecular weight of 1917; a coupling degree of 3 was calculated.

Preparation of novel antibody 16:

Compound 15 (40 eq equivalent concentration) and PD-1 antibody (Pembrolizumab) were mixed in an appropriate amount of DMSO solvent, and the mixture was added with triethylamine 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 0 ° C pure water, shaken, and filtered to remove small molecules by a 10 kD biological filter, and the product was washed with pure water, and the eluate was freeze-dried to obtain a novel antibody 16 (yield 73%). Mass spectrometry identified an increase in molecular weight of 2377; a calculated coupling degree of about 4.

Preparation of novel antibody 18:

Compound 17 (40 eq equivalent concentration) and PD-1 antibody (AMP-224) were mixed in an appropriate amount of DMSO solvent, the mixture was added with triethylamine 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 0 ° C pure water, shaken, and filtered to remove small molecules by a 10 kD biological filter, and the product was washed with pure water, and the eluate was freeze-dried to obtain a novel antibody 18 (yield 57%). Mass spectrometry identified an increase in molecular weight of 1143; the calculated coupling degree was approximately 2.

Experimental method for immunoactivation of novel antibodies of the invention (mouse):

1. Take spleen lymphocytes from Balb/C mice and plate them at 1x10 ⁶ /ml/well.

2. Preferred antibody concentration: stimulation with 0.1 μM for 24 h.

3. Collect the supernatant and detect IL-6/IL-12 by ELISA (effects are shown in Figures 1 and 2).

Experimental method for immunization activation of small molecule immunoagonists in the present invention (mouse):

1. Take spleen lymphocytes from Balb/C mice and plate them at 1x10 ⁶ /ml/well.

2. Select the concentration of small molecule immunoagonist: stimulated by 0.1, 10, 20μM for 24h.

3. The supernatant was collected and IFN-γ/IL-6 was detected by ELISA (effects are shown in Figures 6 and 7).

Experimental method for immune activation of novel antibodies of the invention (human):

1. Peripheral monocytes were taken from volunteers, and T cells (suspended cells) were isolated and cultured at 37 ° C, 5% CO ₂ serum-containing medium for 7 days, and plated at 1×10 ⁶ /ml/well.

2. Preferred antibody concentration: stimulation with 0.1 μM for 24 h.

3. The supernatant was collected and IFN-γ was detected by ELISA (as shown in Figure 3).

The anti-tumor effect test method of the novel antibody of the invention:

6-8 week old Balb/C mice were randomly divided into 7 groups. 2.5x10 ⁵ 4T1 tumor cells were implanted subcutaneously in the back of the mouse. The PBS blank control and 10 mg/kg of PD-1 antibody were administered respectively. The control drug and the novel antibody 10, 12, 14, 16, 18 were administered at a dose of 10 mg/kg and a volume of 100 μL. Intraperitoneal injection. On the first day after tumor implantation, each group was administered on days 7, 15, 22, and 29, respectively. Mice were euthanized when the tumor reached 1500 mm ³ or greater than 15% of body weight. Tumor inhibition results are shown in Figure 4. It can be seen that the novel antibody-treated group of the present invention has a markedly enhanced antitumor effect.

Experimental method for promoting T cell proliferation of the novel antibody of the present invention:

Peripheral monocytes were taken from volunteers, T cells (suspended cells) were isolated and cultured at 37 ° C, 5% CO ₂ serum-containing medium for 7 days, and serum-free medium was added simultaneously with the novel antibody and PD-1 of the present invention. (All concentrations were 1 μM), and CCK-8 cells staining method was detected 3 days later. It can be seen that the novel antibodies of the present invention can promote T cell proliferation by potentiating blocking PD-1 and activating innate immunity. The result is shown in Figure 5.

Experimental method for functional characteristics of PD-1 antibody of the novel antibody of the present invention (detection of PD-1 antibody by specific secondary antibody of PD-1):

1. Extracting peripheral blood from healthy volunteers

2. Obtain peripheral blood in a centrifuge tube, 800g, 30min. Separate serum and blood cells.

3. Obtain blood cells, resuspend in physiological saline, and gently add to the upper layer of peripheral blood lymphocyte separation solution along the wall.

Centrifuge at 4.800 g for 30 min. Absorb the white layer (peripheral lymphocyte layer).

5. The isolated peripheral blood lymphocytes were resuspended in physiological saline at 900 g for 10 min. Wash twice.

6. Count the peripheral blood lymphocytes obtained by separation.

7. 1x106 cells were resuspended in 100 ul of physiological saline, and the novel antibodies 10, 12, 16, and IgG1 (control) of the present invention were incubated at 37 degrees for 30 min with PD-1 antibody, respectively.

8. Wash 3 times with physiological saline, resuspend with 100 ul of physiological saline, add anti-PD-1-FITC secondary antibody, and incubate for 30 min at room temperature.

9. Washing with saline, flow on the machine.

The results are shown in Figures 8, 9, 10, and 11: It was found that the PD-1 antibody functional characteristics of the novel antibody of the present invention remained intact as compared with the control group and the unconjugated group.

The above is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily think of changes or within the technical scope disclosed by the present invention. Alternatives are intended to be covered by the scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims (10)

A novel antibody of a small molecule immunoagonist conjugated to a PD-1 antibody, characterized in that the novel antibody is obtained by coupling a small molecule immunoagonist and a PD-1 antibody, and the novel antibody comprises the following formula [ Compound of I]:

In the 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 moiety and the small molecule immunoagonist component are small molecule immunoagonists The coupled precursor and the antibody are covalently linked to form, m is the number of small molecule agonists (m is defined as the degree of coupling), is a number from 1 to 10; the antibody portion is referred to as a monoclonal antibody against PD-1 ( IgG1, IgG2 or IgG4); the small molecule immunoagonist-coupled precursor is defined as an activated form of a small molecule immunoagonist when covalently bound to an antibody. When the small molecule immunoagonist-coupled precursor is a compound of formula 1:

The X ² represents a thiocarbonyl group:

When the small molecule immunoagonist-coupled precursor is a compound of formula 2-1, 2-2, 2-3:

The X ² represents the following group:

When the small molecule immunoagonist is coupled to a compound of formula 3:

In Formula 3, u is an integer of 0 to 12; X ² represents the following groups:

Where u is an integer from 0 to 12; When the small molecule immunoagonist is coupled to a compound of formula 4:

X ² represents the following groups:

Wherein PEG is a polyethylene glycol group

Such as diethylene glycol groups

Triethylene glycol group is

Tetraethylene glycol group is

The novel antibody of the small molecule immunoagonist-conjugated PD-1 antibody according to claim 1, wherein the PD-1 antibody comprises various monoclonal antibodies against human PD-1 protein (IgG1, IgG2 or IgG4). The sequence of the PD-1 protein is preferentially selected as follows: ^{1 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTS 61 ESFVLNWYR 70 MSPSNQTDKLAAFPEDR 86 SQPGQDCRFRVTQLPNGRDFHMSVVRARRND} 118 SGTYLCGAISLAPKAQIKE 136 SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGS 18 1 LVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP 241 CVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL; The PD-1 antibody is directed against the sequence as an epitope sequence of an antigen. The novel antibody of the small molecule immunoagonist-conjugated PD-1 antibody according to claim 1, wherein the PD-1 antibody is Nivolumab, Pembrolizumab, AMP-514, AMP-224, Pidilizumab or other anti h PD‐1 (h CD279) IgG1. The novel antibody of the small molecule immunoagonist-conjugated PD-1 antibody according to claim 1, wherein the small molecule immunoagonist is represented by the formula 1, 2-1, 2-2, 2-3, and 4 The typical representative structural formula of the novel antibody is as follows:

Where m is a number between 1 and 10. The novel antibody of the small molecule immunoagonist-conjugated PD-1 antibody according to claim 4, wherein the small molecule immunoagonist is represented by the formula 1, 2-1, 2-2, 2-3, and 4 The structural formula of the novel representative of 10, 12, 14, 16, 18 is as follows:

A small molecule immunoagonist-conjugated precursor compound characterized by the typical representative 12, 14, 16, 18 for the preparation of the novel antibody of claim 5, corresponding to the following structural formula 11, 13, 15, 17 compound of:

A small molecule immunoagonist compound for synthesizing a small molecule immunoagonist-conjugated precursor compound according to claim 6, wherein the small molecule immunoagonist compound comprises the structural formula 19, 20- shown below. 2, 20, 21, 22 compounds or their salts:

A novel antibody and a small molecule compound according to any one of claims 1 to 7 or a salt thereof, which is suitable for use in various therapeutic drugs, or in combination with other drugs, a synergistic drug, or a preparation A complex or combination of pharmaceutically acceptable carriers. Use of a medicament as claimed in claim 8 for the elimination and challenge of immunosuppression, antibody preparation, antiviral, diabetes, and tumor immunotherapy. The medicament according to claim 9, which is administered intraperitoneally, subcutaneously, intramuscularly, intravenously or orally; or can be used for immunological cells (such as dendritic cells, natural killer cells NK, lymphocytes, After co-culture of monocytes/macrophages, granulocytes, etc., methods for isolating immune cells in vivo are isolated.

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