Immunostimulatory antibodies and their applications

Abstract

The present invention belongs to the field of pharmaceutical technology, and particularly relates to an immune-activating antibody and its application. The immune-activating antibody provided by the present invention comprises an antibody and an immune activator coupled by a coupling chain, and can be used for preparing anti-tumor drugs, anti-viral drugs, immunomodulatory drugs and / or preparations for eliminating target proteins. By coupling the antibody and the immune activator through the coupling chain, a series of immune-activating antibodies targeting specific tissues, lesions and targets can be formed, which can achieve the effect of locally targeted immune activation, solve the negative impact of TLR activation on normal tissues, and have good specific immune regulation and therapeutic effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, and particularly relates to an immune-activating antibody and its application. Background Art

[0002] Toll-like receptors (TLRs) belong to the classical innate immune system of animals. There are 11 members of TLR receptors in mammals and humans, such as TLR2, TLR3, TLR4, TLR7, TLR9, etc.; each TLR can be activated by specific ligands to produce resistance to various microbial invasions such as bacteria and viruses, as well as anti-tumor effects. Among them, TLR7 can be activated by synthetic small molecule immune activators. Although the activation intensity of TLR7 is positively correlated with the ability of immune cells (such as dendritic cells, macrophages, T cells, B cells, and NK cells, etc.) to kill tumor cells, non-specific killing is also prone to cause side effects of immune storm damage to normal tissues.

[0003] Therefore, how to reduce or avoid the side effects of immune activators on normal tissues when activating the immune system is one of the current research focuses. Summary of the Invention

[0004] The object of the present invention is to provide an immune-activating antibody and its application, aiming to solve the technical problems such as the side effects of existing immune activators on normal tissues when activating the immune system.

[0005] To achieve the above object of the invention, in the first aspect of the present invention, there is provided an immune-activating antibody, which comprises an antibody and an immune activator, and the antibody is coupled with the immune activator through a coupling chain, and the coupling chain comprises at least one of the structures shown in formula (A), formula (B), formula (C), and formula (D):

[0006]

[0007]

[0008] By conjugating an immune activator and an antibody through a conjugate chain, the present invention can form a series of immune-activated antibodies targeting specific tissues, lesions, and targets, achieving the effect of locally targeted immune activation and solving the negative impact of TLR activation on normal tissues. Verified by experiments, the immune-activated antibody can guide the immune activator therein to play a role at the required site or environment (such as the tumor microenvironment), and has multiple functions such as activating target immune cells (such as T cells, B cells, NK cells, etc.), reversing inert immune cells (such as macrophages turning into M1-type anti-tumor macrophages and increasing the M1 / M2 ratio, etc.), and transforming immune cells into immune cells with anti-tumor activity (such as increasing the number of IFN-γ+CD8 cells, etc.), and has better specific immune regulation and therapeutic effects.

[0009] In a second aspect of the present invention, there is provided the use of an immune-activated antibody in the preparation of anti-tumor drugs, anti-viral drugs, immune regulation drugs, and / or preparations for eliminating target proteins.

[0010] Since the immune-activated antibody provided by the present invention has the effect of locally targeted immune activation, when it is used in the preparation of anti-tumor drugs, anti-viral drugs, immune regulation drugs, and / or preparations for eliminating target proteins, it can not only avoid the damage side effects of non-specific killing to normal tissues, but also has multiple functions such as activating target immune cells (such as T cells, B cells, NK cells, etc.), reversing inert immune cells (such as macrophages turning into M1-type anti-tumor macrophages and increasing the M1 / M2 ratio, etc.), and transforming immune cells into immune cells with anti-tumor activity (such as increasing the number of IFN-γ+CD8 cells, etc.), and has good application prospects. Description of the Drawings

[0011] Figure 1 It is for the method and result of the HEK-Blue hTLR7 detection of the compound in Example 1 of the present invention;

[0012] Figure 2 and Figure 3 It is for the detection result of the TLR7 agonist release effect of the compound in Example 2 of the present invention;

[0013] Figure 4 It is for the detection result of the tumor weight on the 25th day after the administration of Compound 15-4 in Example 3 of the present invention;

[0014] Figure 5 It is for the detection result of the tumor volume change on the 25th day after the administration of Compound 34 in Example 3 of the present invention;

[0015] Figure 6Detection results of the regulatory effects of Compounds 28, 30, and 15-4 on tumor microenvironment macrophages in Example 4 of the present invention (relative values of M1 / M2 markers MHC-ClassII / CD206);

[0016] Figure 7 Detection results of the relative change values of IFN-γ+CD8 cells in tumor tissues by Compounds 28, 30, and 15-4 in Example 4 of the present invention;

[0017] Figure 8 Detection results of the inhibitory effect of Compound 35 on SK-BR-3 cells in Example 5 of the present invention;

[0018] Figure 9 Detection results of the inhibitory effect of Compound 36 on A549 cells in Example 5 of the present invention;

[0019] Figure 10 Activation effects of each compound in Example 6 of the present invention on HEK-Blue hTLR7 cells;

[0020] Figure 11 Inactivation effects of each compound in Example 6 of the present invention on HEK-Blue hTLR7 cells;

[0021] Figure 12 Schematic diagram of the degradation of TLR7 agonist by the enzyme (Cathepsin-B) represented by HO-VC-T in the examples of the present invention;

[0022] Figure 13 General formula of immunoreactivating antibody;

[0023] Figure 14 Protein sequence of Trastuzumab;

[0024] Figure 15 Protein sequence of Atezolizumab;

[0025] Figure 16 Schematic diagram of the catalytic cleavage of Cathepsin enzyme to release immunoreactivator;

[0026] Figure 17 TLR7 activation effect diagrams of each compound under the action of Cathepsin-B enzyme;

[0027] Figure 18 TLR7 activation effect diagrams of each compound without the action of Cathepsin-B;

[0028] Figure 19 Inhibitory effect diagram of Antibody 29-1 on human H1299 cells;

[0029] Figure 20 Inhibitory effect diagram of antibody 17-1 on human MDA-MB-453 cells. Detailed implementation manners

[0030] To make the objectives, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below. The following described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Combining the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer; for the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained by purchasing in the market.

[0031] In the description of the present invention, the term "and / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0032] In the description of the present invention, "at least one" means one or more, and "a plurality" means two or more. "At least one (item)" or similar expressions thereof refer to any combination of these items, including any combination of single item (item) or plural items (items). For example, "at least one (item) of a, b, or c", or, "at least one (item) of a, b, and c" can both represent: a, b, c, a-b (that is, a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple respectively.

[0033] It should be understood that the weights of the relevant components mentioned in the embodiments of the present invention not only can refer to the specific contents of each component, but also can represent the proportional relationship of the weights between each component. Therefore, as long as the contents of the relevant components in the embodiments of the present invention are enlarged or reduced in proportion, they are within the scope disclosed in the present invention. Specifically, the weights described in the embodiments of the present invention can be mass units well known in the chemical industry such as μg, mg, g, kg, etc.

[0034] In addition, unless otherwise explicitly stated in the context, the expression of the singular form of a word should be understood to include the plural form of the word. The terms "comprising" or "having" are intended to specify the existence of features, quantities, steps, operations, elements, parts or combinations thereof, but are not used to exclude the existence or possible addition of one or more other features, quantities, steps, operations, elements, parts or combinations thereof.

[0035] An embodiment of the present invention provides an immune-activating antibody, which comprises an antibody and an immune activator. The antibody and the immune activator are coupled by a coupling chain, and the coupling chain comprises at least one of the structures shown in formula (A), the structure shown in formula (B), the structure shown in formula (C), and the structure shown in formula (D):

[0036]

[0037] In the embodiment of the present invention, by coupling the immune activator and the antibody through a coupling chain, a series of immune-activating antibodies targeting specific tissues, lesions, and targets can be formed, which can achieve the effect of locally targeted immune activation and solve the negative impact of TLR activation on normal tissues. Verified by experiments, the immune-activating antibody can guide the immune activator therein to play a role at the required site or environment (such as the tumor microenvironment), and has multiple functions such as activating target immune cells (such as T cells, B cells, NK cells, etc.), reversing inert immune cells (such as converting macrophages into M1-type anti-tumor macrophages and increasing the M1 / M2 ratio), and converting immune cells into immune cells with anti-tumor activity (such as increasing the number of IFN-γ+CD8 cells), etc., and has better specific immune regulation and therapeutic effects.

[0038] The immune-activating antibody provided by the embodiment of the present invention is different from other immune-activating antibodies in that the immune-activating antibody provided by the embodiment of the present invention does not stimulate immune cells expressing TLR7 or TLR8 in the normal immune cell environment, nor does it produce immune cytokines; only in the cell environment of the antibody target (such as the tumor cell environment), or in the environment containing proteases (such as Cathepsin-B) directed by the antibody, can the immune system and immune cells be activated.

[0039] The general structural formula of the immune-activating antibody provided by the embodiment of the present invention is as Figure 13 shown.

[0040] In this general structural formula, the "coupling chain" is composed of a "first linking chain", a "second linking chain", and a "degradable chain". Among them, the "first linking chain" and the "second linking chain" are both structures conventionally used for linking in the art, and will not be elaborated here one by one; the structure of this part of the "degradable chain" is the structure shown in formula (A), and this structure can also be replaced by the structures shown in formula (B), formula (C), and / or formula (D). Accordingly, the general structural formula of the corresponding immune-activating antibody will change, and the changed general formula will not be listed here. For example, some specific structures (Valine-citrulline linker: Val-Cit) in the structure shown in formula (A), by replacing the degradable chain part, some specific structures (Valine-alaninelinker: Val-Ala) of the structure shown in formula (B) can be obtained, as follows:[[]]

[0041]

[0042] The immune-activating antibody provided by the embodiments of the present invention mainly includes three parts: an antibody, a coupling chain, and an immune activator. The following is a specific description of these three parts:

[0043] Coupling chain:

[0044] The coupling chain in the embodiments of the present invention can be selected from at least one of a chain degradable containing a Cathepsin-B region, an alkyl group, an alkoxy group, a nitrogen-containing alkyl group, a heterocycle, and a specific functional chain.

[0045] In some embodiments, the coupling chain includes at least one of compound 5, compound 5-1, compound 5-2, compound GY209, compound 6, compound 6-1, compound 6-2, compound 6-3, compound 7, compound 7-1, compound 7-2, compound 7-3, compound 7-4, compound 7-5, compound 8, compound 8-1, compound 8-2, compound 8-3, compound 8-4, compound 8-5, compound 8-6, compound 8-7, compound 27, compound GY206, compound GY207, compound 5A-GY102, compound VCB-4, compound BVC-T-4, compound Tri-linker-1, and compound Tri-linker-2, and the structural formulas are as follows:

[0046]

[0047] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl 6-amino-2-butoxy-9-(cyanomethyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxylate

[0048]

[0049] 6-amino-2-butoxy-9-(cyanomethyl)-N-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxamide

[0050]

[0051] N-((S)-1-(((S)-1-(6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0052]

[0053] 4-(((S)-1-(((S)-1-((4-(((6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid

[0054]

[0055] 4-(((S)-1-(((S)-1-((4-((6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carboxamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid

[0056]

[0057] 4-(((S)-1-(((S)-1-(6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid

[0058]

[0059] 4-(((S)-1-(((S)-1-(6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid

[0060]

[0061] 4-((2S,5S)-5-isopropyl-17-isothiocyanato-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl

[0062] 6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carboxylate

[0063]

[0064] 6-amino-2-butoxy-N-(4-((2S,5S)-5-isopropyl-17-isothiocyanato-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl)-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carboxamide

[0065]

[0066] 4-((2S,5S)-5-isopropyl-17-isothiocyanato-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl

[0067] 6-amino-2-butoxy-9-(cyanomethyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxylate

[0068]

[0069] (S)-N-((S)-1-(6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)-1-oxo-5-ureidopentan-2-yl)-2-(2-(2-(2-(2-isothiocyanatoethoxy)ethoxy)ethoxy)acetamido)-3-methylbutanamide

[0070]

[0071] (S)-N-(4-(6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carbonyl)phenyl)-2-((S)-2-isopropyl-14-isothiocyanato-4-oxo-6,9,12-trioxa-3-azatetradecanamido)-5-ureidopentanamide

[0072]

[0073] (S)-N-(4-((6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)methyl)phenyl)-2-((S)-14-azido-2-isopropyl-4-oxo-6,9,12-trioxa-3-azatetradecanamido)-5-ureidopentanamide

[0074]

[0075] 4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl

[0076] 6-amino-2-butoxy-9-(cyanomethyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxylate

[0077]

[0078] 6-amino-N-(4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl)-2-butoxy-9-(cyanomethyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxamide

[0079]

[0080] 6-amino-N-(4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl)-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carboxamide

[0081]

[0082] 6-amino-N-(4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl)-2-butoxy-9-((1-cyclooctyl-1H-1,2,3-triazol-4-yl)methyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxamide

[0083]

[0084] (S)-N-((S)-1-(6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)-1-oxopropan-2-yl)-2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetamido)-3-methylbutanamide

[0085]

[0086] N-((S)-1-(((S)-1-(6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0087]

[0088] (S)-N-((S)-1-(6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)-1-oxopropan-2-yl)-2-(2-(2-(2-(2-isothiocyanatoethoxy)ethoxy)ethoxy)acetamido)-3-methylbutanamide

[0089]

[0090] (S)-N-((S)-1-(6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)-1-oxo-5-ureidopentan-2-yl)-2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetamido)-3-methylbutanamide

[0091]

[0092] 4-(((S)-1-(((S)-1-((4-(15-(4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)-1H-1,2,3-triazol-1-yl)-3-oxo-2,7,10,13-tetraoxa-4-azapentadecyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid

[0093]

[0094] 4-(17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl

[0095] 6-amino-2-butoxy-9-((1-cyclooctyl-1H-1,2,3-triazol-4-yl)methyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxylate

[0096]

[0097] 4-(17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl

[0098] 6-amino-2-butoxy-9-((1-(1-((2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)amino)-1-thioxo-5,8,11-trioxa-2-azatridecan-13-yl)-1H-1,2,3-triazol-4-yl)methyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxylate

[0099]

[0100] 4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl

[0101] (2-(2-(2-(2-(4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamate

[0102]

[0103] (S)-N-(4-((6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)methyl)phenyl)-2-((S)-2-isopropyl-14-isothiocyanato-4-oxo-6,9,12-trioxa-3-azatetradecanamido)-5-ureidopentanamide

[0104]

[0105] N2-(4-(((S)-1-(((S)-1-((4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoyl)-N6-diazolysine

[0106]

[0107] N1-(2-(2-(2-(2-(4-(((2-((2-acrylamido-5-methoxy-4-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)(methyl)amino)ethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-(14-(4-((6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)phenyl)-7-(4-azidobutyl)-6,9,12-trioxo-2-thia-5,8,13-triazatetradecyl)-N4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)succinamide

[0108]

[0109] N1-(2-(2-(2-(2-(4-(((2-((2-acrylamido-5-methoxy-4-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)(methyl)amino)ethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-((14S,17S)-22-amino-17-((4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phenyl)carbamoyl)-7-(4-azidobutyl)-14-isopropyl-6,9,12,15,22-pentaoxo-2-thia-5,8,13,16,21-pentaazadocosyl)-N4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)succinamide。

[0110] The embodiments of the present invention also provide a series of intermediate compounds for synthesizing the specific compounds of the above coupling chain, which are at least one of compound 18-2, compound 5A, compound 102-3, compound VC-An4, compound Val5, compound Val6, compound VC100, compound VCB-2, compound POMA-ICO3N3, compound S-POMA-ICO3N3, compound Bi-Linker, compound BVC-T-2, compound HO-VC-T, compound N3-VC-T, compound MA-VC-T, and SVC-T, and the structural formulas are as follows respectively:

[0111]

[0112] 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl

[0113] 6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carboxylate

[0114]

[0115] 4-(17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl(4-nitrophenyl)carbonate

[0116]

[0117] 6-amino-9-((1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-2-butoxy-N-methyl-8-oxo-N-propyl-8,9-dihydro-7H-purine-7-carboxamide

[0118]

[0119] (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carboxamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[0120]

[0121] (S)-N-(4-(aminomethyl)phenyl)-2-((S)-14-azido-2-isopropyl-4-oxo-6,9,12-trioxa-3-azatetradecanamido)-5-ureidopentanamide

[0122]

[0123] 4-nitrophenyl(4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl)carbamate

[0124]

[0125] (S)-2-amino-N-((S)-1-(6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)-1-oxo-5-ureidopentan-2-yl)-3-methylbutanamide

[0126]

[0127] (S)-2-amino-N-((S)-1-(6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-7-yl)-1-oxo-5-ureidopentan-2-yl)-3-methylbutanamide

[0128]

[0129] N1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-2-methylenesuccinamide

[0130]

[0131] 2-(((2-aminoethyl)thio)methyl)-N1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)succinamide

[0132]

[0133] N1-(2-(2-(2-(2-(4-(((2-((2-acrylamido-5-methoxy-4-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)(methyl)amino)ethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-(((2-aminoethyl)thio)methyl)-N4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)succinamide

[0134]

[0135] (S)-N-(4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamide

[0136]

[0137] 4-(((S)-1-(((S)-1-((4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid

[0138]

[0139] (S)-N-(4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-2-((S)-14-azido-2-isopropyl-4-oxo-6,9,12-trioxa-3-azatetradecanamido)-5-ureidopentanamide

[0140]

[0141] N-((S)-1-(((S)-1-((4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0142]

[0143] (S)-N-(4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-2-((S)-2-isopropyl-14-isothiocyanato-4-oxo-6,9,12-trioxa-3-azatetradecanamido)-5-ureidopentanamide。

[0144] Immunostimulants:

[0145] The immunostimulants provided in the embodiments of the present invention may be conventional immunostimulants in the art, including but not limited to at least one of TLR7 agonists, TLR8 agonists, STING agonists, and small molecule immunostimulants. In some embodiments, the small molecule immunostimulants include at least one of Compound 1, Compound 2, Compound 3, and Compound 4, and the structural formulas are as follows:

[0146]

[0147] Antibodies:

[0148] The antibody provided by the embodiments of the present invention can be any antibody targeting a pathogen, and the pathogen is the antigen. In some embodiments, the antigen is selected from HER2, HER3, PD-L1, PD-1, TIGIT, TROP2, EGFR, MUC1, LIV-1, MUC16, CEACAM1 and its various subtypes, URLC10, NY-ESO-1, GAA, OFA, cyclin B1, WT-1, CEF, VEGFR1, VEGFR2, TTK, MUC1, HPV 16E7, CEA, IMA910, KOC1, SL-701, MART-1, gp100, tyrosinase, GSK2302050A, survivin, MAGE-3.1, MAGE-10.A2, gp209-2M, melan-A, NA17.A2, KOC1, CO16, DEPDC1, MPHOSPH1, MAGE12, ONT-10, GD2L, GD3L, GSK2302032A, URLC10, CDCA1, rsPSMA, PSA, MUC-2, TERT, HPV16, HPV18, STF-II, G17DT, ICT-107, Dex2, hTERT, PAP, LRRC15, MSLN, LY6K, CD56, PTK7, FOLR1, DLL3, CD3, BTK, GPC3, EPCAM, CECAM5, cMET, GPA33, ALK, ROS1, BRAF, MEK, RET, CDK4 / 6, BRCA, PARP, BRCA, FLT3, CD19, CD20, BCL2, CD38, Smoothened, GD2, EZH2, MTH1, KRAS, c-MYC, hCA IX, hCA XII, BRD4, HDAC, NYC, TOPK, BCMA, PI3K, PDGFR, TIM3, OX40, CD47, SIRP-ɑ, CD40, CD122, CD160, TGF-β, HIF-1ɑ / 2ɑ, PSGL-1, Frizzled-7, SLC4A7, CCR2, CCR4, CCR5, CXCR4, CXCR5, CCL12, CXCL1, CXCL8, CXCL10, CD30, CD33, CD22, CD79b, Nectin-4, CD45, CD117, PSMA, NKG2D, Claudin18.2. At least one of MG7, ROR1, ROR2, FGFR, FGFR2, FGFR4, WNT2A, WNT3A, WNT5A, WNT9b, WNT7b, HGF, LILRB1, CAF, ANG2, ARG1, CSF1R, TIE2, RXRβ, ASGPR1, Bcl2, HPK1, ENPP1, Sb9, KLRG1, B7-H3, IFI27, REG4, CD161, ST2, RIPK1, Claudin-6. Antibodies against these pathogens can target these pathogens and guide the immune activators in the immune activation antibodies to the corresponding target sites, achieving the effect of local targeted immune activation.

[0149] Immune activation antibody:

[0150] According to the changes in the coupling chain, a series of general formulas of immune activation antibodies with different structures can be obtained, including the conjugate shown in formula (I) The conjugate shown in formula (I-1) The conjugate shown in formula (I-2) The conjugate shown in formula (I-3) The conjugate shown in formula (I-4) The conjugate shown in formula (II) The conjugate shown in formula (II-1) The conjugate shown in formula (II-2) The conjugate shown in formula (II-3) The conjugate shown in formula (II-4) The conjugate shown in formula (III) The conjugate shown in formula (III-1) The conjugate shown in formula (III-2) The conjugate shown in formula (III-3) The conjugate shown in formula (III-4) The conjugate shown in formula (IV) The conjugate shown in formula (IV-1) The conjugate shown in formula (IV-2) The conjugate shown in formula (IV-3) The conjugate shown in formula (IV-4) At least one of them, and n > 0.

[0151] The following lists some relatively typical immune-activating antibodies, including at least one of Compound 15, Compound 15-1, Compound 15-2, Compound 17, Compound 21, Compound 22, Compound 22-2, Compound 24, Compound 24-2, Compound 15-3, Compound 15-4, Compound 15-5, Compound 24-3, Compound 28, Compound 29, Compound 30, Compound 30-1, Compound 31, Compound 32, Compound 33, Compound 34, Compound 35, Compound 36, Compound 37, and their structural formulas are as follows:

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] The synthesis methods of the above-mentioned conjugate chain, small molecule immune agonist and corresponding immune-activating antibody provided by the embodiments of the present invention are specifically as follows:

[0160] The synthesis method of Compound 1 includes the following steps:

[0161] Dissolve 2.37 grams of Compound Pro-1 and 1.2 grams of Compound bromopropionitrile in 50 mL of acetonitrile, add 1.5 grams of K2CO3, and stir at room temperature for 12 hours. Filter, add 2 mL of trimethylsilyl iodide, and stir at room temperature for 10 hours. Add 5 mL of saturated NaHCO3 solution, distill off the solvent under reduced pressure, separate by silica gel column chromatography (methanol:dichloromethane = 1:10 volume ratio), collect the product solution, and concentrate under reduced pressure to remove the dissolution to obtain a solid product, which is Compound 1.

[0162]

[0163] The synthesis methods of Compound 2 and Compound 3 are basically the same as that of Compound 1. The difference is that when synthesizing Compound 2, Compound Pro-1 is replaced by Compound 9; when synthesizing Compound 3, Compound Pro-1 is replaced by Compound 10. Among them, the structural formula of Compound 9 is The structural formula of Compound 10 is

[0164] Synthesis of representative compound 15 of formula III using HER2 antibody as an example:

[0165]

[0166] Dissolve compound 11 (650 mg) and compound 4 (270 mg) in 5 mL of DMF, add 0.5 mL of TEA, and stir the mixture at room temperature for 10 hours. Pour the mixture into 50 mL of water, centrifuge to precipitate the solid product compound 12, and purify it by HPLC to obtain compound 12 (498 mg, 65%). ESI-MS: m / z = 767.3 [M+H]+.

[0167] Using the same method, replacing compound 4 with compound 1, 12-2 can be obtained:

[0168]

[0169] Add compound 12 (200 mg) directly to 10 mL of TFA / DCM (1:3), stir at room temperature for 8 hours, evaporate the solvent under reduced pressure, and dry it under vacuum; add 50 mL of DMSO and compound 13 (80 mg). Then add 36 mg of HOBT, 50 mg of EDC, and 120 μL of DIPEA to the solution, and react at room temperature overnight, monitoring the reaction by LC-MS. After the reaction is completed, purify it by HPLC to obtain white solid compound 14 (119 mg) with a yield of 47.7%. ESI-MS: m / z = 956.5 [M+H]+.

[0170] Add compound 14 (100 mg) to TFA / DCM (1:3) (2 mL), stir at room temperature for 8 hours. Remove the solvent under reduced pressure to dry solid; dissolve it in 5 mL of DMSO, add thiodiimidazole (181 mg) and 300 μL of triethylamine. React at room temperature for 12 hours. Freeze-dry the reaction solution to obtain the crude product, and purify it by HPLC to obtain compound 7 (44 mg, 47%). ESI-MS: m / z = 898.4 [M+H]+.

[0171]

[0172] Take 100 mg of HER2 antibody (deglycosylated molecular weight: 145531), compound 7 (12 mg), and triethylamine (5 μL), dissolve them in a solvent of DMSO and pure water (1:10 volume), react with shaking at 10 °C for 10 hours, and filter through a 10K filter membrane. Elute to obtain the conjugate antibody compound 15; the DAR (drug / antibody, ratio) value measured by mass spectrometry is 5.98.

[0173] Referring to the synthesis of compound 15, replacing compound 4 with compound 1 can obtain compound 7-2:

[0174]

[0175] For the coupling synthesis of reference compound 15, the analogue compound 15-1 can be obtained by replacing compound 7 with compound 7-1:

[0176]

[0177] Furthermore, coupling with compound 7-3 to HER2 antibody can obtain the analogue compound 15-2 of compound 15:

[0178]

[0179] The synthetic route of compound 7-3 is as follows:

[0180]

[0181] Replacing BNCOOH with succinic anhydride and reacting with compound VC100 can directly obtain compound 6-3:

[0182]

[0183] The synthesis steps of compound 5-1 are as follows:

[0184]

[0185] Dissolve 1 micromole of compound val1 and an equivalent amount of compound 16 in 3 mL of DMSO, stir at room temperature for 8 hours, then freeze-dry to remove DMSO. Add 1 mL of TFA / DCM (1:3 by volume), shake and react at room temperature for 2 hours, vacuum decompress and dry the solution, and purify the residue by HPLC to obtain compound MM-VC.

[0186] Mix 300 mg of compound MM-VC with equivalent amounts of HOBT, EDC, and DIPEA and dissolve them in 2 mL of DMSO, and react with a room temperature shaker for 2 h. Add 142 mg of compound BNMA and continue to react overnight. The reaction mixture is freeze-dried, add 1 mL of TFA / DCM (1:3 by volume), shake and react at room temperature for 2 hours, vacuum decompress and dry the solution, and purify the residue by HPLC to obtain 238 mg of compound MM-VCA-NH2, with a yield of 65%. Mass spectrometry: ESI-MS: m / z = 572.5 [M+H] + 。

[0187] Another 1 μmol of Compound 1 and an equivalent amount of Compound 4A were dissolved in 2 mL of DMSO, an equivalent amount of TEA was added, and the reaction was stirred at room temperature for 12 hours and then at 40 °C for 1 hour. The mixture was freeze-dried to obtain the crude product of Compound 4A-1; the crude product was dissolved in dry DMSO, an equivalent amount of MM-VCA-NH2 was added, and the mixture was stirred at room temperature for 12 h. The product was purified by HPLC to obtain Compound 5-1 with a yield of 43%, ESI-MS: m / z = 860.4 [M+H] + 。

[0188] Synthesis of Compound 6-1:

[0189]

[0190] 500 mg of Compound FVC-1 and an equimolar amount of a mixed condensing agent (HOBT, EDC, DIPEA) were dissolved in 20 times the weight of DMSO, and the mixture was stirred at room temperature for 2 hours. An equivalent amount of Compound BNMA was added, and the reaction was continued at room temperature overnight. The reaction mixture was directly freeze-dried, and the residue was dissolved in methanol and purified by HPLC to obtain 453 mg (75%) of pure Compound FVC-2, ESI-MS: m / z = 601.4 [M+H]+.

[0191] 200 mg of Compound 4 and an equimolar amount of Compound 4A were mixed in 2 mL of DMSO, 2 equivalents of TEA were added, and the mixture was shaken at room temperature for 8 hours. An equivalent amount of Compound FVC-2 was added, and the reaction was continued at room temperature overnight. Then, 4 equivalents of piperidine were added, and the mixture was shaken at room temperature for 6 hours. The mixture was freeze-dried. The residue was dissolved in methanol and purified by HPLC to obtain Compound VC-An4 with a yield of 28%, ESI-MS: m / z = 666.30 [M+H] + 。

[0192] 100 mg of Compound VC-An4 was dissolved in 2 mL of DMSO, an equivalent amount of TEA was added, and an equivalent amount of Succinic anhydride was further added. The reaction was shaken at 40 °C overnight. The mixture was freeze-dried, 2 mL of water / methanol (1:1) was added, the pH was adjusted to 5 with acetic acid, and the product was directly purified by HPLC to obtain 94 mg (yield 82%) of Compound 6-1, ESI-MS: m / z = 766.62 [M+H] + 。

[0193] Synthesis of Representative Compound 17 of Formula I:

[0194]

[0195] Compound 12-2 (100 mg) was directly added to 10 mL of TFA / DCM (1:3), and the mixture was stirred at room temperature for 8 hours. The solvent was removed by evaporation under reduced pressure and then dried under vacuum. It was dissolved in 5 mL of DMSO, and 30 μL of triethylamine and compound 16 (45 mg) were added. The mixture was stirred at room temperature for 10 hours. Compound 5 (93 mg) was obtained by HPLC purification. ESI-MS: m / z = 861.4 [M+H]+.

[0196] 100 mg of HS-reduced HER2 antibody, compound 5 (12 mg), and triethylamine (5 μL) were dissolved in 5 mL of pure water, and the mixture was reacted with shaking at 10 °C for 10 hours and then filtered through a 10K filter membrane. The coupled antibody compound 17 was obtained by elution; the DAR (drug / antibody, ratio) value measured by mass spectrometry was 4.

[0197] Preparation of HS-reduced HER2 antibody: The HER2 antibody was dialyzed to remove various additives by dialysis method and dissolved in DPBS (15 mg / mL), and the pH was adjusted to 7.0 with a 5 mM EDTA solution. 5 equivalents of TCEP solution (5 mM TCEP aqueous solution) was added and reduced at room temperature for 2 hours. Small molecules were removed by a 10KD filter membrane, and the antibody was eluted with pure water and freeze-dried under vacuum to obtain the reduced antibody.

[0198] Synthesis of antibody 17-1:

[0199]

[0200] 15 equivalents of 5-2-2 (10 mg) and 0.1 mL of TEA were dissolved in 0.2 mL of DMSO and added to a solution of 100 mg of HS-reduced trastuzumab in DPBS (5 mL). The mixture was reacted with shaking at 20 °C for 10 hours. Small molecules were removed by ultrafiltration through a 20K filter membrane, and the mixture was stirred at room temperature in an open container for 6 hours to obtain a new antibody 17-1 in which Her2 antibody was conjugated with 5-2-2; the conjugation ratio (ADR value) measured by mass spectrometry was 2.

[0201] Preparation of HS-reduced trastuzumab: It was obtained according to the preparation method of HS-reduced HER2.

[0202] Among them, for the protein sequence of Trastuzumab (WHO Drug Information Vol.24, No.2, 2010), please refer to Figure 14 As shown, the protein sequence of Trastuzumab is as shown in SEQ ID NO:1 and SEQ ID NO:2.

[0203] Synthesis of compound 8:

[0204]

[0205] Compound 12-2 (100 mg) was directly added to 10 mL of TFA / DCM (1:3), and the mixture was stirred at room temperature for 8 hours. The solvent was removed by evaporation under reduced pressure and dried under vacuum. Then it was dissolved in 5 mL of DMSO, and 30 μL of triethylamine was added. Compound 19 (30 mg), HOBT (27 mg), EDC (39 mg), and DIPEA (68 μL) were added, and the reaction was carried out overnight at room temperature. The reaction was monitored by LC-MS. After the reaction was completed, it was purified by HPLC to obtain white solid compound 8 (63.6 mg, yield 55.7%). ESI-MS: m / z = 883.3 [M+H]+.

[0206] Synthesis of representative compound 22 of formula IV:

[0207]

[0208] The activated ester 20 of DBCO-acid (20 eq) was mixed with HER2 antibody (1 eq) in pure water containing 10% DMSO, and the reaction was shaken at 10 °C for 12 hours. Mass spectrometry was used to detect that the HER2 antibody reaction was complete. The reaction mixture was filtered through a 10 KD molecular filter membrane to remove small molecules, and the conjugate compound 21 was eluted with DPBS solution (coupling degree 6 was measured). 10% (by volume) DMSO was added to the DPBS solution of compound 21, and 1.5 equivalents of compound 8 were added. The reaction was shaken at 25 °C for 12 hours, and small molecules were removed through a 20 KD molecular filter membrane. The antibody was dissolved in DPBS to obtain the DPBS solution of conjugate antibody compound 22. The average coupling degree of compound 22 was measured by mass spectrometry to be 6.

[0209] Synthesis of compound 22-2:

[0210]

[0211] Equivalent amounts of compound VCB-1 and compound GY100 were dissolved in DMF, 2 equivalents of K2CO3 were added, and the reaction was carried out overnight at room temperature. 10 times the volume of water was added and stirred evenly to obtain a precipitate. The precipitate was separated by silica gel chromatography with 1:10 methanol / DCM to obtain pure compound VCB-2, ESI-MS: m / z = 723.7 [M+H]+. Compound VCB-2 was deprotected by removing the Boc group, and then subjected to a conventional condensation amidation reaction with azido carboxylic acid. The product was purified by HPLC to obtain compound 7-5, ESI-MS: m / z = 838.4 [M+H] + . It was subjected to a click coupling reaction with conjugate compound 21, and purified by molecular filter membrane to obtain compound 22-2. The DAR was measured by mass spectrometry to be 6.

[0212] According to a synthetic method similar to that of 7-5, compound VCB-4 can be synthesized as follows:

[0213]

[0214] VCB-2 (300 mg) was mixed with 10 mL of TFA / DCM (1 / 3 volume), and the mixture was stirred at room temperature for 12 hours. The solvent was removed by distillation under reduced pressure. 20 mL of ethyl acetate and 0.2 mL of TEA were added to the residue. After mixing evenly, the ethyl acetate layer was washed once with 10 mL of water. The organic layer was dried over anhydrous Na2SO4. The desiccant was filtered off, and the ethyl acetate was distilled off under reduced pressure to obtain VCB-3 (210 mg, yield 81%), ESI-MS: m / z = 623.3 [M+H]+.

[0215] 200 mg of VCB-3 was dissolved in 5 mL of DMSO, and 100 mg of BN-PEG-OH, an equivalent amount of EDC, and DIPEA were added. The mixture was stirred at room temperature overnight; the reaction solution was directly freeze-dried, and 10 mL of TFA / DCM (1 / 3 volume) was added and stirred for 12 hours. The solvent was removed by distillation under reduced pressure. 20 mL of ethyl acetate and 0.2 mL of TEA were added to the residue. After mixing evenly, the ethyl acetate layer was washed once with 10 mL of water. The ethyl acetate was distilled off under reduced pressure, and the residue was purified by HPLC to obtain NH2-VCB-3 (124 mg, yield 47%), SI-MS: m / z = 812.4 [M+H]+.

[0216] 100 mg of NH2-VCB-3 was dissolved in 3 mL of DMSO, and 33 mg of DIMS and 1001 μL of TEA were added. The reaction was carried out at room temperature until completion (monitored by MS). After freeze-drying, the residue was separated and purified by HPLC to obtain VCB-4 (51 mg, yield 49%), ESI-MS: m / z = 854.4 [M+H]+.

[0217] Synthesis of compound 5-2-1:

[0218]

[0219] Benzo-acid (150 mg) was dissolved in 5 mL of THF, and NHS (106 mg) and EDC.HCl (211.5 mg) were added. The mixture was stirred at room temperature for 3 hours. NH2-PEG3-N3 (200 mg) and DIPEA (192 μL) were mixed and dissolved in 5 mL of THF solvent, and the solution was slowly added dropwise to the above reaction solution. The resulting mixed reaction was stirred at room temperature for 10 hours. The solvent was removed by distillation under reduced pressure, and the residue was separated and purified by HPLC to obtain Benzo-PEG3-N3 (210 mg, yield 63%); ESI-MS: m / z = 364.1 [M+H]+.

[0220] Dissolve Benzo-PEG3-N3 (100 mg) and NH2-VCB-3 (224 mg) in 7 mL of DMSO, add 2 mL of pure water, 10 mg of copper sulfate and 10 mg of sodium L-ascorbate, and stir the resulting mixture at room temperature for 12 hours. Filter the reactants, freeze-dry the filtrate, dissolve the freeze-dried product in a little DMF, and separate and purify by HPLC to obtain NH2-VCB-209 (175 mg, yield 54%); ESI-MS: m / z = 1175.5 [M+H]+.

[0221] Dissolve M-NHS (25 mg) in 1 mL of DMSO; dissolve NH2-VCB-209 (110 mg) and DIPEA (16 μL) in 2 mL of DMSO, and slowly add dropwise to the M-NHS solution. Stir the resulting reaction mixture at room temperature for 10 hours. Freeze-dry the reactants, dissolve the freeze-dried product in a little DMF, and separate and purify by HPLC to obtain 5-2-1 (82 mg, yield 66%); ESI-MS: m / z = 664.2 [(M / 2)+H]+.

[0222] Synthesis of compound GY209:

[0223]

[0224] Dissolve compound Benzo-PEG3-N3 (100 mg) and GY100 (72 mg) in DMSO-deionized water (volume ratio 3:1, 4 mL), add anhydrous copper sulfate (10 mg), sodium L-ascorbate (12 mg); stir the resulting mixed solution at room temperature overnight. Filter to obtain a clear solution, freeze-dry the filtrate, dissolve the freeze-dried product in a little methanol, and separate and purify by HPLC to obtain GY209 (58 mg, yield 34%); ESI-MS: m / z = 625.2 [M+H]+.

[0225] Synthesis of compound 5-2-2:

[0226]

[0227] Dissolve SZU-128 (100 mg) in 5 mL of DMF, add Boc-Benz-Br (55 mg), 30 mg of K2CO3; stir the resulting mixture at room temperature for 12 hours. Filter the reaction solution, freeze-dry the filtrate, and separate and purify the freeze-dried product by HPLC to obtain NBZ-128 (90 mg, yield 75%); ESI-MS: m / z = 632.3 [M+H]+.

[0228] V-Ala (32 mg), NHS (13 mg), and EDC·HCl (22 mg) were mixed and dissolved in 1 mL of THF, and the mixture was shaken at room temperature for 6 h. NBZ-128 (70 mg) was dissolved in 1 mL of DMSO and slowly added dropwise to the above mixture. After addition, the mixture was shaken at room temperature for 1 h, and DIPEA (20 μL) was added; the mixture was shaken at room temperature for another 6 h. Then, 0.5 mL of TFA was added, and the reaction continued at room temperature for 3 h. The reaction solution was directly separated and purified by HPLC to obtain N-VA-128 (72 mg, yield 81%); ESI-MS: m / z = 802.3 [M+H]+.

[0229] M-PEG3-NHS (35 mg) was dissolved in 1 mL of anhydrous DMSO; N-VA-128 (60 mg) and DIPEA (13 μL) were dissolved in 2 mL of anhydrous DMSO and slowly added dropwise to the M-PEG3-NHS solution. After addition, the mixture was shaken at room temperature for 10 h. The reaction solution was freeze-dried, and the lyophilized product was dissolved in a small amount of methanol and separated and purified by HPLC to obtain 5-2-2 (61 mg, yield 71%); ESI-MS: m / z = 1156.6 [M+H]+.

[0230] Synthesis of Compound 6:

[0231]

[0232] Compound 12 (200 mg) was directly added to 10 mL of TFA / DCM (1:3), and the mixture was stirred at room temperature for 8 h. The solvent was removed under reduced pressure and dried under vacuum; the dried solid was dissolved in water, 40 μL of triethylamine was added, and the product was purified by HPLC to obtain 120 mg of off-white solid of Compound 18-2 (yield 69%); ESI-MS: m / z = 667.3 [M+H]+.

[0233] Compound 18-2 was dissolved in DMSO, an equivalent amount of TEA was added, an equivalent amount of succinic anhydride was added, and the mixture was stirred at room temperature for 6 h. The mixture was freeze-dried to obtain a solid, which was dissolved in an appropriate amount of water, the pH value was adjusted to 5 with acetic acid, the product was precipitated, filtered, and dried to obtain Compound 6. ESI-MS: m / z = 767.3 [M+H]+.

[0234] In the coupling chain represented by Compound 8, the immune activator can be replaced with other TLR7 agonists, such as multifunctional GY159 (the macrocycle can improve cell membrane permeability), GY127 (the anti-cancer effect of immune activation), etc., and multifunctional antibody compounds 24, Compound 24-2, etc. can be prepared:

[0235]

[0236] Dissolve 200 mg of Compound 19, 358 mg of Compound 2A, 176 mg of HOBT, 254 mg of EDC, and 450 μL of DIPEA in 5 mL of DMSO, and react overnight at room temperature while monitoring the reaction by LC-MS. After the reaction is completed, purify by HPLC to obtain 210 mg of a white solid (Compound 3A) with a yield of 41.2%. ESI-MS: m / z = 594.3 [M+H]+.

[0237]

[0238] Dissolve 200 mg of Compound 3A, 113 mg of Compound 4A, and 180 μL of DIPEA in 2 mL of DMSO, and react overnight at room temperature while monitoring the reaction by LC-MS. After the reaction is completed, purify by HPLC to obtain 65 mg of a white solid (Compound 5A) with a yield of 25.4%. ESI-MS: m / z = 759.3 [M+H]+.

[0239]

[0240] Dissolve 30 mg of Compound 5A, 18 mg of Compound GY159, and 21 μL of DIPEA in 1 mL of DMSO, and react overnight at room temperature while monitoring the reaction by LC-MS. After the reaction is completed, purify by HPLC to obtain 8 mg of a white solid (Compound GY206) with a yield of 19.5%. ESI-MS: m / z = 1035.5 [M+H]+.

[0241]

[0242] Dissolve 30 mg of Compound 5A, 42 mg of Compound GY127, and 21 μL of DIPEA in 1 mL of DMSO, and react overnight at room temperature while monitoring the reaction by LC-MS. After the reaction is completed, purify by HPLC to obtain 11 mg of a yellow solid (Compound GY207) with a yield of 17.7%. ESI-MS: m / z = 1586.7 [M+H]+.

[0243] Referring to the HER2 conjugate Compound 22, the immune-activating antibody Compound 24 can be prepared from Compound GY207 and Compound 21 as follows:

[0244]

[0245] Compound GY127 itself has TLR7 activation and tumor cell inhibitory effects. Under the specific targeting guidance of the multifunctional antibody Compound 24, Compound GY127 is released in the tumor microenvironment and tumor cells, achieving local immune activation and enhanced anti-tumor effects.

[0246] Using the same method, replacing the antibody in Compound 22 with the c-Met antibody (abcam, ab51067) and replacing Compound 1 with Compound GY102, the analogue Compound 24-2 can be obtained:

[0247]

[0248] Referring to the synthesis method of Compound 15, replacing the agonist moiety with Compound GY102, Compound 15-4 can be obtained:

[0249]

[0250] Replacing the antibody with the ASGPR1 antibody and the immune agonist with Compound GY102-3, Compound 15-3 can be obtained:

[0251]

[0252] In Compound 15-3, ASGPR1 is the asialoglycoprotein receptor, specifically expressed in liver tissue; the small molecule immune agonist precursor Compound 102-3 is metabolized to Compound 102 in the liver. Therefore, Compound 15-3 is a specifically liver-targeted immune-activated antibody. The synthesis method of the small molecule immune agonist Compound 102-3 is as follows:

[0253]

[0254] Dissolve 480 mg of Compound GY102 in DMSO, add 140 μL of triethylamine, and slowly dropwise add an equivalent amount of tert-butyl chloroformate at 5-10 °C; react at natural room temperature for 4 hours. The reaction mixture is directly freeze-dried to obtain a solid. The solid is added to water at 5 °C, stirred to dissolve and remove salts, filtered to obtain Compound 102-1, and dried in vacuo. The dried product is dissolved in DMSO, 140 μL of triethylamine is added, and an equivalent amount of Compound AC-1 is slowly dropwise added at 5-10 °C, and stirred at natural room temperature overnight. The reaction solution is directly freeze-dried to obtain a solid. The solid is added to water at 5 °C, stirred to dissolve and remove salts, filtered to obtain Compound 102-2. Add 1 mL of TFA and 3 mL of DCM, stir at room temperature for 4 hours, distill off the solvent under reduced pressure in vacuo. The residue is added with water and purified by HPLC to obtain Compound 102-3 (121 mg, yield 21%), ESI-MS: m / z = 579.3 [M+H]+.

[0255] Replacing the antibody in Compound 15-4 with the CD206 antibody (abcam, ab64693), Compound 15-5 can be obtained:

[0256]

[0257] Using the same method, replacing the antibody in Compound 22 with a PD-1 antibody and replacing Compound 8 with Compound GY206, the analogue Compound 24-3 can be obtained:

[0258]

[0259] Synthesis of the representative conjugate antibody Compound 28 of Formula II:

[0260]

[0261] Dissolve Compound 26 and Compound GY102 in DMSO in equimolar amounts, stir the mixed solution at room temperature, and detect by mass spectrometry that the reaction of the raw materials is basically complete. Freeze-dry the reaction solution, dissolve the dried solid in an appropriate amount of TFA / DCM (1:3), and stir at room temperature for 4 hours. Remove TFA by distillation under reduced pressure, separate and purify by HPLC to obtain Compound 27, ESI-MS: m / z = 985.4 [M+H]+.

[0262] Dissolve 100 mg of Compound 27 and an equimolar amount of NHS (N-hydroxysuccinimide) in 1 mL of anhydrous DMSO, add an equimolar amount of EDC, and stir the mixture at 15 °C in a sealed container for 6 hours; dissolve 738 mg of HER2 antibody in 10 mL of pure water, slowly add it to the reaction solution, and after adding, continue to react at room temperature in a sealed container for 10 hours, and add ethanolamine (6 μL); filter the reaction solution through a 20KD molecular filter membrane, and elute small molecules with DPBS to obtain a DPBS solution of the conjugate antibody Compound 28. The average conjugation degree of Compound 28 measured by mass spectrometry is 4.

[0263] Referring to the synthesis of Compound 17, the conjugate antibody Compound 29 can be obtained as follows:

[0264]

[0265] Dissolve 15-fold equivalent of 5-2-1 (12 mg) and 0.1 mL of TEA in 0.2 mL of DMSO, and add it to a DPBS (5 mL) solution of 100 mg of HS-reduced PD-L1 monoclonal antibody (Atezolizumab). The mixture is shaken at 20 °C for 10 hours. Ultrafilter to remove small molecules with a 20K filter membrane, and stir in an open container at room temperature for 6 hours to obtain the new antibody 29-1 conjugated with PD-L1 antibody and 5-2-1; the conjugation ratio (ADR value) measured by mass spectrometry is 2.

[0266] Preparation of HS-reduced PD-L1 antibody: It can be obtained according to the preparation method of HS-reduced HER2 antibody.

[0267] Among them, for the protein sequence of Atezolizumab (WHO Drug Information Vol. 28, No. 4, 2014), please refer to Figure 15 As shown, the protein sequences of Atezolizumab are SEQ ID NO:3 and SEQ ID NO:4.

[0268] Synthesis method of the conjugate antibody compound 30:

[0269]

[0270] Using the same method as for the synthesis of compound 8-4 and replacing compound 19, compounds 8-5 and 8-6 can be obtained:

[0271]

[0272]

[0273] Compounds 5-2 and 7-3 can be obtained in the same way:

[0274]

[0275] Using the same route method as for the synthesis of compound 30, the antibody compound 30-1 can be synthesized:

[0276]

[0277] Synthesis method of compound 8-2:

[0278]

[0279] Equivalent amounts of compound Val1 and compound 1A react in DMSO with HOBT, EDC, and DIPEA to form compound Val2. After treatment with TFA, compound Val3 is formed. Then, it continues to react with compound 6A under the action of HOBT, EDC, and DIPEA to form compound Val4, and deprotection gives compound Val5. Compound Val5 directly reacts with compound 4A to form compound Val6. Compound Val6 and compound 4 react under heating conditions to form compound 8-2.

[0280] Compounds 8-3 and 8-1 can be synthesized using the same method as for compound 8-2:

[0281]

[0282] The synthesis of compound 7-1 can be achieved using a method similar to that of compound 8-2:

[0283]

[0284] Compound VC-An4 (200 mg) was mixed with compound 13 (92 mg) and dissolved in 2 mL of DMSO. An equivalent amount of condensation reagents (HOBt, EDC, DIPEA) was added, and the mixture was reacted at room temperature for 12 hours. It was directly freeze-dried and purified by HPLC to obtain compound 14-1, 209 mg (73%), ESI-MS: m / z = 955.4 [M+H]+.

[0285] 100 mg of compound 14-1 was added to 2 mL of TFA / DCM (1:3 by volume). After the deprotection reaction was completed monitored by mass spectrometry, the reaction solution was distilled under vacuum to remove the solvent. 3 mL of DMF was added to the residue to dissolve it, 100 μL of TEA and 20 mg of thiouronium were added, and the reaction was carried out at room temperature until completion (monitored by MS). The reaction mixture was separated and purified by HPLC to obtain compound 7-1 (52 mg, yield 56%), ESI-MS: m / z = 897.7 [M+H] + 。

[0286] Synthesis of HER2 antibody conjugate compound 31:

[0287]

[0288] Compound BVA-3 was mixed with 1:3 TFA / DCM and stirred at room temperature for 8 hours. The solvent was removed by distillation under reduced pressure. The residue was dried under vacuum, dissolved in an appropriate amount of DMF, and 2 equivalents of TEA were added and mixed evenly. Then, an equivalent amount of succinic anhydride was slowly added at 10 °C in an ice-water bath, and stirring was continued at room temperature for 12 hours. After the reaction was completed, 10 times the amount of water was added to adjust the pH to 3, and the solid was precipitated by freezing at -20 °C. It was filtered and washed with water to obtain the crude product of compound 6-2. The pure product compound 6-2 was obtained by HPLC purification, ESI-MS: m / z = 532.3 [M+H]+.

[0289] 20 equivalents of compound 6-2 were added to an appropriate amount of DMSO, and an equivalent amount of HOBT, EDC, and DIPEA were added at 10 °C. The mixture was stirred and reacted for 2 hours. This mixed solution was slowly added to an aqueous solution containing 1 equivalent of HER2 antibody, and the reaction was carried out at room temperature naturally for 12 hours. The reaction solution was dialyzed through a 10K filter membrane to remove small molecules, and the obtained macromolecular antibody was washed with pure water multiple times. The washing solution was freeze-dried to obtain the conjugate antibody compound 31. The average conjugation degree (DAR = 4) was measured.

[0290] By the same method, when compound 6-2 was replaced with compound 8-5 or compound 8-6, conjugate antibody compounds 32 and 33 could be obtained:

[0291]

[0292] Synthetic route of antibody compound 34:

[0293]

[0294] Dissolve compound BVC-1 and p-nitrophenol in DMSO in equimolar amounts, add equimolar amounts of EDC and DIPEA, and stir at room temperature for 12 hours. Add the reaction product to ether and freeze it. Filter the precipitated solid to obtain BVC-2; dissolve BVC-2 in dry DMF, add equimolar amounts of K2CO3 and equimolar amounts of compound GY100, stir at room temperature for 12 hours, and purify the product by HPLC to obtain compound BVC-3. Perform a click reaction on compound BVC-3 and compound PIP-3, then remove the protecting group with TFA, and purify by HPLC to obtain compound BVC-4; under the action of a condensing agent, compound BVC-4 and compound 19 react to form compound BVC-5, and perform a click reaction with conjugate compound 21 to obtain conjugate antibody compound 34:

[0295]

[0296] The immune activator part of the new antibody compound 34 is compound GY161, which has strong anti-tumor activity. The antibody targeting the tumor releases the immune activator in a targeted manner, which will greatly enhance its anti-tumor effect.

[0297] The immune activator in the examples of the present invention can be replaced with a multifunctional immune activation small molecule, such as a trifunctional small molecule part containing lenalidomide / Osimertinb / SZU-160 at the same time (compound Tri-linker-1):

[0298]

[0299] Synthesis of compound Tri-linker-1:

[0300] Mix itaconic anhydride and compound NO3N3 in equimolar amounts in dry DMSO, add equimolar amounts of TEA, stir at 40 °C overnight, neutralize to pH 4 with HCl, and directly freeze-dry the reaction mixture to obtain compound ICO3N3. React compound ICO3N3 and lenalidomide in equimolar amounts in DMSO with a condensing agent (HOBt / EDC) for condensation reaction, and then purify by HPLC to obtain compound POMA-ICO3N3: ESI-MS: m / z = 572.20 [M+H] + . Dissolve compound POMA-ICO3N3 in DMSO, add equimolar amounts of mercaptoethylamine, seal and stir at room temperature overnight, and directly freeze-dry the reactant to obtain S-POMA-ICO3N3: ESI-MS: m / z = 649.20 [M+H]+ Dissolve 100 mg of compound S-POMA-ICO3N3 and 79 mg of compound Kyne-9291 in a 1:4 water / DMSO mixed solution (0.5 mL). Add 8 mg each of sodium L-ascorbate and anhydrous copper sulfate. The mixture is reacted at room temperature for 5 hours. After monitoring the reaction by LC-MS and completion of the reaction, the product is directly purified by HPLC to obtain 91 mg (51%) of compound Bi-linker. ESI-MS: m / z = 1172.50 [M+H] + Take 70 mg of compound Bi-linker and dissolve it in 1 mL of dry DMSO. Add 36 mg of compound SZU-160 and 1.1 equivalents of HOBt / EDC / DIPEA. The mixture is shaken and reacted overnight at room temperature. The reaction mixture is directly freeze-dried, dissolved in a small amount of isopropanol, and purified by HPLC to obtain 69 mg (yield 66%) of pure compound Tri-linker-1. ESI-MS: m / z = 1752.7 [M+H] + 。

[0301] Synthesis of antibody compound 35:

[0302]

[0303] Add 10% (by volume) DMSO to the DPBS solution of conjugate compound 21, add 1.5 equivalents of compound Tri-linker-1, and shake and react at 25 °C for 12 hours. Remove small molecules using a 20KD molecular filter membrane. Dissolve the antibody in DPBS to obtain a DPBS solution of conjugate antibody compound 35. The average conjugation degree of compound 35 measured by mass spectrometry is 6.

[0304] Referring to the synthetic technical method of compound Tri-linker-1, according to the following synthetic route, compound Tri-linker-2 is obtained:

[0305]

[0306]

[0307] Compound BVC-1 was reacted and condensed with an equivalent amount of compound SZU-163 using HOBt / EDC / DIPEA (equivalent amount) in dry DMSO. After deprotecting with TFA / DCM, compound BVC-T-2 was obtained by HPLC purification. Compound BVC-T-2 and compound 41 were dissolved in DMSO in equivalent amounts and further condensed using HOBt / EDC / DIPEA. Compound BVC-T-3 was obtained by HPLC purification. Compound BVC-T-3 was stirred with 6N hydrochloric acid at room temperature overnight and distilled to dryness under reduced pressure to obtain white solid compound BVC-T-4, which was dried in vacuo.

[0308] Compound BVC-T-4 and compound Bi-linker were mixed in DMSO in an equimolar ratio and subjected to a condensation reaction using HOBt / EDC / DIPEA as the condensing agent. After HPLC purification and LC-MS molecular confirmation, compound Tri-linker-2 was obtained: ESI-MS: m / z = 1993.20 [M+H] + . For the mass spectrometry identification of each intermediate, compound BVC-T-2: ESI-MS: m / z = 585.5 [M+H] + ; compound BVC-T-3: ESI-MS: m / z = 853.5 [M+H] + ; compound BVC-T-4: ESI-MS: m / z = 839.4 [M+H] + .

[0309] Synthesis of compound 41:

[0310]

[0311] Compound HO-N3 was dissolved in anhydrous THF, and a THF solution containing an equivalent amount of TsCl (p-toluenesulfonyl chloride) was slowly added dropwise. The mixture was stirred at room temperature for 30 minutes and refluxed for 2 hours. After cooling to room temperature, the solvent was removed by distillation under reduced pressure to obtain compound Ts-N3, which was an oily substance. ESI-MS: m / z = 270.10 [M+H] + . It was dissolved in dioxane for standby.

[0312] Compound 37 (5.3 g, 19 mmol) was dissolved in 100 mL of anhydrous dioxane. Potassium tert-butoxide (2.36 g, 20.9 mmol) was added. Under nitrogen protection, the reaction was carried out at 60 °C for 2 hours. A solution of compound Ts-N3 (5.1 g, 19 mmol) in anhydrous dioxane (10 mL) was slowly added dropwise. After the addition was complete, the temperature was raised to reflux and the reaction was carried out for 20 hours. The reaction solution was cooled to room temperature, filtered by suction, washed with ether, the filtrate was recovered, concentrated, and then separated by column chromatography (petroleum ether:ethyl acetate = 6:4) to obtain 3.6 g of compound 38 as a yellow oily liquid, with a yield of 51%. ESI-MS: m / z = 373.2 [M+H]+.

[0313] Compound 38 (3 g, 8 mmol) was added to 10 mL of 2N hydrochloric acid, and the mixture was heated to reflux. The reaction was monitored by LC-MS. After the reaction was complete, the solvent was removed by distillation under reduced pressure. The residue was dissolved in saturated Na2CO3 solution, and hydrochloric acid was slowly added dropwise until a solid precipitated. The white solid compound 39 was obtained by filtration, 0.85 g, with a yield of 62%. ESI-MS: m / z = 173 [M+H]+.

[0314] Compound 39 (0.8 g, 4.6 mmol) was mixed in anhydrous methanol (10 mL). Under cooling (5 - 10 °C), 3 mL of SOCl2 was slowly added dropwise, and the mixture was stirred naturally overnight under nitrogen protection. The solvent was removed by distillation under reduced pressure and vacuum-dried to obtain the hydrochloride of compound 40, with a yield of 97%. ESI-MS: m / z = 187.2 [M+H]+.

[0315] The hydrochloride of compound 40 (0.5 g, 2.2 mmol) was dissolved in 5 mL of DMSO. Equivalent amounts of succinic anhydride and 2 equivalents of TEA were added, and the mixture was stirred at 60 °C for 4 hours. The reaction product was directly freeze-dried. The resulting solid was dissolved in saturated Na2CO3 solution, and the pH was adjusted to 4 with hydrochloric acid to precipitate a solid. The solid was filtered and dried in vacuo to obtain compound 41, 0.42 g, with a yield of 67%. ESI-MS: m / z = 287.2 [M+H]+.

[0316] The synthesis and purification of antibody compound 36 were the same as those of compound 35 to obtain antibody compound 36. The average conjugation ratio (DAR = 6) was measured by mass spectrometry:

[0317]

[0318] Synthesis of N3-VC-T:

[0319]

[0320] BVC-T-2 and N3PEGOH were mixed in equimolar amounts in DMSO, and an equivalent amount of condensing agent (HOBt, EDC, DIPEA / DMSO) was added. The mixture was stirred at room temperature for 8 hours. The reaction mixture was freeze-dried, and the residue was dissolved in a small amount of methanol and separated and purified by HPLC to obtain N3-VC-T. ESI-MS: m / z = 800.5 [M+H]+.

[0321] Synthesis of MA-VC-T:

[0322]

[0323] The synthesis method was the same as that of N3-VC-T, except that N3PEGOH was replaced by MA-OH to obtain MA-VC-T. ESI-MS: m / z = 778.4 [M+H]+.

[0324] Synthesis of HO-VC-T:

[0325]

[0326] BVC-T-2 and succinic anhydride were mixed in an equimolar ratio and dissolved in DMF. 2 equivalents of TEA were added, and the mixture was stirred at 60 °C for 4 hours. The reaction mixture was distilled under reduced pressure (≤60 °C) to remove DMF. The residue was dissolved in saturated Na2CO3, filtered, and the clear liquid was adjusted to pH 4 with acetic acid and then filtered to obtain the pure product. ESI-MS: m / z = 685.4 [M+H]+.

[0327] Synthesis of SVC-T:

[0328]

[0329] BVC-T-2 and FPEGOH were mixed in equimolar amounts and dissolved in an appropriate amount of DMSO. An equivalent amount of condensing agent (HOBt, EDC, DIPEA) was added, and the mixture was stirred at room temperature for 8 hours. Then an equivalent amount of piperidine was added, and the mixture was stirred at 60 °C for 4 hours. The reaction mixture was freeze-dried, and the residue was dissolved in a small amount of methanol and separated and purified by HPLC to obtain NVC-T. ESI-MS: m / z = 774.4 [M+H]+.

[0330] NVC-T and an equimolar amount of SC-DIMI were dissolved in a dry appropriate amount of DMSO. 2 equivalents of TEA were added, and the reaction was carried out at room temperature for 12 hours. The reaction solution was directly freeze-dried and purified by HPLC to obtain SVC-T. ESI-MS: m / z = 816.3 [M+H]+.

[0331] Compounds N3-VC-T, MA-VC-T, HO-VC-T, and SVC-T can be specifically used for antibody or targeted drug conjugation, targeting intracellular TLR7 expression, degrading TLR7 agonists, and achieving the elimination of the proliferative effect of TLR7 agonists on such cells. For example, the synthesis of HER2 antibody conjugated with SVC-T, i.e., 37:

[0332]

[0333] To a 100 mL DPBS solution of 1 equivalent of HER2 antibody, 6 equivalents of SVC-T in 5 mL of DMSO solution were added. 12 equivalents of TEA were added to the formed mixture, and the reaction was shaken at room temperature for 12 hours. Small molecules were removed by dialysis to obtain 37, and the average conjugation degree (DAR) measured by mass spectrometry was 4.

[0334] Synthesis of SZU-164:

[0335]

[0336] 1 g of SZU-163 and 0.3 g of succinic anhydride were dissolved in 20 mL of dry DMSO, 1 mL of triethylamine (TEA) was added, and the reaction was stirred at room temperature for 12 hours. The mixture was lyophilized to remove the solvent, the residue was dissolved in water, and the pH was adjusted to 4 with hydrochloric acid. The product was precipitated, filtered, and dried to obtain SZU-164; 1 g, yield 77%, ESI-MS: m / z = 429.2 [M+H]+.

[0337] The above immune activation antibodies and their respective representative compounds provided in the embodiments of the present invention can be used in the preparation of anti-tumor drugs, antiviral drugs, immunomodulatory drugs, and / or preparations for eliminating target proteins.

[0338] The embodiments of the present invention also provide the application of the above immune activation antibodies in the preparation of anti-tumor drugs, antiviral drugs, immunomodulatory drugs, and / or preparations for eliminating target proteins.

[0339] Since the immune activation antibodies provided in the embodiments of the present invention have the effect of locally targeting and activating the immune system, when used in the preparation of anti-tumor drugs, antiviral drugs, immunomodulatory drugs, and / or preparations for eliminating target proteins, it can not only avoid the damage side effects of non-specific killing to normal tissues, but also has multiple effects such as activating target immune cells (such as T cells, B cells, NK cells, etc.), reversing inert immune cells (such as converting macrophages into M1 anti-tumor macrophages and increasing the M1 / M2 ratio), and converting immune cells into anti-tumor active immune cells (such as increasing the number of IFN-γ+CD8 cells), etc., and has good application prospects.

[0340] To enable those skilled in the art to clearly understand the above implementation details and operations of the present invention, and to significantly demonstrate the advanced performance of the immune-activating antibody and its application in the embodiments of the present invention, the following examples are used to illustrate the above technical solutions.

[0341] In the following examples, the HER2 antibody (InVivoMab anti-human / rat HER2 (neu)), PD-1 and PD-L1 antibodies (anti-mouse antibodies) were all purchased from BioXcell; dialysis was used for pure product treatment.

[0342] The preparation steps of the conjugate sample are as follows:

[0343] Take 20 μg of the sample to be tested in an EP tube, add 2 μL of GlycoBuffer 2 (10X), and then add ultrapure water to make the final volume of the system 20 μL. Subsequently, add 3 μL of PNGase F to each sample and react in a water bath at 37 °C for 48 h. Dilute the above sample 10-fold (final concentration 0.1 mg / mL), and test the sample to be tested according to the following detection parameters.

[0344] The antibody detection parameters and mass spectrometry conditions are as follows:

[0345] 1. Instrument and equipment

[0346]

[0347] 2. Reagents, test solutions

[0348]

[0349]

[0350] 3. Liquid chromatography conditions

[0351] (1) Mobile phase gradient

[0352] Time Phase A (aqueous solution containing 0.1% formic acid) Phase B (acetonitrile solution containing 0.1% formic acid) 1 min 80％ 20％ 2 min 10％ 90％ 4 min 10％ 90％ 4.1 min 80％ 20％ 7 min 80％ 20％

[0353] (2) Detection parameters

[0354] Parameter Parameter value Detection wavelength 214 nm Flow rate 0.3 mL / min Injection volume 20 μL Column temperature 80℃ Collection time 7 min Elution mode Fast gradient elution

[0355] 4. Mass spectrometry conditions

[0356] Parameter Parameter value Ion source ESI Scan mode TOF MS Scan range (Da) 1000-5000 Spray gas 45 psi Auxiliary heating gas 45 psi Curtain gas 30 psi Temperature 450℃ Desolvation voltage 300V

[0357] Detection of TLR7 activation of the compound in Example 1 (HEK-BlueTM Detection)

[0358] HEK-Blue™ hTLR7 cells in logarithmic growth phase (purchased from InvivoGen) were used. The growth medium (Gibco, C11995500BT, Invivo Gen, ant-nr) was discarded. An appropriate amount of PBS at 37°C (Hyclone, SH30256.01) was used to gently wash the cells twice, and the PBS was then discarded. 2 - 5 mL of PBS at 37°C was added, and the cells were incubated for 1 - 2 min. The cells were scraped off with a cell scraper and gently pipetted to disperse them into a single-cell suspension. A hemocytometer was used to count the cells and calculate the cell concentration. The cell suspension was adjusted to 2.5×10⁴ / 180 μL per well using HEK-Blue™ Detection solution (purchased from InvivoGen), and the cells were seeded in a 96-well cell culture plate. HEK-Blue™ hTLR7 cells were stimulated according to the compound or drug concentrations (e.g., 0.01 μM, 0.1 μM, 1 μM, 5 μM, 15 μM, 30 μM, 40 μM), and three replicates were set for each concentration. The cells were incubated at 37°C under 5% carbon dioxide for 6 - 16 h. After incubation, the absorbance value was read at a wavelength of 650 nm using a full-wavelength microplate reader (BioTek-Epoch). The results are as Figure 1 shown. The OD value on the vertical axis represents the degree of TLR7 activation, and the horizontal axis represents the concentration of the compound.

[0359] Detection of TLR7 agonist release effect of each immune activation antibody in Example 2

[0360] Each immune activation antibody was taken and added to the RPMI1640 medium of HER2-positive SKBR3 (10 5 cells) according to the concentrations (e.g., 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, 40 μM;). The cells were co-cultured for 12 hours, and each supernatant was taken out, and the activation effect of TLR7 was tested according to the method in Example 1. The results are as Figure 2 and Figure 3 shown.

[0361] Anti-tumor effect experiment of representative immune activation antibodies in Example 3

[0362] Murine Her2 + CT26 tumor cell line and model were prepared according to the reference (“In vivo properties of three human HER2 / neu-expressing murine cell lines in immunocompetent mice”, Lab Anim Sci, 1999 Apr; 49(2):179 - 88.).

[0363] Establishment of a mouse colorectal cancer tumor model: Collect Her2+ CT26 cells in the logarithmic growth phase by digestion and centrifugation, wash twice with PBS and then count. Adjust the cell concentration to 2.3×10 5 cells / mL. The mice used were 6-week-old SPF-grade BALB / c mice. Shave the hair on the right back of the mice with a hair clipper, draw 100 μL of cell suspension with a syringe, expel the air bubbles and inject it subcutaneously on the back of the mice. When the tumor diameter reaches 4 - 5 mm, start administering the drug.

[0364] Evaluation of the treatment effect of tumor-bearing mice and the anti-tumor effects of compounds 15-4 and 34: Randomly divide the tumor-bearing mice (BALB / c) into a Control group, a TLR7 agonist group, a HER2 antibody group, a TLR7 agonist + HER2 antibody mixture group (the equivalent ratio of the two is n:1), and an immune-activating antibody group, with 8 mice in each group. Prepare the injection according to the dosing dose (the solvent components are mixed: 5% DMSO; +40% PEG300; +5% Tween 80; +50% PBS). The dosing dose of the TLR7 agonist is 3 mg / kg, the HER2 antibody is 20 mg / kg, the TLR7 agonist and HER2 antibody mixture group is (3 mg / kg + 20 mg / kg), and the TLR7-HER2 conjugate antibody is 20 mg / kg. Inject around the tumor, with a volume of 100 μL each time. Administer the drug on the 5th, 11th, 17th, and 23rd days after tumor inoculation, for a total of 4 injections, around the tumor. At the same time as administering the drug, first measure the tumor with a vernier caliper and record the survival of the mice, for a total of 7 recordings. The calculation method for tumor volume is: 0.5×a×b 2 , where a is the major axis and b is the minor axis. When the tumor diameter of the mice reaches 2 cm, they should be sacrificed by cervical dislocation according to animal ethics, and the tumor tissue should be dissected to calculate the tumor volume and weight. The results are as Figure 4 and Figure 5 shown. Figure 4 The ordinate of Figure 5 is the tumor weight on the 25th day after drug administration,

[0365] Analysis of intratumoral immune cells in Example 4

[0366] The animals in Example 3 were euthanized 48 hours after drug administration on the 11th day, the tumor tissue was separated, and the ratio of M1 / M2 markers (MHC-II:CD206) in the tumor and the change of CD8+ IFN-γ+ T cells were analyzed by flow cytometry, with PBS or the original antibody as a control. The results are as Figure 6 and Figure 7 shown. Figure 6 is the ratio of M1 macrophages and M2 macrophages in the tumor tissue of each dosing group on the 11th day; Figure 7It is the relative value of the amount of CD8+IFN-γ+ T cells in the tumor tissue on the 11th day of each administration group.

[0367] Example 5 Detection of the inhibitory activity of drugs on tumor cells in vitro by CCK8 method

[0368] The immune activation antibody was directly mixed with HER2-expressing tumor cells and cultured for 48 hours, and the inhibitory activity on tumor cells was detected according to the standard CCK8 technique, as shown in Figure 8 and Figure 9 , and the results showed that in the absence of immune cell assistance, the antibody with a tumor-suppressing active small molecule and an immune agonist had a strong inhibitory effect on tumor cells.

[0369] Example 6

[0370] Detection of the activation effect of compounds SZU-164 and HO-VC-T, N3-VC-T, MA-VC-T, SVC-T on TLR7 (HEK-BlueTM Detection):

[0371] 1) Prepare HO-VC-T, N3-VC-T, MA-VC-T, SVC-T at concentrations of 0.1, 1, 10, 20 μM, and according to the method in Example 1, measure the activation results of these four compounds on TLR7, as shown in Figure 10 .

[0372] 2) Incubate SZU-164 and HO-VC-T, N3-VC-T, MA-VC-T, SVC-T with Cathepsin-B (10 μg, Cat.#: 10483-H08H, SinoBiological) in PBS at concentrations of 1 μM, 5 μM, 10 μM, 20 μM, 40 μM at room temperature for 12 hours; filter the filtrate through a 2K molecular membrane, and measure the activation effect of SZU-164 and each compound on TLR7 by the method in Example 1. The results show that in the presence of Cathepsin-B, the group containing Val-Cit (valyl-citrulline) (such as HO-VC-T and other compounds) can be degraded and lose the activation of TLR7, as shown in Figure 11 .

[0373] Studies have shown that when TLR7 is expressed in tumor cells, activating TLR7 will increase the number of tumor cells. The results of Example 6 illustrate that 1) in cells with normal TLR7 expression, HO-VC-T, N3-VC-T, MA-VC-T, and SVC-T activate the TLR7 pathway, which is beneficial for activating the immune system; 2) in tumor cells expressing TLR7, under the action of Cathepsin-B, HO-VC-T, N3-VC-T, MA-VC-T, and SVC-T are inactivated, which is beneficial for selectively activating the immune microenvironment without increasing the number of tumor cells. Similarly, other antibodies or compounds containing Val-Cit in the present invention can be degraded by the action of Cathepsin-B, and according to the small molecule products after degradation, the activation effect on TLR7 can be generated or lost.

[0374] The schematic diagram of the degradation of TLR7 agonists by the enzyme (Cathepsin-B) represented by HO-VC-T is as Figure 12 shown.

[0375] Example 7.

[0376] Experimental study on the TLR7 pathway activation function of compounds SZU-128, GY209, 5-2-1, 5-2-2, 17-1, and 29-1

[0377] 1) Compounds SZU-128, GY209, 5-2-1, 5-2-2, 17-1, and 29-1 were respectively incubated with Cathepsin-B (10 μg, Cat.#: 10483-H08H, SinoBiological) in PBS at concentrations of 0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 40 μM at room temperature for 3 hours; the filtrate was obtained by filtering through a 2K molecular membrane, and the TLR7 activation effects of each compound in the filtrates of each group were measured by the method of Example 1, with R848 as the standard positive control. Among them, the Cathepsin enzyme catalyzes the cleavage and release of immune agonists as Figure 16 shown, and the TLR7 activation effects of each compound under the action of Cathepsin-B enzyme are shown in Figure 17 .

[0378] The results show that under the action of Cathepsin-B, groups containing Val-Cit or Val-Ala (valyl-citrulline or valyl-alanine) can be degraded to release TLR7 agonists and activate TLR7.

[0379] 2) Prepare three groups with concentrations of 1 μM, 0.1 μM, and 0.01 μM for each of the compounds SZU-128, GY209, 5-2-1, 5-2-2, 17-1, 29-1, and SZU-161 respectively. Directly follow the experimental method steps in Example 1 to obtain the activation effect of each compound on TLR7 without the action of Cathepsin-B enzyme. R848 is used as the standard positive control, see Figure 18 . It can be seen that in the absence of Cathepsin-B, only the compounds (GY209, SZU161, SZU-128, and R848) without the Val-Cit or Val-Ala (valyl-citrulline or valyl-alanine) group can activate TLR7.

[0380] Example 8.

[0381] Experiment on the inhibitory effect of antibody 17-1 and antibody 29-1 on tumor cells

[0382] Add each compound to the corresponding tumor cell culture medium according to the concentration groups (0.6 μM, 1.8 μM, 5.5 μM, 16.6 μM, 25 μM, 50 μM) (10 5 cells), and after co-incubating for 3 hours, add hPBMC with 20 times the number of tumor cells, and continue to co-incubate for 48 hours. Test the cell inhibition rate according to the standard CCK8 method, as Figure 19 , Figure 20 shown. It can be seen that the new antibodies 17-1 and 29-1 significantly improve the effect of immune suppression of tumor cells.

[0383] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

Claims

1. An immune-activating antibody, characterized in that, The immune activation-type antibody is Compound 15-1, Compound 17, Compound 22-2, Compound 24-2, Compound 15-4, Compound 28, Compound 30, Compound 31, Compound 34 or Compound 35. The structural formulas of Compound 15-1, Compound 17, Compound 22-2, Compound 24-2, Compound 15-4, Compound 28, Compound 30, Compound 31, Compound 34 and Compound 35 are as follows respectively: 。 2. The immune-activating antibody according to claim 1, characterized in that: The compound for providing the conjugate chain in Compound 15-1 is Compound 7-1; The compound for providing the conjugate chain in Compound 17 is Compound 5; The compound for providing the conjugate chain in Compound 22-2 is Compound 7-5; The compound for providing the conjugate chain in Compound 24-2 is Compound 5A-GY102; The compound for providing the conjugate chain in Compound 28 is Compound 27; The compound for providing the conjugate chain in Compound 30 is Compound 8-4; The compound for providing the conjugate chain in Compound 31 is Compound 6-2; The compound for providing the conjugate chain in Compound 35 is Compound Tri-linker-1; The structural formulas of Compound 5, Compound 6-2, Compound 7-1, Compound 7-5, Compound 8-4, Compound 27, Compound 5A-GY102 and Compound Tri-linker-1 are as follows respectively: 。 3. The immune-activating antibody according to claim 2, characterized in that: The intermediate compound for providing Compound tri-Linker-1 is Compound Bi-linker; The structural formula of Compound Bi-linker is as follows: 。

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