CN113663067A - Immune activation type antibody and application thereof - Google Patents

Immune activation type antibody and application thereof Download PDF

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CN113663067A
CN113663067A CN202110984030.0A CN202110984030A CN113663067A CN 113663067 A CN113663067 A CN 113663067A CN 202110984030 A CN202110984030 A CN 202110984030A CN 113663067 A CN113663067 A CN 113663067A
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靳广毅
王竹林
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Shenzhen Kangjuzheng Medical Technology Co ltd
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Abstract

The invention belongs to the technical field of medicines, and particularly relates to an immune activation type antibody and application thereof. The immune activation type antibody provided by the invention comprises an antibody and an immune activator which are coupled through a coupling chain, and can be used for preparing antitumor drugs, antiviral drugs, immunoregulation drugs and/or preparations for eliminating target proteins. The invention couples the antibody and the immune activator through the coupling chain, can form a series of immune activation type antibodies targeting specific tissues, focuses and targets, can achieve the effect of locally targeting and activating immunity, solves the negative effect of TLR activation on normal tissues, and has good specific immune regulation and treatment effects.

Description

Immune activation type antibody and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an immune activation type antibody and application thereof.
Background
Toll-like receptors (TLRs) belong to the classical natural immune system of animals. There are 11 members of mammalian and human TLR receptors, such as TLR2, TLR3, TLR4, TLR7, TLR9, etc.; each TLR can be activated by a specific ligand to resist the invasion of various microorganisms such as bacteria, viruses and the like and resist tumors. Wherein TLR7 can be activated by synthetic small molecule immune activator. Although the activation strength of TLR7 is directly related to the ability of immune cells (e.g., dendritic cells, macrophages, T cells, B cells, and NK cells, etc.) to kill tumor cells, non-specific killing also tends to cause the adverse effects of immune storm damage to normal tissues.
Therefore, it is one of the major issues of current research to reduce or avoid the side effects of immune activators on normal tissues when activating the immune system.
Disclosure of Invention
The invention aims to provide an immune activation type antibody and application thereof, and aims to solve the technical problems that the existing immune activator easily causes damage and side effects on normal tissues when activating an immune system and the like.
In order to achieve the above object, according to a first aspect of the present invention, there is provided an immune-activated antibody comprising an antibody and an immune activator, wherein the antibody is coupled to the immune activator via 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):
Figure BDA0003229949260000011
Figure BDA0003229949260000021
the immune activator and the antibody are coupled through the coupling chain, so that a series of immune activated antibodies targeting specific tissues, focuses and targets can be formed, the function of locally targeting and activating immunity can be achieved, and the negative influence of TLR activation on normal tissues is avoided. Experiments prove that the immune activation type antibody can guide the immune activator in the immune activation type antibody to play a role in a required site or environment (such as a tumor microenvironment), has multiple functions of activating target immune cells (such as T cells, B cells, NK cells and the like), reversing inactive immune cells (such as macrophages are converted into M1 type anti-tumor macrophages, the proportion of M1/M2 and the like is improved), converting the immune cells into immune cells with anti-tumor activity (such as the increase of the cell number of IFN-gamma + CD8 and the like), and has better specific immune regulation and treatment effects.
In a second aspect, the invention provides an application of an immune activated antibody in preparation of anti-tumor drugs, antiviral drugs, immunomodulatory drugs and/or preparations for eliminating target proteins.
Because the immune activation antibody provided by the invention has the function of locally targeting activation immunity, the immune activation antibody can be used for preparing anti-tumor drugs, antiviral drugs, immunoregulation drugs and/or target protein elimination preparations, not only can avoid the side effect of nonspecific killing on the damage of normal tissues, but also has various functions of activating target immune cells (such as T cells, B cells, NK cells and the like), reversing inert immune cells (such as macrophages are converted into M1 type anti-tumor macrophages, the proportion of M1/M2 and the like), converting the immune cells into immune cells with anti-tumor activity (such as the increase of the cell number of IFN-gamma + CD8 and the like), and has good application prospect.
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FIG. 1 shows the method and results of HEK-Blue hTLR7 assay for compounds of example 1 of the present invention;
FIGS. 2 and 3 are graphs showing the results of the test of the release effect of the TLR7 agonist released from the compound in example 2 of the present invention;
FIG. 4 shows the results of the measurement of tumor weight on day 25 after the administration of compound 15-4 in example 3 of the present invention;
FIG. 5 shows the results of measuring the change in tumor volume of compound 34 administered for 25 days in example 3 of the present invention;
FIG. 6 shows the results of measurement of macrophage regulating effects of compound 28, compound 30 and compound 15-4 on the tumor microenvironment in example 4 of the present invention (relative values of M1/M2 markers MHC-ClassII/CD 206);
FIG. 7 shows the results of detecting the relative change of IFN-. gamma. + CD8 cells in tumor tissues by the compounds 28, 30 and 15-4 in example 4 of the present invention;
FIG. 8 shows the results of examining the inhibitory effect of compound 35 on SK-BR-3 cells in example 5 of the present invention;
FIG. 9 shows the results of the measurement of the inhibitory effect of Compound 36 on A549 cells in example 5 of the present invention;
FIG. 10 shows the effect of each compound of example 6 of the present invention on the activation of HEK-Blue hTLR7 cells;
FIG. 11 shows the inactivation effect of each compound of example 6 of the present invention on HEK-Blue hTLR7 cells;
FIG. 12 is a schematic diagram showing the degradation of an enzyme (Cathepsin-B) acting on a TLR7 agonist, represented by HO-VC-T in an embodiment of the invention;
FIG. 13 general formula for an immunoactivating antibody;
FIG. 14 protein sequence of Trastuzumab Trastuzumab;
FIG. 15 protein sequence of Atezolizumab monoclonal antibody;
FIG. 16 is a schematic diagram of the release of an immune agonist by enzymatic cleavage of Cathepsin;
FIG. 17 is a graph showing the effect of TLR7 activation by the action of the Cathepsin-B enzyme on various compounds;
FIG. 18 is a graph of the effect of compounds on TLR7 activation in the absence of Cathepsin-B action;
FIG. 19 is a graph showing the inhibitory effect of antibody 29-1 on human H1299 cells;
FIG. 20 is a graph showing the inhibitory effect of antibody 17-1 on human MDA-MB-453 cells.
Detailed Description
In order to make the objects, 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, and the embodiments described below are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention. Those whose specific conditions are not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the description of the present invention, the term "and/or" describing an association relationship of associated objects means that there may be three relationships, for example, a and/or B, may mean: a is present alone, A and B are present simultaneously, and B is present alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
In the description of the present invention, "at least one" means one or more, "a plurality" means two or more. "at least one of the following" or similar expressions refer to any combination of these items, including any combination of the singular or plural items. For example, "at least one (a), b, or c", or "at least one (a), b, and c", may each represent: a. b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, wherein a, b, and c can be single or multiple respectively.
It should be understood that the weight of the related components mentioned in the embodiments of the present invention may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, it is within the scope of the disclosure that the content of the related components is scaled up or down according to the embodiments of the present invention. Specifically, the weight described in the embodiments of the present invention may be a unit of mass known in the chemical field such as μ g, mg, g, kg, etc.
In addition, unless the context clearly uses otherwise, an expression of a word in the singular is to be understood as including the plural of the word. The terms "comprises" or "comprising" are intended to specify the presence of stated features, quantities, steps, operations, elements, portions, or combinations thereof, but are not intended to preclude the presence or addition of one or more other features, quantities, steps, operations, elements, portions, or combinations thereof.
The embodiment of the invention provides an immune activation type antibody, which comprises an antibody and an immune activator, wherein the antibody and the immune activator are coupled through a coupling chain, and the coupling chain comprises at least one of a structure shown as a formula (A), a structure shown as a formula (B), a structure shown as a formula (C) and a structure shown as a formula (D):
Figure BDA0003229949260000041
according to the embodiment of the invention, the immune activator and the antibody are coupled through the coupling chain, so that a series of immune activated antibodies targeting specific tissues, focuses and targets can be formed, the function of locally targeting and activating immunity can be achieved, and the negative influence of TLR activation on normal tissues is solved. Experiments prove that the immune activation type antibody can guide the immune activator in the immune activation type antibody to play a role in a required site or environment (such as a tumor microenvironment), has multiple functions of activating target immune cells (such as T cells, B cells, NK cells and the like), reversing inactive immune cells (such as macrophages are converted into M1 type anti-tumor macrophages, the proportion of M1/M2 and the like is improved), converting the immune cells into immune cells with anti-tumor activity (such as the increase of the cell number of IFN-gamma + CD8 and the like), and has better specific immune regulation and treatment effects.
The immune activation type antibody provided by the embodiment of the invention is different from other immune activation type antibodies in that the immune activation type antibody provided by the embodiment of the invention does not stimulate immune cells expressing TLR7 or TLR8 in a common immune cell environment, and does not produce immune cytokines; the immune system and immune cells are activated only in the cellular environment of the antibody target (e.g., tumor cell environment), or in an antibody-directed environment containing proteases (e.g., Cathepsin-B).
The general structural formula of the immune activation antibody provided by the embodiment of the invention is shown in figure 13.
In the structural general formula, the "coupling chain" is composed of a "first connecting chain", a "second connecting chain" and a "degradable chain", wherein the "first connecting chain" and the "second connecting chain" are structures conventionally used in the art for connection, and are not described herein again; the structure of the "degradable chain" portion is the structure represented by formula (A), and the structure may be replaced by the structure represented by formula (B), formula (C) and/or formula (D), and the structural formula of the corresponding immune-activating antibody may be changed, and the changed formula is not shown here. For example, some specific structures (Valine-alanine linker: Val-Ala) of the structure of formula (B) can be obtained by replacing the degradable chain moiety among the specific structures (Valine-citrulline linker: Val-Cit) of formula (A), as follows:
Figure BDA0003229949260000051
the immune activation type antibody provided by the embodiment of the invention mainly comprises three parts: antibodies, conjugate chains and immune activators. The three sections are specifically described below:
coupling chain:
the coupling chain in the embodiment of the present invention may be at least one selected from the group consisting of a degradable chain containing a Cathepsin-B region, an alkyl group, an alkoxy group, a nitrogenous alkyl group, a heterocycle, and a specific functional chain.
In some embodiments, the coupling chain comprises at least one of compound 5, compound 5-1, compound 5-2, compound GY209, compound 6-1, compound 6-2, compound 6-3, compound 7-1, compound 7-2, compound 7-3, compound 7-4, compound 7-5, 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, compound Tri-linker-2, the structural formulas are respectively as follows:
Figure BDA0003229949260000061
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
Figure BDA0003229949260000062
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
Figure BDA0003229949260000063
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
Figure BDA0003229949260000071
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
Figure BDA0003229949260000081
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
Figure BDA0003229949260000082
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
Figure BDA0003229949260000083
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
Figure BDA0003229949260000084
4-((2S,5S)-5-isopropyl-17-isothiocyanato-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl
6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carboxylate
Figure BDA0003229949260000091
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
Figure BDA0003229949260000092
4-((2S,5S)-5-isopropyl-17-isothiocyanato-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl
6-amino-2-butoxy-9-(cyanomethyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxylate
Figure BDA0003229949260000093
(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
Figure BDA0003229949260000101
(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
Figure BDA0003229949260000102
(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
Figure BDA0003229949260000103
4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl
6-amino-2-butoxy-9-(cyanomethyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxylate
Figure BDA0003229949260000104
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
Figure BDA0003229949260000111
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
Figure BDA0003229949260000112
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
Figure BDA0003229949260000113
(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
Figure BDA0003229949260000114
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
Figure BDA0003229949260000121
(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
Figure BDA0003229949260000122
(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
Figure BDA0003229949260000123
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
Figure BDA0003229949260000131
4-(17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl
6-amino-2-butoxy-9-((1-cyclooctyl-1H-1,2,3-triazol-4-yl)methyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxylate
Figure BDA0003229949260000132
4-(17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl
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
Figure BDA0003229949260000141
4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl
(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
Figure BDA0003229949260000142
(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
Figure BDA0003229949260000143
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
Figure BDA0003229949260000151
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
Figure BDA0003229949260000152
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。
the embodiment of the invention also provides a series of intermediate compounds for synthesizing the specific coupled chain compounds, which are at least one of the compounds 18-2, 5A, 102-3, VC-An4, Val5, Val6, VC100, VCB-2, POMA-ICO3N3, S-POMA-ICO3N3, Bi-Linker, BVC-T-2, HO-VC-T, N3-VC-T and MA-VC-T, SVC-T, wherein the structural formulas are respectively as follows:
Figure BDA0003229949260000161
4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl
6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carboxylate
Figure BDA0003229949260000162
4-(17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl(4-nitrophenyl)carbonate
Figure BDA0003229949260000163
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
Figure BDA0003229949260000171
(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
Figure BDA0003229949260000172
(S)-N-(4-(aminomethyl)phenyl)-2-((S)-14-azido-2-isopropyl-4-oxo-6,9,12-trioxa-3-azatetradecanamido)-5-ureidopentanamide
Figure BDA0003229949260000173
4-nitrophenyl(4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl)carbamate
Figure BDA0003229949260000174
(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
Figure BDA0003229949260000181
(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
Figure BDA0003229949260000182
N1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-2-methylenesuccinamide
Figure BDA0003229949260000183
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
Figure BDA0003229949260000184
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
Figure BDA0003229949260000191
(S)-N-(4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamide
Figure BDA0003229949260000192
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
Figure BDA0003229949260000193
(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
Figure BDA0003229949260000194
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
Figure BDA0003229949260000201
(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。
immune activator:
the immune activator provided by the embodiment of the invention can be an immune activator conventional in the field, and includes but is not limited to at least one of a TLR7 agonist, a TLR8 agonist, a STING agonist and a small molecule immune activator. In some embodiments, the small molecule immune activator comprises at least one of compound 1, compound 2, compound 3, compound 4, having the following structural formulas:
Figure BDA0003229949260000202
antibody:
the antibodies provided by the embodiments of the present invention can be any antibody that targets a pathogen, i.e., an antigen. In some embodiments, the antigen is selected from the group consisting of HER2, HER3, PD-L1, PD-1, TIGIT, TROP2, EGFR, MUC1, LIV-1, MUC16, CEACAM1 and subtypes thereof, 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, GSK 1, survivin, MAGE-3.1, MAGE-10. A1, gp209-2 1-A, NANAN 17.A2, KOC1, CO1, DEBTCLK 1, MPGE H1, MAGE-10, MAGE-1, GPCRK 1, PSRCA 1, PSRCD 1, PSRCA 1, PSRCD 1, PSRCA 1, PSRCD 1, PSRCA 1, PSRCK 1, PSRCD 1, PSRCK 1, PSRC, At least one of FLT, CD, BCL, CD, Smoothened, GD, EZH, MTH, KRAS, c-MYC, hCA IX, hCA XII, BRD, HDAC, NYC, TOPK, BCMA, PI3, PDGFR, TIM, OX, CD, SIRP-alpha, CD122, CD160, TGF-beta, HIF-1/2 alpha, PSGL-1, Frizled-7, SLC4A, CCR, CXCR, CCL, CXCL, CD79, Nectin-4, CD117, PSMA, NKG2, Claudin18.2, MG, ROR, FGFR, WNT2, WNT3, WNT5, WNT9, LITT 7, HGF, LIB, ARF, ANG, ARG, ARRG, RAKE, HRR, HRACK, HREP, HRACK, CRP, HRTF, CRH-6, HRTF, CRAS, HRSP, HRG, HRRB, HRR, HRRB, and KL-6. The antibody of the pathogens can lead the immune activator in the immune activation type antibody to a corresponding target site by targeting the pathogens, thereby achieving the effect of locally and targetedly activating immunity.
Immune-activating antibodies:
according to the change of the coupling chain, a series of immune activation antibodies with different structures can be obtained, including the conjugate shown as the formula (I)
Figure BDA0003229949260000211
A pair shown as a formula (I-1)Things linked together
Figure BDA0003229949260000212
A conjugate represented by the formula (I-2)
Figure BDA0003229949260000213
A conjugate represented by the formula (I-3)
Figure BDA0003229949260000214
A conjugate represented by the formula (I-4)
Figure BDA0003229949260000221
A conjugate of formula (II)
Figure BDA0003229949260000222
A conjugate represented by the formula (II-1)
Figure BDA0003229949260000223
A conjugate represented by the formula (II-2)
Figure BDA0003229949260000224
A conjugate represented by the formula (II-3)
Figure BDA0003229949260000225
A conjugate represented by the formula (II-4)
Figure BDA0003229949260000231
A conjugate of formula (III)
Figure BDA0003229949260000232
A conjugate represented by the formula (III-1)
Figure BDA0003229949260000233
A conjugate represented by the formula (III-2)
Figure BDA0003229949260000234
A conjugate represented by the formula (III-3)
Figure BDA0003229949260000235
Is represented by the formula (III-4)Conjugates
Figure BDA0003229949260000241
A conjugate of formula (IV)
Figure BDA0003229949260000242
A conjugate represented by the formula (IV-1)
Figure BDA0003229949260000243
A conjugate represented by the formula (IV-2)
Figure BDA0003229949260000244
A conjugate represented by the formula (IV-3)
Figure BDA0003229949260000245
A conjugate represented by the formula (IV-4)
Figure BDA0003229949260000251
And n is greater than 0.
Some more typical immune-activating antibodies are listed below, including at least one of compound 15, compound 15-1, compound 15-2, compound 17, compound 21, compound 22-2, compound 24-2, compound 15-3, compound 15-4, compound 15-5, compound 24-3, compound 28, compound 29, compound 30-1, compound 31, compound 32, compound 33, compound 34, compound 35, compound 36, compound 37, and having the following structural formulas:
Figure BDA0003229949260000252
Figure BDA0003229949260000261
Figure BDA0003229949260000271
Figure BDA0003229949260000281
Figure BDA0003229949260000291
Figure BDA0003229949260000301
Figure BDA0003229949260000311
the synthesis method of the coupling chain, the small molecule immune agonist and the corresponding immune activation antibody provided by the embodiment of the invention comprises the following steps:
the synthesis method of the compound 1 comprises the following steps:
2.37 g of Pro-1, the compound, and 1.2 g of bromopropionitrile are dissolved in 50mL of acetonitrile, and 1.5 g of K are added2CO3The mixture was stirred at room temperature for 12 hours. After filtration, 2mL of iodotrimethylsilane was added and the mixture was stirred at room temperature for 10 hours. 5mL of saturated NaHCO was added3And distilling the solution under reduced pressure to remove the solvent, separating by silica gel column chromatography (methanol: dichloromethane: 1: 10 by volume), collecting the product solution, and concentrating under reduced pressure to remove the solvent to obtain a solid product, namely the compound 1.
Figure BDA0003229949260000312
The synthesis methods of compound 2 and compound 3 are substantially the same as the synthesis method of compound 1, except that compound 2 is synthesized by replacing compound Pro-1 with compound 9; when compound 2 was synthesized, compound Pro-1 was replaced with compound 10. Wherein the structural formula of the compound 9 is
Figure BDA0003229949260000313
The structural formula of the compound 10 is
Figure BDA0003229949260000314
Synthesis of representative compound 15 of formula III exemplified by HER2 antibody:
Figure BDA0003229949260000321
compound 11(650mg) and compound 4(270mg) were dissolved in 5mL of DMF, 0.5mL of TEA was added, and the mixture was stirred at room temperature for 10 hours. The mixture was poured into 50mL of water and the solid product compound 12 precipitated by centrifugation and purified by HPLC to give compound 12(498mg, 65%) ESI-MS: M/z 767.3[ M + H ] +.
In the same manner, compound 1 is substituted for compound 4 to give 12-2:
Figure BDA0003229949260000322
compound 12(200mg) was added directly to 10mL of TFA/DCM (1:3), stirred at room temperature for 8 hours, the solvent was evaporated under reduced pressure, and vacuum-dried; 50mL of DMSO, Compound 13(80mg) was added. 36mg HOBT, 50mg EDC and 120. mu.L DIPEA were added to the solution and reacted overnight at room temperature, and the reaction was monitored by LC-MS. After the reaction was complete, purification by HPLC gave compound 14(119mg) as a white solid in 47.7% yield. ESI-MS, M/z 956.5[ M + H ] +.
Compound 14(100mg) was added to TFA/DCM (1:3) (2mL) and stirred at room temperature for 8 h. Removing the solvent under reduced pressure to a dry solid; dissolved in 5mL of DMSO, and thiodiimidazole (181mg) and 300. mu.L of triethylamine were added. The reaction was carried out at room temperature for 12 hours. The reaction was lyophilized to give crude product, which was purified by HPLC to give Compound 7(44mg, 47%). ESI-MS, M/z 898.4[ M + H ] +.
Figure BDA0003229949260000331
100mg of HER2 antibody (deglycosylated molecular weight: 145531), Compound 7(12mg), and triethylamine (5. mu.L) were dissolved in a solvent of DMSO and pure water (1: 10 volumes), reacted at 10 ℃ with shaking for 10 hours, and filtered through a 10K filter. Eluting to obtain a conjugated antibody compound 15; the DAR (drug/antibody, ratio) value was 5.98 as determined by mass spectrometry.
Referring to the synthesis of compound 15, substituting compound 1 for compound 4 gives compounds 7-2:
Figure BDA0003229949260000332
coupling synthesis of reference compound 15, substitution of compound 7 with compound 7-1 gives the analog compound 15-1:
Figure BDA0003229949260000341
further conjugation of HER2 antibody with compound 7-3 gave compound 15 analogue compound 15-2:
Figure BDA0003229949260000342
the synthetic route of compound 7-3 is as follows:
Figure BDA0003229949260000351
replacing BNCOOH with succinic anhydride, and reacting with compound VC100 to directly obtain compound 6-3:
Figure BDA0003229949260000352
the synthesis procedure of compound 5-1 is as follows:
Figure BDA0003229949260000361
dissolving 1 micromole of compound val1 and an equivalent amount of compound 16 in 3mL of DMSO, stirring at room temperature for 8 hours, freeze-drying to remove DMSO, adding 1mL of TFA/DCM (1:3 volume), shaking at room temperature for 2 hours, vacuum-drying to remove the solvent, and purifying the residue by HPLC to obtain compound MM-VC.
300mg of compound MM-VC was dissolved in 2mL of DMSO by mixing with an equivalent amount of HOBT, EDC and DIPEA, and reacted for 2h with a shaker at room temperature. 142mg of compound BNMA was added and the reaction was continued overnight. The reaction mixture was lyophilized, 1mL of TFA/DCM (1:3 vol.) was added and the reaction was stirred at room temperature for 2h, then the solution was removed under reduced pressure and the residue was purified by HPLC to give the compound MM-VCA-NH2, 238mg, 65% yield, MS: ESI-MS: M/z 572.5[ M + H ]]+。
Separately, 1. mu. mol of Compound 1 and an equivalent amount of Compound 4A were dissolved in 2mL of DMSO, an equivalent amount of TEA was added thereto, and the mixture was stirred at room temperature for 12 hours and reacted at 40 ℃ for 1 hour. Freeze drying to obtain crude product of compound 4A-1; dissolving the crude product in dry DMSO, adding MM-VCA-NH2 with equivalent weight, stirring at room temperature for 12H, and purifying by HPLC to obtain compound 5-1 with yield of 43%, ESI-MS: M/z 860.4[ M + H ] of]+。
Synthesis of Compound 6-1:
Figure BDA0003229949260000371
500mg of the compound FVC-1 and an equimolar amount of a mixed condensing agent (HOBT, EDC, DIPEA) were dissolved in 20-fold by weight of DMSO, stirred at room temperature for 2 hours, and an equivalent amount of the compound BNMA was added to continue the reaction at room temperature overnight. The reaction mixture was directly freeze-dried, and the residue was dissolved in methanol and purified by HPLC to obtain 453mg, 75% pure compound FVC-2, ESI-MS: M/z 601.4[ M + H ] +.
Mixing 200mg of compound 4 and an equimolar amount of compound 4A in 2mL of DMSO, adding 2 equivalents of TEA, shaking at room temperature for 8 hours, adding an equivalent amount of compound FVC-2, continuing the reaction at room temperature overnight, adding 4 times of equivalent amount of piperdine, and shaking at room temperature for 6 hours. The mixture was freeze-dried. Dissolving in methanol, and purifying by HPLC to obtain compound VC-An4 with yield of 28%, ESI-MS (ESI-MS) with M/z 666.30[ M + H ]]+。
100mg of VC-An4 compound was dissolved in 2mL of DMSO, An equivalent of TEA was added, An equivalent of Succinic anhydride was added, and the mixture was reacted with shaking at 40 ℃ overnight. The mixture was freeze dried, 2mL of water/methanol (1: 1) was added, pH was adjusted with acetic acid 5 and HPLC was used directly to purify the mixture to give 6-1, 94mg (82% yield) of the compound ESI-MS: M/z 766.62[ M + H ], (M + H) ]]+。
Synthesis of representative compound 17 of formula I:
Figure BDA0003229949260000381
directly adding compound 12-2(100mg) into 10mL of TFA/DCM (1:3), stirring at room temperature for 8 hours, evaporating the solvent under reduced pressure, and vacuum-drying; dissolved in 5mL of DMSO, and 30. mu.L of triethylamine and Compound 16(45mg) were added. The mixture was stirred at room temperature for 10 hours. Purification by HPLC afforded Compound 5(93 mg). ESI-MS, M/z 861.4[ M + H ] +.
100mg of HS-reduced HER2 antibody, Compound 5(12mg), and triethylamine (5. mu.L) were dissolved in 5mL of purified water, reacted at 10 ℃ with shaking for 10 hours, and filtered through a 10K filter. Eluting to obtain a coupled antibody compound 17; the DAR (drug/antibody, ratio) value was 4 as determined by mass spectrometry.
Preparation of HS-reduced HER2 antibody: the HER2 antibody was dialyzed by dialysis to remove various additives, dissolved in DPBS (15mg/mL), and adjusted to pH 7.0 with a 5mM EDTA solution. 5 equivalents of TCEP solution (5 mM TCEP in water) were added and the mixture was reduced at room temperature for 2 hours. Removing small molecules with 10KD filter membrane, eluting antibody with pure water, and vacuum freeze drying to obtain reduced antibody.
Synthesis of antibody 17-1:
Figure BDA0003229949260000391
15-fold equivalents of 5-2-2(10mg) and 0.1mL of TEA were dissolved in 0.2mL of DMSO and added to 100mg of DPBS (5mL) solution of HS-reduced trastuzumab. The mixture was reacted for 10 hours with shaking at 20 OC. Ultrafiltering with 20K filter membrane to remove small molecules, and stirring in an open container at room temperature for 6 hr to obtain new antibody 17-1 of Her2 antibody coupled with 5-2-2; the coupling ratio (ADR value) was 2 by mass spectrometry.
Preparing HS-reduced trastuzumab: is prepared according to the preparation method of HS-reduced HER 2.
As shown in FIG. 14, the protein sequence of Trastuzumab Trastuzumab (WHO Drug Information Vol.24, No.2,2010) is shown in SEQ ID NO:1 and SEQ ID NO: 2.
Synthesis of compound 8:
Figure BDA0003229949260000401
compound 12-2(100mg) was directly added to 10mL of TFA/DCM (1:3), stirred at room temperature for 8 hours, the solvent was evaporated under reduced pressure, dried under vacuum, dissolved in 5mL of DMSO, and 30. mu.L of triethylamine was added. Compound 19(30mg), HOBT (27mg), EDC (39mg), DIPEA (68. mu.L) were reacted at room temperature overnight. The reaction was monitored by LC-MS. After the reaction was completed, purification was performed by HPLC to obtain Compound 8(63.6mg, yield 55.7%) as a white solid. ESI-MS, M/z 883.3[ M + H ] +.
Synthesis of representative compound 22 of formula IV:
Figure BDA0003229949260000411
activated ester 20(20eq) of DBCO-acid and HER2 antibody (1eq) were mixed in 10% DMSO-containing purified water and reacted with shaking at 10 ℃ for 12 hours. Mass spectrometry detection was complete for HER2 antibody response. The reaction mixture was freed of small molecules using a 10kD molecular filter and the conjugate compound 21 was eluted with DPBS solution (degree of coupling 6 was measured). The DPBS solution of compound 21 was added with 10% DMSO by volume, added with 1.5 equivalents of compound 8, reacted at 25 ℃ with shaking for 12 hours, and small molecules were removed with a 20KD molecular filter. The DPBS dissolves the antibody to give a solution of DPBS conjugated to antibody compound 22. The average degree of coupling of compound 22 was 6 as determined by mass spectrometry.
Synthesis of Compound 22-2:
Figure BDA0003229949260000421
mixing and dissolving an equivalent amount of compound VCB-1 and compound GY100 in DMF, adding 2 equivalents of K2CO3And reacted with room temperature overnight. Adding 10 times of water, and stirring uniformly to obtain a precipitate. The precipitate was purified by filtration using 1: separating with 10 methanol/DCM silica gel chromatography to obtain pure compound VCB-2, ESI-MS, M/z-723.7 [ M + H ]]+. The compound VCB-2 is subjected to Boc protection removal, then is subjected to conventional condensation amidation reaction with azido carboxylic acid, and a product is purified by HPLC to obtain a compound 7-5, ESI-MS, wherein M/z is 838.4[ M + H ]]+. And (3) carrying out click coupling reaction with a coupler compound 21, purifying by a molecular filter membrane to obtain a compound 22-2, and measuring DAR (DAR 6) by mass spectrum.
In analogy to the synthesis of 7-5, compound VCB-4 can be synthesized:
Figure BDA0003229949260000431
VCB-2(300mg) was mixed with 10mL of TFA/DCM (1/3 vol.), stirred at room temperature for 12 hours, the solvent was distilled off under reduced pressure, 20mL of ethyl acetate and 0.2mL of TEA were added to the residue, and after mixing well, ethyl acetate was washed with 10mL of water 1 time. The organic layer was dried over anhydrous Na2SO 4. The drying agent was filtered off, and ethyl acetate was evaporated under reduced pressure to give VCB-3(210mg, yield 81%) and ESI-MS: M/z 623.3[ M + H ] +.
200mg of VCB-3 was dissolved in 5mL of DMSO, and 100mg of BN-PEG-OH, an equivalent amount of EDC, DIPEA, was added. Stirring at room temperature overnight; the reaction was directly lyophilized and 10mL of TFA/DCM (1/3 vol.) was added and mixed with stirring for 12 h. The solvent was distilled off under reduced pressure, and 20mL of ethyl acetate and 0.2mL of TEA were added to the residue and mixed well, followed by washing of ethyl acetate 1 time with 10mL of water. Ethyl acetate was evaporated under reduced pressure and the residue was purified by HPLC to give NH2-VCB-3(124mg, yield 47%), SI-MS: M/z 812.4[ M + H ] +.
100mg of NH2-VCB-3 was dissolved in 3mL of DMSO, and 33mg of DIMS and 1001. mu.L of TEA were added to complete the reaction at room temperature (MS mass spectrometry monitoring). After lyophilization, the residue was purified by HPLC to give VCB-4(51mg, 49% yield), ESI-MS: M/z 854.4[ M + H ] +.
Synthesis of Compound 5-2-1:
Figure BDA0003229949260000441
benzo-acid (150mg) was dissolved in 5mL of THF, and NHS (106mg), EDC.HCl (211.5mg) was added to stir the reaction at room temperature for 3 hours. NH2-PEG3-N3(200mg) and DIPEA (192. mu.L) were mixed and dissolved in 5mL of THF solvent and slowly added dropwise to the above reaction mixture, and the resulting mixture was stirred at room temperature for 10 hours. The solvent was removed by distillation under the reduced pressure, and the residue was separated and purified by HPLC to give benzoPEG 3-N3(210mg, yield 63%); ESI-MS: 364.1[ M + H ] +.
Benzo-PEG3-N3(100mg) and NH2-VCB-3(224mg) were mixed and dissolved in 7mL of DMSO, 2mL of pure water, 10mg of copper sulfate and 10mg of sodium L-ascorbate were added, and the resulting mixture was stirred at room temperature for 12 hours. Filtering the reactant, freeze-drying the filtrate, adding a little DMF into the freeze-dried substance to dissolve, and separating and purifying by HPLC to obtain NH2-VCB-209(175mg, yield 54%); ESI-MS, M/z 1175.5[ M + H ] +.
M-NHS (25mg) was dissolved in 1mL of DMSO; NH2-VCB-209(110mg) and DIPEA (16. mu.L) were dissolved in 2mL of DMSO and slowly added dropwise to the M-NHS solution. The resulting reaction mixture was stirred at room temperature for 10 hours. Freeze-drying the reactant, adding a little DMF into the freeze-dried product for dissolving, and separating and purifying by HPLC to obtain 5-2-1(82mg, yield 66%); ESI-MS: M/z 664.2[ (M/2) + H ] +.
Synthesis of Compound GY 209:
Figure BDA0003229949260000451
the compounds Benzo-PEG3-N3(100mg) and GY100(72mg) were dissolved in DMSO-deionized water (3: 1 vol: 4mL), anhydrous copper sulfate (10mg), sodium L-ascorbate (12mg) were added; the resulting mixed solution was stirred at room temperature overnight. Filtering to obtain clear solution, freeze-drying the filtrate, adding a little methanol into the freeze-dried substance for dissolving, and separating and purifying by HPLC to obtain GY209(58mg, yield 34%); ESI-MS, M/z 625.2[ M + H ] +.
Synthesis of Compound 5-2-2:
Figure BDA0003229949260000452
SZU-128(100mg) was dissolved in 5mL of DMF, and Boc-Benz-Br (55mg) and 30mg of K2CO3 were added; the resulting mixture was stirred at room temperature for 12 hours. The reaction was filtered, the filtrate was lyophilized, and purified NBZ-128(90mg, yield 75%) was HPLC-isolated as a lyophilizate; ESI-MS, M/z 632.3[ M + H ] +.
V-Ala (32mg), NHS (13mg), and EDC.HCl (22mg) were mixed and dissolved in 1mL of THF, and the mixture was reacted at room temperature with shaking for 6 hours. Dissolving NBZ-128(70mg) in 1mL DMSO, slowly dropwise adding into the above mixed solution, continuing to shake reaction at room temperature for 1 hr after adding, and adding DIPEA (20 μ L); the reaction was continued at room temperature with shaking for 6 hours. Then 0.5mL of TFA was added and the reaction was continued at room temperature for 3 hours. The reaction solution was directly subjected to HPLC to separate purified N-VA-128(72mg, yield 81%); ESI-MS, M/z 802.3[ M + H ] +.
M-PEG3-NHS (35mg) was dissolved in 1mL of anhydrous DMSO; N-VA-128(60mg) and DIPEA (13 mu L) were dissolved in 2mL of anhydrous DMSO, and slowly added dropwise to M-PEG3-NHS solution, followed by reaction with shaking at room temperature for 10 hours. 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(61mg, yield 71%); ESI-MS, M/z 1156.6[ M + H ] +.
Synthesis of Compound 6:
Figure BDA0003229949260000461
compound 12(200mg) was added directly to 10mL of TFA/DCM (1:3), stirred at room temperature for 8 hours, the solvent was evaporated under reduced pressure, and vacuum-dried; the dried solid was dissolved in water, 40. mu.L of triethylamine was added, and HPLC purification was performed to obtain 120mg (yield 69%) of compound 18-2 as a white solid, ESI-MS: M/z 667.3[ M + H ] +.
Compound 18-2 was dissolved in DMSO, an equivalent of TEA was added, an equivalent of succinic anhydride was added, and the mixture was stirred at room temperature for 6 hours. Freeze drying the mixed solution to obtain solid, adding proper amount of water to dissolve, regulating pH value to 5 with acetic acid, precipitating product, filtering, and drying to obtain compound 6. ESI-MS, M/z 767.3[ M + H ] +.
In the coupling chain represented by the compound 8, the immune activator can be replaced by other TLR7 agonists, such as multifunctional GY159 (macrocyclic lipid can improve cell membrane permeability), GY127 (immune activated anticancer effect) and the like, and multifunctional antibody compounds 24, compounds 24-2 and the like can be prepared:
Figure BDA0003229949260000471
200mg of compound 19, 358mg of compound 2A, 176mg of HOBT,254mg of EDC, 450. mu.L of DIPEA were dissolved in 5mL of DMSO and reacted overnight at room temperature, and the reaction was monitored by LC-MS. After the reaction was completed, purification was performed by HPLC to obtain 210mg of a white solid (Compound 3A) in 41.2% yield. ESI-MS, M/z 594.3[ M + H ] +.
Figure BDA0003229949260000472
200mg of Compound 3A, 113mg of Compound 4A, 180. mu.L of DIPEA were dissolved in 2mL of DMSO and reacted overnight at room temperature, and the reaction was monitored by LC-MS. After the reaction was completed, purification was performed by HPLC to obtain 65mg of a white solid (Compound 5A) in a yield of 25.4%. ESI-MS, M/z 759.3[ M + H ] +.
Figure BDA0003229949260000481
30mg of compound 5A, 18mg of compound GY159, 21. mu.L of DIPEA were dissolved in 1mL of DMSO and reacted overnight at room temperature, and the reaction was monitored by LC-MS. After the reaction was completed, purification was performed by HPLC to obtain 8mg of a white solid (Compound GY206) in 19.5% yield. ESI-MS, M/z 1035.5[ M + H ] +.
Figure BDA0003229949260000482
30mg of Compound 5A, 42mg of Compound GY127, 21. mu.L of DIPEA were dissolved in 1mL of DMSO and reacted overnight at room temperature, and the reaction was monitored by LC-MS. After the reaction was completed, purification was performed by HPLC to obtain 11mg of a yellow solid (Compound GY207) in 17.7% yield. ESI-MS, M/z 1586.7[ M + H ] +.
Referring to HER2 conjugate compound 22, an immune-activating antibody compound 24 can be prepared from compound GY207 and compound 21 as follows:
Figure BDA0003229949260000491
the compound GY127 has TLR7 activating effect and tumor cell inhibiting effect. The multifunctional antibody compound 24 releases the compound GY127 in the tumor microenvironment and tumor cells under the specific targeting guidance of the antibody, so as to achieve the anti-tumor effects of local immune activation and enhancement.
In the same manner, when the antibody in compound 22 was replaced with the c-Met antibody (abcam, ab51067) and compound 1 was replaced with compound GY102, the analogue compound 24-2:
Figure BDA0003229949260000492
referring to the synthesis of compound 15, substituting the agonist moiety with compound GY102, compound 15-4 can be obtained:
Figure BDA0003229949260000501
substitution of the antibody for ASGPR1 antibody and the immune agonist for compound GY102-3 gave compound 15-3:
Figure BDA0003229949260000502
in compound 15-3, ASGPR1 is a asialoglycoprotein receptor, specifically expressed in liver tissue; the small molecule immune agonist precursor compound 102-3 is metabolized to the compound 102 in the liver, so that the compound 15-3 is a specific liver targeting immune activation type antibody, and the synthesis method of the small molecule immune agonist compound 102-3 is as follows:
Figure BDA0003229949260000503
480mg of compound GY102 is dissolved in DMSO, 140 mu L of triethylamine is added, and equal equivalents of tert-butyl chloride are slowly dropped at 5-10 ℃; reacting at room temperature for 4 hours, directly freezing and drying the reaction mixture to obtain a solid, adding water with the temperature of 5 ℃ into the solid, stirring and dissolving the solid to remove salt, filtering to obtain a compound 102-1, and drying in vacuum. The dried product was dissolved in DMSO, 140. mu.L of triethylamine was added, an equivalent amount of compound AC-1 was slowly dropped at 5 to 10 ℃ and stirred at room temperature overnight. The reaction solution was directly freeze-dried to obtain a solid, the solid was dissolved in 5 ℃ water under stirring to remove the salt, and the mixture was filtered to obtain compound 102-2, 1mL of TFA and 3mL of DCM were added, the mixture was stirred at room temperature for 4 hours, the solvent was removed by vacuum distillation under reduced pressure, and the residue was added with water and purified by HPLC to obtain compound 102-3(121mg, yield 21%), ESI-MS: M/z-579.3 [ M + H ] +.
By substituting the antibody in compound 15-4 with CD206 antibody (abcam, ab64693), compound 15-5:
Figure BDA0003229949260000511
in the same manner, the antibody in compound 22 was replaced with PD-1 antibody and compound 8 was replaced with compound GY206, giving the analogue compound 24-3:
Figure BDA0003229949260000512
synthesis of conjugated antibody representative compound 28 of formula II:
Figure BDA0003229949260000521
dissolving compound 26 and compound GY102 in DMSO in equivalent, stirring the mixed solution at room temperature, and detecting by mass spectrometry that the raw material reaction is almost complete. The reaction was lyophilized, the dried solid was dissolved in the appropriate amount of TFA/DCM (1:3) and stirred at room temperature for 4 hours. TFA was removed by distillation under reduced pressure, and the resulting product was separated and purified by HPLC to obtain compound 27, ESI-MS: M/z 985.4[ M + H ] +.
Dissolving 100mg of compound 27 and an equivalent amount of NHS (N-hydroxysuccinimide) in 1mL of anhydrous DMSO, adding an equivalent amount of EDC, and reacting the mixture at 15 ℃ for 6 hours under sealed stirring; 738mg of HER2 antibody was dissolved in 10mL of purified water, and slowly added to the reaction mixture, after which the reaction was continued at room temperature for 10 hours under sealed conditions, and ethanolamine (6. mu.L) was added; and (3) eluting the small molecules of the reaction solution by using a 20KD molecular filter membrane and DPBS, and dissolving the antibody by the DPBS to obtain a DPBS solution of the coupled antibody compound 28. The average degree of coupling of compound 28 was 4 as determined by mass spectrometry.
With reference to the synthesis of compound 17, conjugated antibody compound 29 can be obtained, as shown in the following formula:
Figure BDA0003229949260000531
15-fold equivalent of 5-2-1(12mg) and 0.1mL of TEA of the antibody were dissolved in 0.2mL of DMSO and added to a 100mg solution of HS-reduced PD-L1 mab (Atezolizumab) in DPBS (5 mL). The mixture was reacted for 10 hours with shaking at 20 OC. Ultrafiltering with 20K filter membrane to remove small molecules, and stirring in an open container at room temperature for 6 hr to obtain new antibody 29-1 coupled with PD-L1 antibody 5-2-1; the coupling ratio (ADR value) was 2 by mass spectrometry.
Preparation of HS-reduced PD-L1 antibody: the preparation method of the HS-reduced HER2 antibody is adopted.
Among them, refer to FIG. 15 which shows the protein sequence of Atezolizumab (protein sequence of human Drug Information Vol.28, No.4,2014), and the protein sequence of Atezolizumab is shown in SEQ ID NO:3 and SEQ ID NO: 4.
Method for synthesizing conjugated antibody compound 30:
Figure BDA0003229949260000541
in the same manner as in the synthesis of compound 8-4, compound 19 was substituted to give compound 8-5, compound 8-6:
Figure BDA0003229949260000542
Figure BDA0003229949260000551
the same procedure gave compounds 5-2 and 7-3:
Figure BDA0003229949260000552
in the same route as the synthesis of compound 30, antibody compound 30-1:
Figure BDA0003229949260000561
the synthesis method of the compound 8-2 comprises the following steps:
Figure BDA0003229949260000571
an equivalent amount of compound Val1 and compound 1A in DMSO of HOBT, EDC and DIPEA to produce compound Val2, acting on TFA to produce compound Val3, and continuing to react with compound 6A in DMSO of HOBT, EDC and DIPEA to produce compound Val4, and deprotecting to produce compound Val 5. Compound Val5 is reacted directly with compound 4A to give compound Val 6. Compound Val6 and compound 4 under heating form compound 8-2.
Compound 8-3 and Compound 8-1 can be synthesized in the same manner as Compound 8-2:
Figure BDA0003229949260000581
the synthesis of compound 7-1 can be achieved analogously to compound 8-2:
Figure BDA0003229949260000591
the compound VC-An4(200mg) and the compound 13(92mg) were mixed and dissolved in 2mL of DMSO, and An equivalent of a condensation reagent (HOBt, EDC, DIPEA) was added to react the mixture at room temperature for 12 hours. Direct lyophilization and HPLC purification gave 14-1, 209mg (73%), ESI-MS: M/z 955.4[ M + H ] +.
After 100mg of Compound 14-1 was added to 2mL of TFA/DCM (1:3 vol.) and the deprotection reaction was completed as monitored by mass spectrometry, the reaction solution was distilled under vacuum to remove the solvent, the residue was dissolved in 3mL of DMF, 100. mu.L of TEA and 20mg of thiodiimidazole were added and the reaction was completed at room temperature (MS mass spectrometry). The reaction mixture was separated and purified by HPLC to give compound 7-1(52mg, 56% yield), ESI-MS: M/z 897.7[ M + H ]]+。
Synthesis of HER2 antibody conjugate compound 31:
Figure BDA0003229949260000601
compound BVA-3 was mixed with 1:3 of TFA/DCM was added and the reaction was stirred at room temperature for 8 hours, and the solvent was removed by distillation under the reduced pressure. Vacuum drying the residue, adding appropriate amount of DMF to dissolve, adding 2 equivalents of TEA, mixing, slowly adding equivalent amount of Succinic acid liver (Succinic anhydride) in ice water bath at 10 deg.C, and stirring at room temperature for 12 hr. After the reaction is finished, 10 times of water is added, the pH is adjusted to 3, the mixture is frozen at minus 20 ℃ to separate out solid, and the solid is filtered and washed by water to obtain a crude compound 6-2. Purifying by HPLC to obtain pure compound 6-2, ESI-MS, M/z is 532.3[ M + H ] +.
20 equivalents of compound 6-2 were added to an appropriate amount of DMSO, and equal equivalents of HOBT, EDC and DIPEA were added at 10 deg.C, and the mixture was stirred for 2 hours. The mixture was slowly added to a solution containing 1 equivalent of the antibody HER2 in water, and allowed to react naturally at room temperature for 12 hours. And dialyzing the reaction solution by using a 10K filter membrane to remove small molecules, washing the obtained macromolecular antibody by using pure water for multiple times, and freeze-drying the washing solution to obtain the coupled antibody compound 31. The average degree of coupling (DAR ═ 4) was measured.
In the same manner, by replacing compound 6-2 with compound 8-5 or compound 8-6, the conjugated antibody compounds 32 and 33:
Figure BDA0003229949260000611
synthetic route for antibody compound 34:
Figure BDA0003229949260000621
the compounds BVC-1 and p-nitrophenol were dissolved in DMSO in equal amounts, EDC and DIPEA in equal amounts were added and stirred at room temperature for 12 hours. Adding diethyl ether into the reaction product for freezing, separating out a solid, and filtering to obtain BVC-2; BVC-2 was dissolved in dry DMF and an equivalent of K was added2CO3And an equivalent amount of compound GY100, stirring at room temperature for 12 hours, and separating and purifying the product by HPLC to obtain compound BVC-3. Carrying out click reaction on a compound BVC-3 and a compound PIP-3, then removing a protecting group from TFA, and carrying out HPLC purification to obtain a compound BVC-4; the compound BVC-4 and the compound 19 generate a compound BVC-5 under the action of a condensing agent, and react with a conjugate compound 21click to obtain a conjugate antibody compound 34:
Figure BDA0003229949260000631
the immune activator part of the novel antibody compound 34 is a compound GY161 which has strong anti-tumor activity, and the targeted release of the tumor antibody can greatly enhance the anti-tumor effect.
The immune activator in the embodiment of the invention can be replaced by multifunctional immune activation small molecules, such as a trifunctional small molecule part (compound Tri-linker-1) simultaneously containing lenalidomide/osimertinib/SZU-160:
Figure BDA0003229949260000641
synthesizing a compound Tri-linker-1:
the compound ICO3N3 is obtained by mixing isocolar itaconic anhydride with the compound NO3N3 in dry DMSO, adding equimolar TEA, stirring overnight at 40 ℃, neutralizing to pH4 with HCl and direct freeze drying of the reaction mixture. Performing condensation reaction on compounds ICO3N3 and lenalidomide in DMSO by using a condensing agent (HOBt/EDC), and then separating and purifying by HPLC to obtain a compound POMA-ICO3N 3: ESI-MS: M/z 572.20[ M + H ]]+. Dissolving a compound POMA-ICO3N3 in DMSO, adding mercaptoethylamine with the same mole, sealing, stirring at room temperature overnight, and directly freeze-drying a reactant to obtain S-POMA-ICO3N 3: ESI-MS: M/z 649.20[ M + H ]]+. 100mg of the compound S-POMA-ICO3N3 was mixed with 79mg of the compound Kyne-9291 and dissolved in 1: 4 (0.5mL), 8mg each of sodium L-ascorbate and anhydrous copper sulfate was added to the mixture at room temperature for 5 hours, and after the reaction was monitored by LC-MS, the product was purified directly by HPLC to give the compound Bi-linker,91mg (51%), ESI-MS: M/z-1172.50 [ M + H ], (51%)]+. 70mg of the compound Bi-linker was dissolved in dry 1mL of DMSO, and 36mg of the compound SZU-160 and 1.1 equivalent of HOBt/EDC/DIPEA were added and reacted with shaking at room temperature overnight. The reaction mixture was directly freeze-dried, dissolved in a small amount of isopropanol and purified by HPLC to give Tri-linker-1 as a pure compound, 69mg (66% yield), ESI-MS: M/z-1752.7 [ M + H ]]+。
Synthesis of antibody compound 35:
Figure BDA0003229949260000651
adding 10% DMSO by volume into DPBS solution of the conjugate compound 21, adding 1.5 equivalent of compound Tri-linker-1, reacting at 25 deg.C with shaking for 12 hr, and removing small molecules with 20KD molecular filter membrane. The DPBS dissolves the antibody to give a DPBS solution of conjugated antibody compound 35. The average degree of coupling of compound 35 was 6 as determined by mass spectrometry.
According to a synthesis technical method of a compound Tri-linker-1, obtaining a compound Tri-linker-2 according to the following synthesis route:
Figure BDA0003229949260000661
Figure BDA0003229949260000671
the compound BVC-1 and an equivalent of compound SZU-163 are reacted and condensed in dry DMSO by HOBt/EDC/DIPEA (equivalent), then the protecting group is removed by TFA/DCM, and the compound BVC-T-2 is obtained by HPLC purification. Dissolving the compound BVC-T-2 and the compound 41 in DMSO in an equivalent, continuing to perform condensation reaction by HOBt/EDC/DIPEA, and purifying by HPLC to obtain the compound BVC-T-3. Stirring the compound BVC-T-3 and 6N hydrochloric acid at room temperature overnight, distilling under reduced pressure to dryness to obtain a white solid compound BVC-T-4, and drying in vacuum.
Mixing a compound BVC-T-4 and a compound Bi-linker in an equal molar ratio in DMSO, performing condensation reaction by using a HOBt/EDC/DIPEA condensing agent, and performing HPLC purification and LC-MS molecule confirmation to obtain a compound Tri-linker-2: ESI-MS: M/z 1993.20[ M + H ]]+. Mass spectrum identification of each intermediate, and the mass spectrum identification of the compound BVC-T-2: ESI-MS: M/z 585.5[ M + H ]]+(ii) a Compound BVC-T-3: ESI-MS: M/z 853.5[ M + H ]]+(ii) a Compound BVC-T-4: ESI-MS: M/z 839.4[ M + H ]]+。
Synthesis of Compound 41:
Figure BDA0003229949260000681
the 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 thereto, stirred at room temperature for 30 minutes, and reacted at reflux for 2 hours. Cooled to room temperature and the solvent was distilled off under reduced pressure to give compound Ts-N3 as an oil. ESI-MS: 270.10[ M + H ] M/z]+. Dissolving in dioxane for use.
Dissolving compound 37(5.3g,19mmol) in 100mL anhydrous dioxane, adding potassium tert-butoxide (2.36g,20.9mmol), and reacting at 60 ℃ for 2 hours under nitrogen protection; slowly dropwise adding a solution of a compound Ts-N3(5.1g,19mmol) in anhydrous dioxane (10mL), heating to reflux after the dropwise adding is finished, and reacting for 20 hours; the reaction solution was cooled to room temperature, filtered under suction, washed with ether, the filtrate was recovered, and after concentration, the compound was separated by column chromatography (petroleum ether: ethyl acetate: 6: 4) to give 3.6g of a compound 38 as a yellow oily liquid in 51% yield. ESI-MS, M/z 373.2[ M + H ] +.
Compound 38(3g,8mmol) was added to 10mL of 2N hydrochloric acid, heated under reflux, the reaction was monitored by LC-MS, after completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was dissolved in saturated Na2CO3Hydrochloric acid was slowly added dropwise to the solution to precipitate a solid, which was then filtered to obtain 39, 0.85g of a white solid compound in 62% yield. ESI-MS: M/z 173[ M + H ]]+。
Compound 39(0.8g, 4.6mmol) was mixed in dry methanol (10mL) and 3mL of SOCl was slowly added dropwise with cooling (5- -10 ℃ C.)2And stirring naturally overnight under the protection of nitrogen. The solvent was removed by distillation under the reduced pressure, and the extract was dried under vacuum to give the hydrochloride of compound 40 in 97% yield, ESI-MS: M/z 187.2[ M + H ]]+。
The hydrochloride salt of Compound 40 (0.5g, 2.2mmol) was dissolved in 5mL of DMSO, an equivalent amount of succinic anhydride and 2 equivalents of TEA were added, the reaction was stirred at 60 ℃ for 4 hours, the reaction was directly lyophilized, and the resulting solid was dissolved in saturated Na2CO3In the solution, the pH value is adjusted to 4 by hydrochloric acid, solid is separated out, the filtration and the vacuum drying are carried out to obtain the compound 41, 0.42g,the yield thereof was found to be 67%. ESI-MS: M/z 287.2[ M + H ]]+。
Synthesis and purification of antibody compound 36 the same procedure as for compound 35 was followed to obtain antibody compound 36, and the average coupling ratio (DAR ═ 6) was measured by mass spectrometry:
Figure BDA0003229949260000691
N3-VC-T synthesis:
Figure BDA0003229949260000701
BVC-T-2 and N3PEGOH are mixed into DMSO in equal mole, condensing agent (HOBt, EDC, DIPEA/DMSO) in equal weight is added, the mixture is stirred for 8 hours at room temperature, the reaction mixture is frozen and dried, the residue is dissolved in a small amount of methanol, and the mixture is separated and purified by HPLC to obtain N3-VC-T, ESI-MS, M/z is 800.5[ M + H ] +.
MA-VC-T synthesis:
Figure BDA0003229949260000702
the synthesis method is the same as that of N3-VC-T, except that N3PEGOH is replaced by MA-OH to obtain MA-VC-T, ESI-MS, wherein M/z is 778.4[ M + H ] +.
HO-VC-T synthesis:
Figure BDA0003229949260000711
BVC-T-2 and Succinic anhydride were mixed and dissolved in DMF at an equal molar ratio, 2 equivalents of TEA were added and the mixture was stirred at 60 ℃ for 4 hours. The reaction mixture was distilled under reduced pressure (60 ℃ C.) to remove DMF, and saturated Na was added to the residue2CO3Dissolving, filtering to obtain clear liquid, regulating pH with acetic acid 4, and filtering to obtain pure product ESI-MS (ESI-MS) with M/z 685.4[ M + H ]]+。
SVC-T synthesis:
Figure BDA0003229949260000721
mixing and dissolving BVC-T-2 and FPEGOH in equal molar amount in a proper amount of DMSO, adding condensing agent (HOBt, EDC, DIPEA) in equal amount, stirring for 8 hours at room temperature, adding piperidine in equal amount, and stirring for reaction for 4 hours at 60 ℃. The reaction mixture was lyophilized, 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 ] +.
NVC-T and equimolar SC-DIMI are dissolved in a proper amount of dried DMSO, 2 equivalents of TEA are added, the mixture reacts for 12 hours at room temperature, the reaction solution is directly frozen and dried, and HPLC purification is carried out to obtain SVC-T, ESI-MS, wherein M/z is 816.3[ M + H ] +.
The compound N3-VC-T, MA-VC-T, HO-VC-T, SVC-T can be specially used for antibody or targeted drug coupling, targets the cells expressing TLR7, degrades TLR7 agonist, and achieves the effect of eliminating the proliferation of the TLR7 agonist on the cells. For example, HER2 antibody conjugation to SVC-T, synthesis of 37:
Figure BDA0003229949260000731
to a mixture of 100mL of DPBS solution containing 1 equivalent of HER2 antibody and 5mL of DMSO solution containing 6 equivalents of SVC-T, 12 equivalents of TEA was added and the mixture was shaken at room temperature for 12 hours. The small molecule was removed by dialysis to give 37 and the average degree of coupling (DAR) was measured by mass spectrometry to be 4.
SZU-164 Synthesis:
Figure BDA0003229949260000732
1g of SZU-163 and 0.3 g of Succinic anhydride (Succinic anhydride) were dissolved in dry 20mL of DMSO, 1mL of Triethylamine (TEA) was added, and the reaction was stirred at room temperature for 12 hours. Freeze drying the mixture to remove solvent, dissolving the residue in water, adjusting pH to 4 with hydrochloric acid, precipitating product, filtering, and drying to obtain SZU-164; 1g, 77% yield, ESI-MS: M/z 429.2[ M + H ] +.
The immune activation type antibody and corresponding representative compounds provided by the embodiment of the invention can be used for preparing antitumor drugs, antiviral drugs, immunoregulation drugs and/or preparations for eliminating target proteins.
The embodiment of the invention also provides application of the immune activation type antibody in preparation of antitumor drugs, antiviral drugs, immunoregulation drugs and/or preparations for eliminating target proteins.
Because the immune activation type antibody provided by the embodiment of the invention has the function of locally targeting and activating immunity, when the immune activation type antibody is used for preparing anti-tumor drugs, antiviral drugs, immunoregulation drugs and/or target protein elimination preparations, the immune activation type antibody not only can avoid the side effect of nonspecific killing on normal tissues, but also has various functions of activating target immune cells (such as T cells, B cells, NK cells and the like), reversing inert immune cells (such as macrophages are converted into M1 type anti-tumor macrophages, the proportion of M1/M2 and the like), converting the immune cells into immune cells with anti-tumor activity (such as the increase of the cell number of IFN-gamma + CD8 and the like), and the like, and has good application prospect.
In order to clearly understand the details and operation of the above-mentioned embodiments of the present invention and to clearly show the advanced performance of the immune-activated antibody and its application in the embodiments of the present invention, the above-mentioned technical solutions are illustrated by the following examples.
In the following examples, HER2 antibody (InVivoMab anti-human/rat HER2(neu)), PD-1 and PD-L1 antibodies (anti-mouse antibodies) were purchased from BioXcell; the pure product is treated by dialysis.
The preparation of the conjugate samples was as follows:
20 μ g of a sample to be measured was put in an EP tube, and after 2 μ L of GlycoBuffer 2(10X) was added, ultrapure water was added to make the final volume of the system 20 μ L. Subsequently, 3. mu.L of PNGase F was added to each sample and reacted in a water bath at 37 ℃ for 48 hours. The above samples were diluted 10-fold (final concentration 0.1mg/mL) and the samples to be tested were tested according to the following test parameters.
Antibody detection parameters and mass spectrometry conditions were as follows:
1. instrumentation and equipment
Figure BDA0003229949260000741
2. Reagent and test solution
Figure BDA0003229949260000742
Figure BDA0003229949260000751
3. Conditions of liquid chromatography
(1) Gradient of mobile phase
Time Phase A (aqueous solution containing 0.1% formic acid) Phase B (acetonitrile solution containing 0.1% formic acid)
1min 80％ 20％
2min
10％ 90％
4min
10％ 90％
4.1min 80％ 20％
7min
80％ 20％
(2) Detecting parameters
Parameter(s) Parameter value
Detection wavelength 214nm
Flow rate of flow 0.3mL/min
Sample volume 20μL
Column temperature
80℃
Time of acquisition 7min
Elution mode Rapid gradient elution
4. Conditions of Mass Spectrometry
Parameter(s) Parameter value
Ion source ESI
Scanning mode TOF MS
Scanning Range (Da) 1000-5000
Spray mist 45psi
Auxiliary heating gas 45psi
Air curtain 30psi
Temperature of 450℃
Declustering voltage 300V
EXAMPLE 1 Detection of TLR7 activation by Compounds (HEK-blue Detection)
HEK-Blue TMh TLR7 cells (purchased from InvivoGen) in logarithmic growth phase were taken, growth medium (Gibco, C11995500BT, Invivo Gen, ant-nr) was discarded, appropriate amount of 37 ℃ PBS (Hyclone, SH30256.01) was gently rinsed 2 times, and PBS was discarded. Adding 2-5mL of PBS (phosphate buffer solution) at 37 ℃, incubating for 1-2 min, scraping cells by using a cell scraper, and then gently blowing and beating the cells to disperse the cells into single cell suspension. Cells were counted and cell concentrations were calculated using a hemocytometer and cell suspensions were adjusted to 2.5 × 104/180 μ L per well using HEK-blue (tm) precipitation solution (purchased from Invivo Gen) for cell plating on 96-well cell culture plates. HEK-BlueTM hTLR7 cells were stimulated according to compound or drug concentrations (e.g., 0.01. mu.M, 0.1. mu.M, 1. mu.M, 5. mu.M, 15. mu.M, 30. mu.M, 40. mu.M), with 3 duplicate wells set at each concentration. Incubating for 6-16 h at 37 ℃ under the condition of 5% carbon dioxide. After the incubation, the absorbance was read at 650nm using a full-wavelength microplate reader (BioTek-Epoch). The results are shown in figure 1 with OD values on the ordinate representing the degree of TLR7 activation and the abscissa representing the concentration of compound.
Example 2 detection of TLR7 agonist Release Effect of various Immunoactive antibodies
Each of the immunoactive antibodies was added to HER2 positive SKBR3 (10. mu.M) at a concentration (e.g., 0.1. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M;)5Individual) cells in RPMI1640 medium. After 12 hours of mixed culture, each supernatant was removed and the activation effect of TLR7 was tested according to the method in example 1. The results are shown in FIGS. 2 and 3.
Example 3 anti-tumor Effect test of representative Immunoactivating antibody
Murine Her2+CT26 tumor cells and models, preparation method reference ("In vivo properties of three human HER2/neu-expressing hormone cell lines In immunological competence mice", Lab Anim Sci, 1999 Apr; 49(2): 179-88.).
Establishing a colorectal cancer tumor model of a mouse: her2+ CT26 cells were collected at logarithmic growth phase by digestion and centrifugation, washed twice with PBS and counted to adjust the cell concentration to 2.3X 105one/mL. The mice were SPF-grade BALB/c mice 6 weeks old. Shaving the hair on the right back of the mouse by using a hair shaver, sucking 100 mu L of cell suspension by using an injector, discharging bubbles, injecting the cell suspension into the subcutaneous surface of the back of the mouse, and starting to perform the operation when the diameter of the tumor reaches 4-5 mmAnd (4) administration.
Evaluation of the therapeutic effect in tumor-bearing mice and the antitumor effect of compounds 15-4 and 34: tumor-bearing mice (BALB/c) were randomly divided into a Control group, a TLR7 agonist group, a HER2 antibody group, a TLR7 agonist + HER2 antibody mixed group (equivalent ratio of the two is n: 1), and an immune-activated antibody group, with 8 mice per group. Injections (solvent component is mixed with 5% DMSO; 40% PEG 300; 5% Tween 80; 50% PBS) are prepared according to the administration dosage, the administration dosage is 3mg/kg of TLR7 agonist, 20mg/kg of HER2 antibody, 20mg/kg of TLR7 agonist and HER2 antibody mixed group (3mg/kg +20mg/kg), 20mg/kg of TLR7-HER2 coupling antibody are administered, and the injection is injected in the tumor week, and the volume is 100 muL each time. The injection is respectively administered on the 5 th day, the 11 th day and the 17 th day after the tumor implantation, and is administered on the 23 rd day, and the total injection is 4 times and is injected in the tumor periphery. While dosing, tumors were measured with vernier caliper and survival of mice was recorded for 7 times. The tumor volume calculation method comprises the following steps: 0.5 × a × b2Wherein a is the major diameter and b is the minor diameter. When the tumor diameter of the mouse reaches 2cm, the mouse should be killed according to ethical cervical dislocation of the animal, and tumor tissues of the mouse are stripped to calculate the tumor volume and the tumor weight. The results are shown in FIGS. 4 and 5. The ordinate of fig. 4 is the tumor weight at 25 days of administration, and fig. 5 is a graph of tumor volume versus time.
Example 4 Intra-tumor immune cell assay
The animals of example 3 were euthanized 48 hours after the 11 th day of administration, tumor tissue was isolated, and the intratumoral M1/M2 marker ratio (MHC-II: CD206) was analyzed by flow cytometry; and CD8+ IFN- γ + T cell changes, PBS or primary antibody control, with results shown in figures 6 and 7. FIG. 6 is the ratio of M1 macrophages to M2 macrophages in tumor tissue at day 11 of each administration group; FIG. 7 shows the relative amounts of CD8+ IFN-. gamma. + T cells in tumor tissues at day 11 of each administration group.
Example 5 CCK8 method for in vitro detection of tumor cell inhibitory Activity of drugs
The immune activation type antibody is directly mixed with a tumor cell expressed by HER2 and cultured for 48 hours, the inhibitory activity to the tumor cell is detected according to the standard CCK8 technology, and the result is shown in figure 8 and figure 9, and the result shows that the antibody with the tumor inhibitory activity micromolecule and the immune agonist has stronger inhibitory effect to the tumor cell under the environment without the assistance of immune cells.
Example 6
Compounds SZU-164 and HO-VC-T, N3-VC-T, MA-VC-T, SVC-T were tested for TLR7 activation (HEK-blue Detection):
1) HO-VC-T, N3-VC-T, MA-VC-T, SVC-T was formulated at concentrations of 0.1, 1, 10, 20. mu.M, and the results of TLR7 activation of the four compounds were measured as in example 1, and are shown in FIG. 10.
2) SZU-164 and HO-VC-T, N3-VC-T, MA-VC-T, SVC-T were incubated with Cathepsin-B (10. mu.g, Cat. #:10483-H08H, SinoBiological) in PBS at 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M concentration for 12 hours at room temperature, respectively; 2K was filtered through a molecular membrane to obtain a filtrate, and the activation effect of SZU-164 and each compound on TLR7 was measured by the method of example 1, which shows that groups containing Val-Cit (valyl-citryl) (such as HO-VC-T, etc.) can be degraded under the action of Cathepsin-B, and the activation on TLR7 is lost, as shown in FIG. 11.
It has been shown that in the case of tumor cells expressing TLR7, activation of TLR7 increases the number of tumor cells. The results of example 6 demonstrate that 1) cells expressed in normal TLR7, HO-VC-T, N3-VC-T, MA-VC-T, SVC-T, activate the TLR7 pathway, which is beneficial for activating the immune system; 2) in the tumor cells expressing TLR7, HO-VC-T, N3-VC-T, MA-VC-T, SVC-T is inactivated under the action of Cathepsin-B, thus being beneficial to selectively activating immune microenvironment without growing the tumor cells. Similarly, other antibodies or compounds of the invention containing Val-Cit may be degraded by the action of Cathepsin-B and, depending on the small molecule product after degradation, may produce or lose activation of TLR 7.
The degradation of an enzyme represented by HO-VC-T (Cathepsin-B) acting on a TLR7 agonist is schematically shown in FIG. 12.
Example 7.
TLR7 pathway activation function test of compounds SZU-128, GY209, 5-2-1, 5-2-2, 17-1 and 29-1
1) Compounds SZU-128, GY209, 5-2-1, 5-2-2, 17-1 and 29-1 were co-incubated with Cathepsin-B (10. mu.g, Cat. #:10483-H08H, SinoBiological) in PBS at 0.01. mu.M, 0.1. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M concentrations for 3 hours at room temperature, respectively; 2K molecular membrane filtration to obtain filtrate, and the activation effect of each compound on TLR7 is measured by the method of example 1 for each group of filtrate, and R848 is a standard positive control. Wherein, the Cathepsin enzyme catalyzes the cleavage to release the immune agonist is shown in figure 16, and the activation effect of TLR7 of each compound under the action of the Cathepsin-B enzyme is shown in figure 17.
The results show that under the action of Cathepsin-B, a group containing Val-Cit or Val-Ala (valyl-citryl or valyl-alanyl) can be degraded to release a TLR7 agonist, and activate TLR 7.
2) Three groups of compounds SZU-128, GY209, 5-2-1, 5-2-2, 17-1, 29-1 and SZU-161 were prepared at concentrations of 1. mu.M, 0.1. mu.M and 0.01. mu.M, respectively, and the activation effect of each compound on TLR7 without the action of the Cathepsin-B enzyme was obtained directly according to the procedure of the experimental method in example 1, and R848 was a standard positive control, as shown in FIG. 18. It can be seen that without the addition of Cathepsin-B, only compounds that do not contain Val-Cit or Val-Ala (valyl-citryl or valyl-alanyl) groups (GY209, SZU161, SZU-128 and R848) are able to activate TLR 7.
Example 8.
Experiment on inhibitory Effect of antibody 17-1 and antibody 29-1 on tumor cells
Each compound was added to the corresponding tumor cell culture medium (10. mu.M) in concentration groups (0.6. mu.M, 1.8. mu.M, 5.5. mu.M, 16.6. mu.M, 25. mu.M, 50. mu.M)5Individual cells), after 3 hours of co-incubation, hPBMC at 20 times the number of tumor cells was added and co-incubation was continued for 48 hours, and the cell inhibition rate was measured according to the standard CCK8 method, as shown in fig. 19 and fig. 20. It can be seen that the novel antibodies 17-1 and 29-1 significantly improved the effect of immunosuppressing tumor cells.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. An immune activation type antibody is characterized by comprising an antibody and an immune activator, wherein the antibody is coupled with the immune activator through a coupling chain, and the coupling chain comprises at least one of a structure shown as a formula (A), a structure shown as a formula (B), a structure shown as a formula (C) and a structure shown as a formula (D):
Figure FDA0003229949250000011
2. the immune-activated antibody of claim 1, wherein the conjugated chain is at least one selected from the group consisting of a degradable chain containing a Cathepsin-B region, an alkyl group, an alkoxy group, a nitrogenous alkyl group, a heterocycle, and a specific functional chain.
3. The immune-activated antibody of claim 1 or2, wherein the conjugate chain comprises compound 5, compound 5-1, compound 5-2-1, compound 5-2-2, compound GY209, compound 6-1, compound 6-2, compound 6-3, compound 7-1, compound 7-2, compound 7-3, compound 7-4, compound 7-5, 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, At least one of the compound VCB-4, the compound BVC-T-4, the compound Tri-linker-1, the compound Tri-linker-2, the compound 5-1, the compound 5-2-1, the compound 5-2-2, the compound 6-1, the compound 6-2, the compound 6-3, the compound 7-1, the compound 7-2, the compound 7-3, the compound 7-4, the compound 7-5, the compound 8-1, the compound 8-2, the compound 8-3, the compound 8-4, The structural formulas of the compound 8-5, the compound 8-6, the compound 8-7, the compound 27, the compound GY206, the compound GY207, the compound 5A-GY102, the compound VCB-4, the compound BVC-T-4, the compound Tri-linker-1 and the compound Tri-linker-2 are respectively as follows:
Figure FDA0003229949250000021
Figure FDA0003229949250000031
Figure FDA0003229949250000041
Figure FDA0003229949250000051
Figure FDA0003229949250000061
Figure FDA0003229949250000071
Figure FDA0003229949250000081
Figure FDA0003229949250000091
4. the immunoactive antibody of claim 3 wherein said intermediate compounds used for the synthesis of said coupling chain 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 N3-VC-T, compound HO-VC-T, compound MA-VC-T, compound SVC-T, said compound 18-2, said compound 5A, said compound 102-3, said compound VC-An4, said compound Val5, said compound Val6, said compound VC100 Val4, The structural formulas of the compound VCB-2, the compound POMA-ICO3N3, the compound S-POMA-ICO3N3, the compound Bi-Linker, the compound BVC-T-2, the compound N3-VC-T, the compound HO-VC-T, the compound MA-VC-T and the compound SVC-T are respectively as follows:
Figure FDA0003229949250000092
Figure FDA0003229949250000101
Figure FDA0003229949250000111
Figure FDA0003229949250000121
5. the immune activated antibody of claim 1 or2, wherein the immune activator comprises at least one of a TLR7 agonist, a TLR8 agonist, a STING agonist, a small molecule immune activator.
6. The immune-activated antibody of claim 5, wherein the small molecule immune activator comprises at least one of compound 1, compound 2, and compound 3, and the structural formulas of compound 1, compound 2, and compound 3 are as follows:
Figure FDA0003229949250000131
7. the immunoactive antibody of claim 1 or2, wherein said antibody targets an antigen selected from the group consisting of HER, PD-L, PD-1, TIGIT, TROP, EGFR, MUC, LIV-1, MUC, CEACAM and subtypes thereof, URLC, NY-ESO-1, GAA, OFA, cyclin B, WT-1, CEF, VEGFR, TTK, MUC, HPV 16E, CEA, IMA910, KOC, SL-701, MART-1, gp100, tyrosinase, GSK, survin, MAGE-3.1, MAGE-10.A, gp 209-2-A, NA17.A2, KOC, CO, DEPDC, MPSPH, MAGE, ONT-10, GD2, GD3, GSK, URLC, CDCA, rsA, PSA, MUC-2, TERT, PLLR, HPV, FORCL-107, FORCL-17, FORCL, FORCA, FORC-17, FORCL, FORCA, FORC-1, FORC, FORCA, FORC-1, FORCA, and other, FORCA, TAR, CECAM, cMUT, GPA, ALK, ROS, BRAF, MEK, RET, CDK/6, BRCA, PARP, BRCA, FLT, CD, BCL, CD, Smoothened, GD, EZH, MTH, KRAS, c-MYC, hCA IX, hCA XII, BRD, HDAC, NYC, TOPK, BCMA, PI3, PDGFR, TIM, OX, CD, SIRP-alpha, CD122, CD160, TGF-beta, HIF-1 alpha/2 alpha, PSGL-1, Frizzled-7, CXCR 4A, CCR, CXCR, CCL, CXCL, CD79, Nectin-4, CD117, PSMA, NKG2, Claudin18.2, MGR, ROR, FGFR, WNT, FGFR, WNT2, BRAF, MEK, REK, RET, RER, RGF-7, CRAB, CRAT-7, CRAT, RG, CRAT, CRS, CRAT, CR.
8. The immune-activated antibody of claim 1 or2, wherein the immune-activated antibody comprises a conjugate of formula (I)
Figure FDA0003229949250000132
A conjugate represented by the formula (I-1)
Figure FDA0003229949250000141
A conjugate represented by the formula (I-2)
Figure FDA0003229949250000142
A conjugate represented by the formula (I-3)
Figure FDA0003229949250000143
A conjugate represented by the formula (I-4)
Figure FDA0003229949250000144
A conjugate of formula (II)
Figure FDA0003229949250000145
A conjugate represented by the formula (II-1)
Figure FDA0003229949250000151
A conjugate represented by the formula (II-2)
Figure FDA0003229949250000152
A conjugate represented by the formula (II-3)
Figure FDA0003229949250000153
A conjugate represented by the formula (II-4)
Figure FDA0003229949250000154
A conjugate of formula (III)
Figure FDA0003229949250000155
Is represented by the formula (III-1)Conjugates
Figure FDA0003229949250000161
A conjugate represented by the formula (III-2)
Figure FDA0003229949250000162
A conjugate represented by the formula (III-3)
Figure FDA0003229949250000163
A conjugate represented by the formula (III-4)
Figure FDA0003229949250000164
A conjugate of formula (IV)
Figure FDA0003229949250000165
A conjugate represented by the formula (IV-1)
Figure FDA0003229949250000171
A conjugate represented by the formula (IV-2)
Figure FDA0003229949250000172
A conjugate represented by the formula (IV-3)
Figure FDA0003229949250000173
A conjugate represented by the formula (IV-4)
Figure FDA0003229949250000174
And n is greater than 0.
9. The immune-activated antibody of claim 1 or2, wherein the immune-activated antibody comprises at least one of compound 15, compound 15-1, compound 15-2, compound 17-1, compound 21, compound 22-2, compound 24-2, compound 15-3, compound 15-4, compound 15-5, compound 24-3, compound 28, compound 29-1, compound 30-1, compound 31, compound 32, compound 33, compound 34, compound 35, compound 36, compound 37, said compound 15-1, said compound 15-2, said compound 17, compound 17-1, compound 17-2, The structural formulas of the compound 21, the compound 22-2, the compound 24-2, the compound 15-3, the compound 15-4, the compound 15-5, the compound 24-3, the compound 28, the compound 29-1, the compound 30-1, the compound 31, the compound 32, the compound 33, the compound 34, the compound 35, the compound 36 and the compound 37 are respectively as follows:
Figure FDA0003229949250000181
Figure FDA0003229949250000191
Figure FDA0003229949250000201
Figure FDA0003229949250000211
Figure FDA0003229949250000221
Figure FDA0003229949250000231
Figure FDA0003229949250000241
10. use of an immune-activated antibody according to any one of claims 1 to 9 in the manufacture of a formulation for anti-tumor, anti-viral, immunomodulatory and/or elimination of a target protein.
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