CN112957463A - Immune activation type antibody and application thereof - Google Patents

Immune activation type antibody and application thereof Download PDF

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CN112957463A
CN112957463A CN202110196237.1A CN202110196237A CN112957463A CN 112957463 A CN112957463 A CN 112957463A CN 202110196237 A CN202110196237 A CN 202110196237A CN 112957463 A CN112957463 A CN 112957463A
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compound
formula
immune
antibody
conjugate
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靳广毅
王竹林
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Shenzhen Kangjuzheng Medical Technology Co ltd
Shenzhen University
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Shenzhen Kangjuzheng Medical Technology Co ltd
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Priority to CN202110984030.0A priority patent/CN113663067A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators

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) 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 BDA0002946744030000011
Figure BDA0002946744030000021
the immune activator and the antibody are coupled through the coupling chain, so that a series of immune activation type antibodies targeting specific tissues, focuses and targets can be formed, the effect 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 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, immune regulation 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, anti-virus drugs, immune regulation drugs and/or target protein elimination preparations, 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 is improved), converting 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.
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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 of the degradation of an enzyme (Cathepsin-B) acting on a TLR7 agonist, represented by HO-VC-T, according to an embodiment of the invention;
fig. 13 is a schematic diagram of a general structural formula of an immune-activating antibody provided in the embodiment of the present invention.
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. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer; the reagents or instruments used are not indicated by the manufacturer, and are 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 may 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 of each component, 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 BDA0002946744030000041
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 activation type antibodies targeting specific tissues, focuses and targets can be formed, the effect 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 the environment of common immune cells 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 for connection in the art, 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 listed 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 BDA0002946744030000061
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 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 BDA0002946744030000071
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 BDA0002946744030000072
6-amino-2-butoxy-9-(cyanomethyl)-N-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)h exanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-8-oxo-8,9-dihydro-7H-purine-7-ca rboxamide
Figure BDA0002946744030000073
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)hex anamide
Figure BDA0002946744030000074
4-(((S)-1-(((S)-1-((4-(((6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carbo nyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amin o)-4-oxobutanoic acid
Figure BDA0002946744030000081
4-(((S)-1-(((S)-1-((4-((6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carbo xamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino) -4-oxobutanoic acid
Figure BDA0002946744030000082
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-oxopr opan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid
Figure BDA0002946744030000083
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 BDA0002946744030000091
4-((2S,5S)-5-isopropyl-17-isothiocyanato-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazahep tadecanamido)benzyl
6-amino-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carboxylate
Figure BDA0002946744030000092
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-ca rboxamide
Figure BDA0002946744030000093
4-((2S,5S)-5-isopropyl-17-isothiocyanato-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazahep tadecanamido)benzyl
6-amino-2-butoxy-9-(cyanomethyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxylate
Figure BDA0002946744030000094
(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-urei dopentan-2-yl)-2-(2-(2-(2-(2-isothiocyanatoethoxy)ethoxy)ethoxy)acetamido)-3-methylbutanamide
Figure BDA0002946744030000101
(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-ureidopentanam ide
Figure BDA0002946744030000102
(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 BDA0002946744030000103
4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecana mido)benzyl
6-amino-2-butoxy-9-(cyanomethyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxylate
Figure BDA0002946744030000104
6-amino-N-(4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diaza heptadecanamido)benzyl)-2-butoxy-9-(cyanomethyl)-8-oxo-8,9-dihydro-7H-purine-7-carboxamide
Figure BDA0002946744030000111
6-amino-N-(4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diaza heptadecanamido)benzyl)-2-butoxy-8-oxo-9-(prop-2-yn-1-yl)-8,9-dihydro-7H-purine-7-carboxamid e
Figure BDA0002946744030000112
6-amino-N-(4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diaza heptadecanamido)benzyl)-2-butoxy-9-((1-cyclooctyl-1H-1,2,3-triazol-4-yl)methyl)-8-oxo-8,9-dihyd ro-7H-purine-7-carboxamide
Figure BDA0002946744030000113
(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 BDA0002946744030000121
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-oxopr opan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide
Figure BDA0002946744030000122
(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 BDA0002946744030000123
(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-urei dopentan-2-yl)-2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetamido)-3-methylbutanamide
Figure BDA0002946744030000124
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 BDA0002946744030000131
4-(17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)be nzyl
6-amino-2-butoxy-9-((1-cyclooctyl-1H-1,2,3-triazol-4-yl)methyl)-8-oxo-8,9-dihydro-7H-purine-7-c arboxylate
Figure BDA0002946744030000132
4-(17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)be nzyl
6-amino-2-butoxy-9-((1-(1-((2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-meth yl-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 BDA0002946744030000133
4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecana mido)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 BDA0002946744030000141
(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-ureidopentanam ide
Figure BDA0002946744030000142
N2-(4-(((S)-1-(((S)-1-((4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phenyl)amino)-1-o xo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoyl)-N6-diazolysine
Figure BDA0002946744030000143
N1-(2-(2-(2-(2-(4-(((2-((2-acrylamido-5-methoxy-4-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)am ino)phenyl)(methyl)amino)ethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethox y)ethyl)-2-(14-(4-((6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)phenyl)-7-(4-a zidobutyl)-6,9,12-trioxo-2-thia-5,8,13-triazatetradecyl)-N4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxois oindolin-4-yl)succinamide
Figure BDA0002946744030000151
N1-(2-(2-(2-(2-(4-(((2-((2-acrylamido-5-methoxy-4-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)am ino)phenyl)(methyl)amino)ethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethox y)ethyl)-2-((14S,17S)-22-amino-17-((4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phen yl)carbamoyl)-7-(4-azidobutyl)-14-isopropyl-6,9,12,15,22-pentaoxo-2-thia-5,8,13,16,21-pentaazado cosyl)-N4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)succinamide。
the embodiment of the invention also provides a series of intermediate compounds for synthesizing the coupling chain specific 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 BDA0002946744030000152
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 BDA0002946744030000161
4-(17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)be nzyl(4-nitrophenyl)carbonate
Figure BDA0002946744030000162
6-amino-9-((1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-2-buto xy-N-methyl-8-oxo-N-propyl-8,9-dihydro-7H-purine-7-carboxamide
Figure BDA0002946744030000163
(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-carboxa mido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbama
Figure BDA0002946744030000164
(S)-N-(4-(aminomethyl)phenyl)-2-((S)-14-azido-2-isopropyl-4-oxo-6,9,12-trioxa-3-azatetradecana mido)-5-ureidopentanamide
Figure BDA0002946744030000171
4-nitrophenyl
(4-((2S,5S)-17-azido-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecan amido)benzyl)carbamate
Figure BDA0002946744030000172
(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-o xo-5-ureidopentan-2-yl)-3-methylbutanamide
Figure BDA0002946744030000173
(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-o xo-5-ureidopentan-2-yl)-3-methylbutanamide
Figure BDA0002946744030000174
N1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo lin-4-yl)-2-methylenesuccinamide
Figure BDA0002946744030000181
2-(((2-aminoethyl)thio)methyl)-N1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N4-(2-(2,6-dioxo piperidin-3-yl)-1,3-dioxoisoindolin-4-yl)succinamide
Figure BDA0002946744030000182
N1-(2-(2-(2-(2-(4-(((2-((2-acrylamido-5-methoxy-4-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)am ino)phenyl)(methyl)amino)ethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethox y)ethyl)-2-(((2-aminoethyl)thio)methyl)-N4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)s uccinamide
Figure BDA0002946744030000183
(S)-N-(4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-2-((S)-2-amino-3-methylbu tanamido)-5-ureidopentanamide
Figure BDA0002946744030000184
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 BDA0002946744030000191
(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 BDA0002946744030000192
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)hex anamide
Figure BDA0002946744030000193
(S)-N-(4-((6-amino-2-butoxy-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-2-((S)-2-isopropyl-14-isoth iocyanato-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 BDA0002946744030000194
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, HPV16E7, CEA, IMA910, KOC1, SL-701, MART-1, gp100, tyrosinase, GSK 1, survivin, MAGE-3.1, MAGE-10. A1, gp209-2 1-A, NAGE 17. Aa2, KOC1, CO1, DEBTOCH 1, MPGE HOR 1, MAGE-10. ONT 1, MUC1, PGR 3, PSRCK-1, PSRCK 1, PSRCK 1, PSRCK 1, PSRCK 1, 36, At least one of 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, PSGL-1, Frizzled-7, SLC4A, CCR, CXCR, CCL, CXCL, CD79, Nectin-4, CD117, PSMA, NKG2, Claudin18.2, MG, ROR, FGFR, WNT2, WNT3, WNT5, WNT9, WNT7, HGF, LIGB, CAUD, RADG 18.2, REG, HRPG, HRTF, HREP, HRTF, HREP, RG, HRTF, RG, HREP-6, HREP, RG, HRRB, HRTF-6, and HRRB. 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 targeting and 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 BDA0002946744030000201
A conjugate represented by the formula (I-1)
Figure BDA0002946744030000211
A conjugate represented by the formula (I-2)
Figure BDA0002946744030000212
A conjugate represented by the formula (I-3)
Figure BDA0002946744030000213
A conjugate represented by the formula (I-4)
Figure BDA0002946744030000214
A conjugate of formula (II)
Figure BDA0002946744030000215
A conjugate represented by the formula (II-1)
Figure BDA0002946744030000221
A conjugate represented by the formula (II-2)
Figure BDA0002946744030000222
A conjugate represented by the formula (II-3)
Figure BDA0002946744030000223
A conjugate represented by the formula (II-4)
Figure BDA0002946744030000224
A conjugate of formula (III)
Figure BDA0002946744030000225
A conjugate represented by the formula (III-1)
Figure BDA0002946744030000231
A conjugate represented by the formula (III-2)
Figure BDA0002946744030000232
A conjugate represented by the formula (III-3)
Figure BDA0002946744030000233
A conjugate represented by the formula (III-4)
Figure BDA0002946744030000234
A conjugate of the formula (IV)
Figure BDA0002946744030000235
A conjugate represented by the formula (IV-1)
Figure BDA0002946744030000241
A conjugate represented by the formula (IV-2)
Figure BDA0002946744030000242
A conjugate represented by the formula (IV-3)
Figure BDA0002946744030000243
A conjugate represented by the formula (IV-4)
Figure BDA0002946744030000244
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 BDA0002946744030000251
Figure BDA0002946744030000261
Figure BDA0002946744030000271
Figure BDA0002946744030000281
Figure BDA0002946744030000291
Figure BDA0002946744030000301
Figure BDA0002946744030000311
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 compound and 1.2 g of bromopropyne were dissolved in 50mL of DMF, and 1.5 g of K was added2CO3The mixture was stirred at room temperature for 12 hours. Filtration was carried out, the solvent was removed from the filtrate by distillation under the reduced pressure (less than 60 ℃ C.), and 25mL of concentrated hydrochloric acid was added to the residue, followed by stirring at room temperature for 10 hours. K for reactants2CO3Neutralizing to pH7, precipitating to obtain crude solid product, dissolving in methanol, separating with silica gel column chromatography (methanol: dichloromethane: 1: 10 volume ratio), collecting product solution, concentrating under reduced pressure to remove solvent to obtain white solid product (compound 1), ESI-MS: M/z 263.10[ M + H ] M-MS]+。
The reaction scheme for the synthesis of compound 1 from compound Pro-1 is as follows:
Figure BDA0002946744030000312
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 BDA0002946744030000313
The structural formula of the compound 10 is
Figure BDA0002946744030000321
Synthesis of representative compound 15 of formula III exemplified by HER2 antibody:
Figure BDA0002946744030000322
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 was precipitated by centrifugation and purified by HPLC to give compound 12(498mg, 65%) and ESI-MS: M/z 767.3[ M + H ] +.
In the same manner, compound 1 is substituted for compound 4 to give 12-2:
Figure BDA0002946744030000331
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 BDA0002946744030000332
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 BDA0002946744030000341
coupling synthesis of reference compound 15, substitution of compound 7 with compound 7-1 gives the analog compound 15-1:
Figure BDA0002946744030000342
further conjugation of HER2 antibody with compound 7-3 gave compound 15 analogue compound 15-2:
Figure BDA0002946744030000343
the synthetic route of compound 7-3 is as follows:
Figure BDA0002946744030000351
replacing BNCOOH with succinic anhydride, and reacting with compound VC100 to directly obtain compound 6-3:
Figure BDA0002946744030000352
the synthesis procedure of compound 5-1 is as follows:
Figure BDA0002946744030000361
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 under reduced pressure to dissolve, 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 and added to a 1mL TFA/DCM (1:3 vol.) chamberThe reaction was shaken for 2 hours, then vacuum-dried and dissolved, and the residue was purified by HPLC to obtain MM-VCA-NH2, 238mg, yield 65%, 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 BDA0002946744030000371
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 ] +.
200mg of compound 4 and an equimolar amount of compound 4A were mixed in 2mL of DMSO, 2 equivalents of TEA were added, shaking was carried out at room temperature for 8 hours, an equivalent amount of compound FVC-2 was added, the reaction was continued overnight at room temperature, 4-fold equivalent amount of piperdine was added, and the reaction was carried out 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 BDA0002946744030000381
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 compound 8:
Figure BDA0002946744030000391
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 BDA0002946744030000401
the activated ester 20(20eq) of DBCO-acid and HER2 antibody (1eq) were mixed in pure water containing 10% DMSO and reacted with shaking at 10 ℃ for 12 hours. Mass spectrometry detection was complete for HER2 antibody response. The reaction mixture was filtered through a 10kD molecular filter to remove small molecules and the conjugate compound 21 was eluted with DPBS solution (degree of coupling was measured to be 6). 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 BDA0002946744030000411
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 BDA0002946744030000421
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 6:
Figure BDA0002946744030000431
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 BDA0002946744030000441
200mg of compound 19, 358mg of compound 2A, 176mg of HOBT,254mg of EDC, 450. mu.L of DIPEA were dissolved in 5ml of DMMSO 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) with a yield of 41.2%. ESI-MS, M/z 594.3[ M + H ] +.
Figure BDA0002946744030000442
200mg of Compound 3A, 113mg of Compound 4A, 180. mu.L of DIPEA were dissolved in 2ml of DMMSO, 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), with a yield of 25.4%. ESI-MS, M/z 759.3[ M + H ] +.
Figure BDA0002946744030000451
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 BDA0002946744030000452
30mg of compound 5A, 42mg of compound GY127, 21. mu.L of DIPEA were dissolved in 1ml of DMMSO, 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 immunoactive antibody compound 24 can be prepared from compound GY207 and compound 21 as follows:
Figure BDA0002946744030000461
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 BDA0002946744030000462
referring to the synthesis of compound 15, substituting the agonist moiety with compound GY102, compound 15-4 can be obtained:
Figure BDA0002946744030000471
substitution of the antibody for ASGPR1 antibody and the immune agonist for compound GY102-3 gave compound 15-3:
Figure BDA0002946744030000472
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 antibody, and the synthesis method of the small molecule immune agonist compound 102-3 is as follows:
Figure BDA0002946744030000473
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 BDA0002946744030000481
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 BDA0002946744030000482
synthesis of conjugated antibody representative compound 28 of formula II:
Figure BDA0002946744030000491
the compound 26 and the compound GY102 are dissolved in DMSO in an equivalent manner, the mixed solution is stirred at room temperature, and the mass spectrum detection shows 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 conjugated 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 BDA0002946744030000501
method for synthesizing conjugated antibody compound 30:
Figure BDA0002946744030000502
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 BDA0002946744030000511
the same procedure gave compounds 5-2 and 7-3:
Figure BDA0002946744030000512
in the same route as the synthesis of compound 30, antibody compound 30-1:
Figure BDA0002946744030000521
the synthesis method of the compound 8-2 comprises the following steps:
Figure BDA0002946744030000531
an equivalent amount of compound Val1 and compound 1A generates compound Val2 in DMSO of HOBT, EDC and DIPEA, and generates compound Val3 after acting on TFA, and then generates compound Val4 with compound 6A under the action of HOBT, EDC and DIPEA, and generates compound Val5 after deprotection. 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 BDA0002946744030000541
the synthesis of compound 7-1 can be achieved analogously to compound 8-2:
Figure BDA0002946744030000551
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 the deprotection reaction was completed by adding 100mg of Compound 14-1 to 2mL of TFA/DCM (1:3 vol), and monitoring by mass spectrometry, the reaction solution was distilled off in vacuo 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 (monitoring by 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 BDA0002946744030000561
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 BDA0002946744030000571
synthetic route for antibody compound 34:
Figure BDA0002946744030000581
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, and then removing TFAProtecting group, purifying by HPLC to obtain 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 BDA0002946744030000591
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 pomalidomide/osimertinib/SZU-160:
Figure BDA0002946744030000601
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. The compounds of ICO3N3 and pomalidomide are condensed in DMSO by a condensing agent (HOBt/EDC), and then separated and purified by HPLC to obtain the compound POMA-ICO3N 3. Dissolving the compound POMA-ICO3N3 in DMSO, adding mercaptoethylamine with the same mole, sealing, stirring overnight at room temperature, and directly freeze-drying the reactant to obtain S-POMA-ICO3N 3. 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 by HPLC directly to give the compound Bi-linker,91mg (51%), ESI-MS: M/z: 1186.50[ M + H ], []+. 70mg of the compound Bi-linker was dissolved in dry 1mL of DMSO, and 36mg of the compound SZU-160, and 1.1 equivalents of H were addedOBt/EDC/DIPEA, concusse the reaction overnight at room temperature. The reaction mixture was directly freeze-dried, dissolved in a small amount of isopropanol, and purified by HPLC to give Tri-linker, a pure compound, 69mg (66% yield), ESI-MS: M/z-1766.79 [ M + H ]]+。
Synthesis of antibody compound 35:
Figure BDA0002946744030000621
10% DMSO by volume is added into DPBS solution of the conjugate compound 21, 1.5 equivalent of compound Tri-linker-1 is added, the mixture is shaken at 25 ℃ for reaction for 12 hours, and small molecules are removed by a 20KD molecular filter. The DPBS dissolves the antibody to give a solution of DPBS 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 BDA0002946744030000631
Figure BDA0002946744030000641
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 2007.20[ M + H ]]+(ii) a 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 BDA0002946744030000651
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 give 0.85g of a white solid compound 39 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 ]]+。
Hydrochloride salt of compound 40 (0.5g, 2.2mmol) was dissolved in 5mL of DMSO,adding succinic anhydride equivalent and TEA equivalent 2, stirring at 60 deg.C for 4 hr, freeze drying the reaction product, and dissolving the resultant solid in saturated Na2CO3In the solution, the pH was adjusted with hydrochloric acid 4 to precipitate a solid, which was filtered and vacuum-dried to obtain compound 41, 0.42g, yield 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 BDA0002946744030000661
N3-VC-T synthesis:
Figure BDA0002946744030000671
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 BDA0002946744030000672
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 BDA0002946744030000681
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 is distilled under reduced pressure (60 ℃ C.) to remove DMF, and the residue is addedSaturated Na2CO3Dissolving, 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 BDA0002946744030000691
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 BDA0002946744030000701
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 BDA0002946744030000702
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, the immune activation type antibody can be used for preparing anti-tumor drugs, antiviral drugs, immunoregulation drugs and/or target protein elimination preparations, can avoid the side effect of non-specific killing on the damage of normal tissues, and 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 make the above implementation details and operation of the present invention clearly understood by those skilled in the art and to make the progress of the immune activated antibody and its application obvious in the embodiments of the present invention, the above technical scheme is 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 mu g of a sample to be tested was put into an EP tube, 2 mu L of GlycoBuffer 2(10X) was added thereto, and ultrapure water was added thereto so that the final volume of the system became 20 mu L. Subsequently, 3. mu.L of LPNGase 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 BDA0002946744030000711
2. Reagent and test solution
Figure BDA0002946744030000712
Figure BDA0002946744030000721
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 TOFMS
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 plate and cell suspensions were plated onto 96-well cell culture plates using HEK-blue (tm) precipitation solution (purchased from Invivo Gen) adjusted to 2.5 × 104/180 μ L per well. 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 ofhree human HER2/neu-expressing hormone cell lines In immunological competence mics", Lab Anim Sci, 1999 Apr; 49(2): 179-88.).
Establishing a colorectal cancer tumor model of a mouse: digestion and separationHer2+ CT26 cells were collected from the heart at the logarithmic growth phase, 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 air bubbles, injecting the cell suspension under the back skin of the mouse, and starting to administer the drug when the diameter of the tumor reaches 4-5 mm.
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 both n: 1), and an immune-activated antibody group, with 8 mice per group. Injections (solvent component: 5% DMSO; + 40% PEG 300; + 5% Tween 80; + 50% PBS) were formulated at dosing doses of 3mg/kg for the TLR7 agonist, 20mg/kg for the HER2 antibody, 20mg/kg for the TLR7 agonist and HER2 antibody (3mg/kg +20mg/kg), 20mg/kg for the TLR7-HER2 conjugate antibody, injected peritumorally at a volume of 100. mu.L each. 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, results are 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 is a graph showing the relative amounts of CD8+ IFN-. gamma. + T cells in tumor tissues at day 11 of each administration group.
Example 5CCK8 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
Detection of the activation effect of compounds SZU-164 and HO-VC-T, N3-VC-T, MA-VC-T, SVC-T on TLR7 (HEK-BlueTMDetection):
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 may produce or lose activation of TLR7 depending on the small molecule product after degradation.
The degradation of an enzyme represented by HO-VC-T (Cathepsin-B) acting on a TLR7 agonist is schematically shown in FIG. 12.
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 FDA0002946744020000011
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, 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 bvtr-4, compound x-linker-1, compound y-2, compound y-4, compound y-2, compound y, At least one of the compounds Tri-linker-2, the compound 5-1, the compound 5-2, 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 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 5, The structural formulas of 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 FDA0002946744020000021
Figure FDA0002946744020000031
Figure FDA0002946744020000041
Figure FDA0002946744020000051
Figure FDA0002946744020000061
Figure FDA0002946744020000071
Figure FDA0002946744020000081
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 FDA0002946744020000082
Figure FDA0002946744020000091
Figure FDA0002946744020000101
Figure FDA0002946744020000111
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 FDA0002946744020000121
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, cyclinB, WT-1, CEF, VEGFR, TTK, MUC, HPV16E, 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, FOLR-II, FORCL-107, FORCL-17, FORCL, FORCA, FORCL-17, FORCA, FORCL, FORCA, FO, At least one of GPA, ALK, ROS, BRAF, MEK, RET, CDK/6, BRCA, PARP, BRCA, FLT, CD, BCL, CD, Smoothened, GD, EZH, MTH, KRAS, c-MYC, hCAIX, hCAXII, 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, BRC 4A, CCR, CXCR, CCL, CXCL, CD, WN, CD79, Nenn-4, CD117, PSMA, NKG2, Claudin18.2, Claudin, RORR, ROR, FGFR, WNT2, RET, CDK/6, CRF, RG, RGF, RG-7, CRYb, RG-7, CRZ, RG-7, RG, and RG-7.
8. The immune-activated antibody of claim 1 or2, wherein the immune-activated antibody comprises a conjugate of formula (I)
Figure FDA0002946744020000122
A conjugate represented by the formula (I-1)
Figure FDA0002946744020000131
A conjugate represented by the formula (I-2)
Figure FDA0002946744020000132
A conjugate represented by the formula (I-3)
Figure FDA0002946744020000133
A conjugate represented by the formula (I-4)
Figure FDA0002946744020000134
A conjugate of formula (II)
Figure FDA0002946744020000135
A conjugate represented by the formula (II-1)
Figure FDA0002946744020000141
A conjugate represented by the formula (II-2)
Figure FDA0002946744020000142
A conjugate represented by the formula (II-3)
Figure FDA0002946744020000143
A conjugate represented by the formula (II-4)
Figure FDA0002946744020000144
A conjugate of formula (III)
Figure FDA0002946744020000145
A conjugate represented by the formula (III-1)
Figure FDA0002946744020000151
A conjugate represented by the formula (III-2)
Figure FDA0002946744020000152
A conjugate represented by the formula (III-3)
Figure FDA0002946744020000153
A conjugate represented by the formula (III-4)
Figure FDA0002946744020000154
A conjugate of formula (IV)
Figure FDA0002946744020000155
A conjugate represented by the formula (IV-1)
Figure FDA0002946744020000161
A conjugate represented by the formula (IV-2)
Figure FDA0002946744020000162
A conjugate represented by the formula (IV-3)
Figure FDA0002946744020000163
A conjugate represented by the formula (IV-4)
Figure FDA0002946744020000164
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, 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, compound 15-1, compound 15-2, compound 17, compound 21, compound 22-2, compound 15-2, compound 17, compound 21, compound 22-2, compound 15-2, or compound 37, The structural formulas of the compound 24, 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, 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 FDA0002946744020000171
Figure FDA0002946744020000181
Figure FDA0002946744020000191
Figure FDA0002946744020000201
Figure FDA0002946744020000211
Figure FDA0002946744020000221
Figure FDA0002946744020000231
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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CN114533895A (en) * 2021-10-25 2022-05-27 深圳市康居正医药科技有限公司 Antibody-conjugated compound and application thereof
WO2023104214A1 (en) * 2021-12-10 2023-06-15 苏州泽璟生物制药股份有限公司 Multi-specific t cell engagers comprising lrrc15 antigen-binding domain

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