Immunoconjugation drug, and pharmaceutical composition and application thereof

Abstract

The invention belongs to the field of precise diagnosis and treatment, and particularly relates to an immunoconjugation medicine, a medicine composition based on the immunoconjugation medicine and application of the immunoconjugation medicine in the field of tumor treatment. Specifically, the immunoconjugation drug comprises a targeted tumor fragment (TTF) combined with a tumor marker in a targeted manner and a targeted immune cell fragment (ITF) combined with an immune cell surface molecule, the two fragments are connected through a linker fragment (Lk), the tumor cells can be targeted and the immune cells can be connected at the same time, the immune cells do not need to be transfected, and the immunoconjugation drug can be applied to the immunoconjugation drug. The immune cells can be activated, and the capabilities of targeting tumors and killing the tumors are obtained.

Description

Technical Field

[0001] The present invention belongs to the field of precision diagnosis and treatment, and particularly relates to an immunoconjugate drug, a pharmaceutical composition based on the immunoconjugate drug, and its application in the field of tumor treatment. Background Art

[0002] Malignant tumors are a class of diseases that seriously endanger human physical and mental health and affect the social labor force, bringing a heavy economic burden to numerous families and society. According to the "Cancer Statistics Report for the Year 2023" released by the American Cancer Society (ACS), it is estimated that there will be approximately 1,958,310 new cancer cases in the United States in 2023, equivalent to approximately 5,370 new cases per day. And the number of new cancer death cases will reach 609,820, equivalent to 1,670 people dying from cancer every day. Therefore, cancer remains a major killer threatening human life and health.

[0003] Currently, the treatment methods for tumors include traditional radiotherapy and chemotherapy, as well as targeted therapy and immunotherapy that have emerged in recent years. Immunotherapy refers to the treatment concept of specifically killing tumors by activating the body's own immune system. Compared with other treatment methods, the advantage of immunotherapy lies in establishing a memory effect against tumors and prolonging the cure time of patients. In existing immunotherapy, NK cells and T cells are the immune cells commonly used to activate and attack tumors.

[0004] Natural Killer (NK) cells are part of the human immune system. They are a type of large granular lymphocytes that can quickly recognize and kill infected pathogens or abnormal cells, including cancer cells and virus-infected cells.

[0005] The following are some important characteristics of NK cells:

[0006] Natural cytotoxicity: NK cells have spontaneous cytotoxic activity and do not require prior activation. They can directly recognize and kill target cells without the need to adapt after prior exposure to pathogens.

[0007] Recognition mechanism: NK cells recognize targets by interacting with receptors and ligands on the surface of target cells. They can recognize cells infected by certain viruses, mutated cells, and certain cancer cells and mount an immune response against them.

[0008] Immune regulation: NK cells play an important role in anti-pathogen and anti-tumor immunity. They can produce cytokines, such as interferon γ (IFN-γ), thereby enhancing the immune response. At the same time, NK cells can also interact with other immune cells to regulate the function of the entire immune system.

[0009] Activating and inhibitory signals: NK cells determine whether a target cell should be killed through activating and inhibitory receptors on their surface. When activating receptors bind to ligands, they release toxins and cytokines, leading to the death of the target cell.

[0010] CD16 is a cell surface molecule, also known as FcγRIII (Fcγ receptor III), which plays an important role in the human immune system. CD16 belongs to the Fcγ receptor family in the immunoglobulin superfamily and can bind to the Fc region of immunoglobulin G (IgG). CD16 is mainly present on the surface of natural killer cells (NK cells) and macrophages. It mediates the effector functions of cells, such as cytotoxicity, phagocytosis, and cell activation, by binding to IgG antibodies. There are two types of CD16 in the human body, namely CD16a and CD16b. CD16a is an activating receptor that can trigger the activation response of immune cells through cell signal transduction mechanisms. CD16b is a low-affinity Fcγ receptor that mainly participates in immune regulation and the clearance of immune complexes in the circulation. CD16 is of great significance in the study of immune-related diseases, infections, and cancer immunotherapy. It can be used as a marker to identify different types of immune cells and to evaluate their functional status. In addition, immunotherapy strategies targeting CD16 are also under investigation to enhance the activity and effector functions of immune cells.

[0011] PSMA (Prostate-Specific Membrane Antigen) is a prostate-specific membrane antigen that is highly expressed in prostate tissue. It is a membrane-bound enzyme that is mainly present on the cell membrane of prostate epithelial cells. PSMA is widely used clinically as a biomarker for prostate cancer, especially in early diagnosis, disease assessment, and treatment monitoring. Since the expression level of PSMA in normal tissues is relatively low, while it is significantly upregulated in prostate cancer cells, the diagnosis and staging of prostate cancer can be assisted by detecting serum PSMA levels or using PSMA-targeted imaging techniques. In addition, PSMA is also a target in treatment strategies for prostate cancer. PSMA-based targeted therapies include radionuclide therapy and immunotherapy. Radionuclide therapy uses anti-PSMA antibodies or small molecule ligands conjugated to radioactive isotopes to deliver radiation directly to prostate cancer cells to achieve therapeutic effects. Immunotherapy uses immune cells or antibodies against PSMA to identify and eliminate prostate cancer cells.

[0012] Currently, the genetic modification of immune cells used clinically requires the use of engineered viruses to randomly integrate payload DNA into the genome of donor cells, which has the possibility of causing unregulated cell growth and carcinogenesis. The most effective method of delivering this DNA is through adeno-associated virus (AAV), but the production of AAV is expensive and complex. Additionally, the co-delivery of double-stranded DNA templates with CRISPR / Cas has been shown to be toxic to primary cells. Although these novel genome editing techniques can provide precise manipulation of the immune cell genome, they may produce off-target effects or exhibit errors in genome rearrangements, so more research is needed to accurately determine the risks of these editing errors.

[0013] One of the greatest challenges in the manufacturing of immune cell therapies is the use of viral transduction to deliver DNA payloads, which is a complex process with a high failure rate. To overcome this challenge, non-viral methods of gene delivery in primary human immune cells are being developed, such as mRNA transfection or transposon-based genome engineering tools.

[0014] Cell source variability is another challenge, especially when using patient-derived cells. The medical history or stage of the patient's disease can lead to T cell dysfunction and alter the immune cell composition, resulting in suboptimal T cell products during the manufacturing process. Many resources have been used to develop allogeneic "off-the-shelf" cell products, either through alternative cell types (e.g., NK cells, iPSC-derived immune cells), or through genome editing tools to disrupt proteins that cause allogeneic rejection (e.g., β2 microglobulin) and GvHD (e.g., endogenous TCR). However, these methods have their unique challenges. For example, NK cells are less suitable for viral transduction than T cells, and regulatory issues related to genome editing specificity have not been resolved. Summary of the Invention

[0015] Problems to be Solved by the Invention

[0016] In view of the above, the present invention aims to provide an immunoconjugate drug, a pharmaceutical composition based on the immunoconjugate drug, and its application in the field of tumor (especially prostate cancer) treatment.

[0017] Solutions for Solving the Problems

[0018] <First aspect>

[0019] The present invention provides an immunoconjugate drug or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled compound, active metabolite or prodrug thereof, and the immunoconjugate drug may comprise:

[0020] A tumor-targeting fragment that targets and binds to a tumor marker; and

[0021] A targeting immune cell fragment that binds to a surface molecule of an immune cell.

[0022] In one embodiment of the present invention, the targeting tumor fragment in the above immunoconjugate drug can target and bind to PSMA.

[0023] In one embodiment of the present invention, the targeting tumor fragment in the above immunoconjugate drug is in the form of an antibody.

[0024] In one embodiment of the present invention, the targeting tumor fragment in the above immunoconjugate drug is in the form of a ligand.

[0025] In one embodiment of the present invention, when the targeting tumor fragment in the above immunoconjugate drug is in the form of a ligand, the ligand can be formed by a polypeptide or a small molecule compound.

[0026] In one embodiment of the present invention, the targeting immune cell fragment in the above immunoconjugate drug can be in the form of an antibody.

[0027] In one embodiment of the present invention, the targeting immune cell fragment in the above immunoconjugate drug can be in the form of a monoclonal antibody.

[0028] In one embodiment of the present invention, the targeting tumor fragment and the targeting immune cell fragment in the above immunoconjugate drug can be connected by a linker fragment. At this time, the above immunoconjugate drug can include a targeting tumor fragment, a targeting immune cell fragment, and a linker fragment.

[0029] In one embodiment of the present invention, the above immunoconjugate drug can be composed of a targeting tumor fragment, a targeting immune cell fragment, and a linker fragment.

[0030] In one embodiment of the present invention, the targeting immune cell fragment in the above immunoconjugate drug can be connected to a targeting tumor fragment through a linker fragment.

[0031] In one embodiment of the present invention, the targeting immune cell fragment in the above immunoconjugate drug can be connected to multiple (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) targeting tumor fragments through multiple (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) linker fragments.

[0032] In one embodiment of the present invention, when the above immunoconjugate drug contains multiple linker fragments, the multiple linker fragments can be completely the same, partially the same, or completely different from each other.

[0033] In one embodiment of the present invention, when the above-mentioned immunoconjugate drug contains multiple tumor-targeted fragments, the multiple tumor-targeted fragments may be completely identical, partially identical, or completely different from each other.

[0034] In one embodiment of the present invention, the above-mentioned immunoconjugate drug may have a structure shown in Formula I,

[0035]

[0036] wherein

[0037] TTF represents a tumor-targeted fragment;

[0038] Lk represents a linking fragment;

[0039] ITF represents an immunocyte-targeted fragment;

[0040] n can be 2 - 12, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or any value within the above range.

[0041] In one embodiment of the present invention, n in the above Formula I can be 6 - 12, such as 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 or any value within the above range.

[0042] In one embodiment of the present invention, n in the above Formula I can be 11 - 12, such as 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12 or any value within the above range.

[0043] In one embodiment of the present invention, n in the above Formula I can be 12. At this time, the immunoconjugate drug may have a structure shown in Formula I-1,

[0044]

[0045] wherein

[0046] TTF represents a tumor-targeted fragment;

[0047] Lk represents a linking fragment;

[0048] ITF represents an immunocyte-targeted fragment.

[0049] In one embodiment of the present invention, each TTF in the above formula I or formula I-1 can independently target and bind to a tumor marker (TM).

[0050] In one embodiment of the present invention, each TTF in the above formula I or formula I-1 can independently target and bind to the tumor marker PSMA.

[0051] In one embodiment of the present invention, each TTF in the above formula I or formula I-1 can independently be in the form of an antibody.

[0052] In one embodiment of the present invention, each TTF in the above formula I or formula I-1 can independently be in the form of a ligand.

[0053] In one embodiment of the present invention, when the TTF in the above formula I or formula I-1 is in the form of a ligand, each TTF can independently be formed from a polypeptide or a small molecule compound.

[0054] In one embodiment of the present invention, the above-mentioned multiple tumor-targeting fragments or each TTF in the above formula I or formula I-1 can independently be formed from a targeting ligand compound represented by the following formula,

[0055] Preferably

[0056] wherein

[0057] PG1 is hydrogen or a C1-C6 alkyl group, preferably a C1-C6 alkyl group, more preferably a tert-butyl group;

[0058] R Lg1 is hydrogen or wherein R1 is a hydroxyl group (i.e., -OH) or an amino-substituted C1-C6 alkylamino group (for example, 6-aminohexylamino, i.e., ).

[0059] In one embodiment of the present invention, the above-mentioned multiple tumor-targeting fragments or each TTF in the above formula I or formula I-1 can independently be formed from any one of the following targeting ligand compounds.

[0060]

[0061] In one embodiment of the present invention, the linker fragment in the above immunoconjugate drug (or, Lk in formula I or formula I-1) can serve as an intermediate fragment to connect the tumor-targeting immune cell fragment (or, ITF) and the tumor-targeting fragment (or, TTF), and it can be either cleavable (or degradable) or non-cleavable (or non-degradable), preferably non-cleavable (or non-degradable).

[0062] In one embodiment of the present invention, each of the plurality of linker fragments or each Lk in Formula I or Formula I-1 above may be independently formed from a linker compound represented by the following formula,

[0063]

[0064] wherein

[0065] R Lk1 is a hydroxyl group or wherein R2 is hydrogen or

[0066] In one embodiment of the present invention, each of the plurality of linker fragments or each Lk in Formula I or Formula I-1 above may be independently formed from any one of the following linker compounds.

[0067]

[0068] In one embodiment of the present invention, the ITF in Formula I or Formula I-1 above may be in the form of an antibody.

[0069] In one embodiment of the present invention, the ITF in Formula I or Formula I-1 above may be in the form of a monoclonal antibody.

[0070] In one embodiment of the present invention, the ITF in Formula I or Formula I-1 above may be in the form of an anti-CD16 monoclonal antibody.

[0071] Except for having a group capable of covalently binding to a linker compound, the present invention does not specifically limit the structure of the above monoclonal antibody (especially the anti-CD16 monoclonal antibody). For example, the monoclonal antibody (especially the anti-CD16 monoclonal antibody) can covalently bind to the carbon-carbon double bond (i.e., CH=CH) within the maleimide ring in the structure of other compounds (such as a separate linker compound or an intermediate compound formed by a linker compound and a targeting ligand compound) through a Michael addition reaction using the sulfhydryl group (i.e., -SH) in its own structure.

[0072] In the present invention, the TTF formed by the above-mentioned targeting ligand compound can be of various types, and the present invention does not make specific limitations thereon. For example, it can be a monovalent group obtained by removing a hydrogen atom from a primary amino group in the structure (preferably a primary amino group located at the end of the main chain), that is, a secondary amino group, which can be linked to the carboxyl group of other structural fragments in the conjugate drug precursor to form an amide group. Alternatively, it can be a monovalent group obtained by removing a hydroxyl group from a carboxyl group in the structure (preferably a carboxyl group located at the end of the main chain), that is, a carboxyl group, which can be linked to the secondary amino group of other structural fragments in the conjugate drug precursor to form an amide group.

[0073] In the present invention, the Lk formed by the above-mentioned linker compound can be of various types, and the present invention does not make specific limitations thereon. For example, it can be an amide group formed by the condensation reaction of the primary amino group in the linker compound (preferably a primary amino group located at the end of the main chain) with the carboxyl group in the targeting ligand compound, or it can be an amide group formed by the condensation reaction of the carboxyl group in the linker compound (preferably a carboxyl group located at the end of the main chain) with the primary amino group in the targeting ligand compound.

[0074] In the present invention, the TTF and Lk used to construct the immunoconjugate drug can be linked to each other through an amide group, and the amide group can be formed by the condensation reaction between a primary amino group and a carboxyl group. Generally, the above-mentioned condensation reaction can be carried out in the presence of a coupling reagent (such as, but not limited to, HATU, etc.) to activate the carboxylic acid into a better electrophilic reagent, thereby promoting the forward progress of the reaction.

[0075] <Second aspect>

[0076] The present invention provides a specific immunoconjugate drug or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled substance, active metabolite or prodrug thereof. The immunoconjugate drug can have a structure as shown in Formula II.

[0077]

[0078] Wherein, ITF represents a targeting immunocyte fragment.

[0079] In one embodiment of the present invention, the above-mentioned immunoconjugate drug can have a structure as shown in Formula II'.

[0080]

[0081] Wherein, ITF represents a targeting immunocyte fragment.

[0082] In one embodiment of the present invention, n in the above-mentioned Formula II or Formula II' can be 2-12, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or any value within the above range.

[0083] In one embodiment of the present invention, n in the above formula II or formula II' can be 6 - 12, such as 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 or any value within the above range.

[0084] In one embodiment of the present invention, n in the above formula II or formula II' can be 11 - 12, such as 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12 or any value within the above range.

[0085] In one embodiment of the present invention, n in the above formula II can be 12, and at this time the immunoconjugate drug can have the structure shown in formula II - 1,

[0086]

[0087] wherein, ITF represents a targeting immunocyte fragment.

[0088] In one embodiment of the present invention, n in the above formula II' can be 12, and at this time the immunoconjugate drug can have the structure shown in formula II - 1',

[0089]

[0090] wherein, ITF represents a targeting immunocyte fragment.

[0091] In one embodiment of the present invention, ITF in the above formula II, formula II', formula II - 1 or formula II - 1' can be in the form of an antibody.

[0092] In one embodiment of the present invention, ITF in the above formula II, formula II', formula II - 1 or formula II - 1' can be in the form of a monoclonal antibody.

[0093] In one embodiment of the present invention, ITF in the above formula II, formula II', formula II - 1 or formula II - 1' can be in the form of an anti - CD16 monoclonal antibody.

[0094] <Third aspect>

[0095] The present invention provides a pharmaceutical composition, which comprises the immunoconjugate drug or its pharmaceutically acceptable salt, ester, solvate, polymorph, isotope - labeled substance, active metabolite or prodrug described in <First aspect> or <Second aspect>.

[0096] Preferably, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0097] <Fourth aspect>

[0098] The present invention provides the use of the immunoconjugate drug or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled compound, active metabolite or prodrug thereof described in <First aspect> or <Second aspect> or the pharmaceutical composition described in <Third aspect> in the preparation of a drug for preventing and / or treating a disease or disorder.

[0099] <Fifth aspect>

[0100] The present invention provides the immunoconjugate drug or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled compound, active metabolite or prodrug thereof described in <First aspect> or <Second aspect> or the pharmaceutical composition described in <Third aspect>, which is used for preventing and / or treating a disease or disorder.

[0101] <Sixth aspect>

[0102] The present invention provides a method for preventing and / or treating a disease or disorder, the method comprising: administering a prophylactically and / or therapeutically effective amount of the immunoconjugate drug or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled compound, active metabolite or prodrug thereof described in <First aspect> or <Second aspect> or the pharmaceutical composition described in <Third aspect> to an individual in need thereof.

[0103] Preferably, in <Fourth aspect>, <Fifth aspect> and / or <Sixth aspect>, the disease or disorder is cancer, preferably prostate cancer.

[0104] <Seventh aspect>

[0105] The present invention provides a method for preparing an immunoconjugate drug, the preparation method comprising the following steps:

[0106] 1) Covalently connecting a targeting ligand compound with a linker compound to obtain a targeting ligand-linker intermediate;

[0107] 2) Covalently connecting the targeting ligand-linker intermediate in step 1) with an antibody (such as a monoclonal antibody) to obtain the immunoconjugate drug.

[0108] In one embodiment of the present invention, the targeting ligand compound in the above preparation method has the meaning as defined above.

[0109] In one embodiment of the present invention, the linker compound in the above preparation method has the meaning as defined above.

[0110] In one embodiment of the present invention, the antibody (such as a monoclonal antibody) in the above preparation method has the meaning defined above.

[0111] In one embodiment of the present invention, step 1) in the above preparation method is completed by a condensation reaction, preferably by a condensation reaction between an amino group (such as a primary amino group) and a carboxyl group.

[0112] In one embodiment of the present invention, step 2) in the above preparation method is completed by a Michael addition reaction, preferably by a Michael addition reaction between a thiol group and a carbon-carbon double bond (such as the carbon-carbon double bond in a maleimide ring).

[0113] Effects of the Invention

[0114] The immunoconjugate drug of the present invention (or its pharmaceutically acceptable derivative form, such as an addition salt) can connect immune cells while targeting tumor cells, and can activate immune cells and endow them with the ability to target and kill tumors without the need for transfection of immune cells. Description of the Drawings

[0115] Figure 1 1H NMR spectrum of compound 5 1

[0116] Figure 2 1H NMR spectrum of compound 9 1

[0117] Figure 3 TOF-MS spectrum of the immunoconjugate drug (compound CR)

[0118] Figure 4 Comparison chart of the proportion of NK cells before and after sorting

[0119] Figure 5 Effect diagram of the immunoconjugate drug (compound CR) promoting NK cells to kill tumor cells

[0120] Figure 6 Effect diagram of the immunoconjugate drug (compound CR) on tumor cells in the absence of NK cells

[0121] Figure 7 Effect diagram of the immunoconjugate drug (compound CR) promoting NK cells to aggregate around tumor cells and mediate the killing effect Detailed Description of the Invention

[0122] General Terms and Definitions

[0123] Unless otherwise stated, the terms used in the present invention have the following meanings.​​

[0124] In the present invention, a "target" or "drug target" refers to the binding site of a drug or ligand to a biological macromolecule in an organism, mainly involving receptors, enzymes, ion channels, transporters, the immune system, genes, etc. Among existing drugs, more than 50% of drugs or ligands target receptors, making receptors the most important and crucial targets; more than 20% of drugs or ligands target enzymes, especially enzyme inhibitors, which have a special status in clinical applications; about 6% of drugs or ligands target ion channels; 3% of drugs or ligands target nucleic acids; and the targets of 20% of drugs or ligands remain to be further studied.

[0125] In the present invention, a "conjugated drug" or "drug conjugate" refers to a pharmaceutically acceptable compound formed by connecting different functional parts through a linker part and capable of exerting corresponding activities, such as antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs), small molecule-drug conjugates (SMDCs), radioimmunoconjugates (RDCs), etc.

[0126] In the present invention, a "radioimmunoconjugate" refers to a conjugate compound formed by connecting an immune cell targeting part and a tumor cell targeting part through a connecting part (e.g., a linker fragment), which kills tumor cells by recruiting and stimulating immune cells; among them, the tumor cell targeting part can bind to a tumor target antigen, and the immune cell targeting part can bind to an antigen on effector cells / immune cells, thereby activating the effector cells / immune cells and enabling them to re-recognize and eliminate tumor cells; T cells and NK cells are relatively commonly used types of effector cells / immune cells, and the most common / commonly used surface targets are CD3 and CD16.

[0127] In the present invention, a "targeting ligand" refers to any molecule or moiety capable of targeting a target site, target tissue, target organ, target cell, or intracellular region of a target cell. In some embodiments, compared to a non-target site, non-target tissue, non-target organ, non-target cell, or intracellular region of a non-target cell, the targeting ligand causes a moiety linked to the targeting ligand to be distributed more in the target site, target tissue, target organ, target cell, or intracellular region of the target cell, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more. In some embodiments, compared to a conjugate compound or reagent without a targeting ligand, a conjugate compound with a targeting ligand is distributed more in the target site, target tissue, target organ, target cell, or intracellular region of the target cell, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more. In some embodiments, the targeting ligand is capable of triggering or promoting the specific binding of a conjugate compound containing such a targeting ligand to a target molecule, triggering or promoting the endocytosis of the conjugate compound by a target cell, triggering or promoting the enrichment of the conjugate compound around the target cell and / or entry into the target cell.

[0128] In the present invention, a "ligand" may include a variety of chemical molecules or polypeptides that have specific binding affinity for a selected target, which may be a cell surface protein (such as a cell surface receptor or cell surface antigen), a specific protein, a cell, a tissue, an organ, etc. In some embodiments, the ligand may specifically bind to a cell surface receptor. In some embodiments, the ligand may specifically bind to a cell surface antigen. In some embodiments, the ligand may specifically bind to a specific protein, and the specific protein may be disease-causing. In some embodiments, overexpression of the specific protein may cause a disease or the specific protein is a mutant protein that causes a disease. In some embodiments, the ligand of the present application binds to the target with an affinity of 10 -6 ~10 -11 M (K d value). In some embodiments, the ligand of the present application binds to the target with an affinity of at least 10 -6 、at least 10 -7 、at least 10 -8 or at least 10 -9 M (K dThe affinity of the [value] binds to the target. In some embodiments, the ligand of the present application binds to the target with a certain affinity, and the certain affinity means that the affinity of the ligand for the target is at least two times, three times, four times, five times, six times, eight times, ten times, twenty times, fifty times, one hundred times or more times higher than the affinity for non-targets (such as other cell surface receptors, cell surface antigens or specific proteins, etc.). In some embodiments, the expression of the cell surface receptor, cell surface antigen, or specific protein of the present application on the surface or inside of target cells (such as cancer cells or cells with abnormal physiological functions) is significantly higher than that in normal cells. The term "significant" used in the present application refers to a statistically significant difference or a significant difference that can be recognized by those skilled in the art.

[0129] In the present invention, "PSMA", namely prostate-specific membrane antigen, refers to a type II transmembrane glycoprotein present in the prostate epithelial cell membrane, consisting of 750 amino acids, with 19 intracellular region amino acids, 24 transmembrane region amino acids, and 707 extracellular region amino acids. Prostate-specific membrane antigen is expressed in normal prostate epithelial cells, but its expression level in prostate cancer cells is much higher. Compared with the prostate-specific antigen conventionally used for clinical detection, prostate-specific membrane antigen is a more sensitive and specific prostate cancer tumor marker, especially highly expressed in hormone-refractory prostate cancer and prostate cancer metastases, and has high sensitivity and specificity in differentiating prostate cancer from other types of malignant tumors. At the same time, in various solid tumors of non-prostate origin (such as lung cancer, bladder cancer, gastric cancer, pancreatic cancer, kidney cancer, and colorectal cancer, etc.), prostate-specific membrane antigen is also highly specifically expressed on tumor vascular endothelial cells.

[0130] In the present invention, "pharmaceutical composition" refers to a pharmaceutically acceptable composition, which contains a small molecule drug, polypeptide, antibody (or antibody-like ligand) or its conjugate as the active pharmaceutical ingredient (API), and other components (such as pharmaceutically acceptable excipients). The pharmaceutical composition can be prepared by any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, and / or freeze-drying processes.

[0131] In the present invention, "pharmaceutically acceptable excipients" refer to auxiliary materials widely used in the field of drug production. The main purposes of using excipients are to provide a drug composition that is safe to use, stable in nature, and / or has specific functionality, and also to provide a method so that after the drug is administered to a subject, the active ingredient can dissolve at a desired rate or promote the effective absorption of the active ingredient in the subject receiving the drug administration. Pharmaceutically acceptable excipients can be inert fillers or functional ingredients that provide a certain function for the drug composition (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient in the composition). Examples of "pharmaceutically acceptable excipients" include but are not limited to binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, glidants, wetting agents, gelling agents, absorption retardants, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners, etc.

[0132] In the present invention, for immunoconjugate drugs, the number of targeting tumor fragments (TTFs) linked to the same targeting immune cell fragment (ITF) is one of the important factors affecting their effectiveness and safety. For the manufacture of immunoconjugate drugs, in order to make the number of linkages of the drug a fixed number, generally the reaction conditions such as the amounts of raw materials and reagents for the reaction are specified and then implemented. However, different from the reaction of small molecule compounds, conjugate drugs are usually obtained in the form of a mixture of compounds linked with different numbers of functional fragments. Therefore, in the present invention, the average value of the number of linkages (i.e., the average number of linkages) is also used to represent the number of targeting tumor fragments (TTFs) linked to the same targeting immune cell fragment (ITF). In the present invention, the number of linkages of the conjugate drug (i.e., n in the general formula structure) refers to the theoretical value calculated based on the usage amounts of raw materials, reagents, etc. of the reaction, while the number of targeting tumor fragments (TTFs) in each conjugate drug molecule will vary according to the actual reaction situation and there is a certain deviation from the theoretical value. For example, in the present invention, the theoretical value of the number of linkages is 12, while the average value of the number of linkages of the actually obtained product is 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, or 11.9. It should be noted that those skilled in the art understand and recognize this deviation, and can design the necessary number of linkages or obtain the corresponding actual number of linkages according to the description of the present invention. In principle, unless otherwise specified, the number of linkages of the drug refers to the theoretical value, that is, 1 targeting immune cell fragment can be linked to 2 - 12, preferably 6 - 12, more preferably 11 - 12 targeting tumor fragments.

[0133] The technical solution of the present invention will be described below in conjunction with specific embodiments. The following embodiments are provided to further illustrate the present invention, rather than to limit the scope of the present invention. For those skilled in the art, various changes and improvements to the specific embodiments of the present invention will be obvious without departing from the spirit and scope of the present invention.

[0134] The starting materials used in the present invention can be synthesized by methods known in the art or purchased through conventional commercial means. The separation and purification of the compounds of the present invention can be achieved by methods well-known to those skilled in the art, including but not limited to thin-layer chromatography, high-performance liquid chromatography, etc. The structural identification of the compounds of the present invention can be achieved by methods well-known to those skilled in the art, including but not limited to nuclear magnetic resonance (NMR), mass spectrometry (MS), etc.

[0135] List of Abbreviations

[0136]

[0137] Example: Synthesis of Immunoconjugate Drug (Compound CR)

[0138] 1. Synthesis of Compound 3

[0139]

[0140] S1: Add Compound 1 (3.85 g, 10.00 mmol, 1.00 eq) and N-Boc-1,6-hexanediamine (2.38 g, 11.00 mmol, 1.10 eq) to a 250 mL three-necked flask, and then add DCM (80 mL) to dissolve. Under nitrogen protection, add HATU (5.70 g, 15.00 mmol, 1.50 eq), and then slowly dropwise add (completed within 5 min) DIEA (2.58 g, 20.00 mmol, 2.00 eq). Stir the reaction magnetically at room temperature (25 °C) for 1 h. After monitoring the reaction by TLC (DCM:MeOH = 10:1, v / v) to completion, quench with 5 wt% aqueous citric acid solution (30 mL), extract and separate the layers. The organic phase is washed with an appropriate amount of saturated brine, dried, and concentrated under reduced pressure to obtain Compound 2 (5.60 g, yield: 96.05%, purity: 92.64%); MS: m / z 584.73 [M+H] + ;

[0141] S2: Add compound 2 (5.60 g, 9.60 mmol, 1.00 eq) into a 250 mL three-necked flask, dissolve it with DCM (10 mL), and then slowly add dropwise (completed within 10 min) an aqueous solution of 20 wt% TFA (60 mL, 1.50 eq). Stir magnetically at room temperature (25 °C) for 1 - 2 h; Concentrate under reduced pressure, purify by reverse HPLC (aqueous solution of 0.1 wt% TFA:ACN = 9:1, v / v), and lyophilize to obtain compound 3 (4.61 g, two-step yield: 79.48%, purity: 99.81%); MS: m / z 484.61 [M+H] + 。

[0142] 2. Synthesis of compound 5

[0143]

[0144] S3: Add compound 4 (6.00 g, 8.84 mmol, 1.00 eq) and compound 3 (4.61 g, 9.54 mmol, 1.08 eq) into a 250 mL three-necked flask, and then add DCM (100 mL) to dissolve; Under nitrogen protection, add HATU (5.14 g, 13.26 mmol, 1.50 eq), and then slowly add dropwise (completed within 5 min) DIEA (3.34 g, 26.52 mmol, 3.00 eq). Stir magnetically at room temperature (25 °C) for 1 - 2 h; After monitoring the reaction by HPLC until completion, add an aqueous solution of 5 wt% sodium bicarbonate (30 mL) to quench the reaction, extract and separate layers. Wash the organic phase with an appropriate amount of saturated brine, dry, and concentrate under reduced pressure to obtain an oily compound 5 (10.05 g, yield: 92.59%, purity: 87.54%); MS: m / z 1145.0; 1 1H NMR (400 MHz, DMSO-d6) spectrum is as Figure 1 shown.

[0145] 3. Synthesis of compound 8

[0146]

[0147] S4 & S5: Add compound 5 (4.10 g, 3.58 mmol, 1.00 eq) into a 250 mL three-necked flask, and then add DCM (100 mL) to dissolve it. Under nitrogen protection and an ice-water bath, slowly add dropwise (completed within 5 min) DBU (0.55 g, 3.58 mmol, 1.00 eq), and stir magnetically at 10 °C for 1 - 1.5 h. After monitoring by HPLC that the reaction of compound 5 is completed (converted to compound 6), add compound 7 (0.98 g, 3.94 mmol, 1.10 eq) and HATU (2.04 g, 5.37 mmol, 1.50 eq) to the reaction system, and then slowly add dropwise (completed within 5 min) DIEA (0.93 g, 7.16 mmol, 2.50 eq), and stir magnetically at room temperature (25 °C) for 1 - 2 h. After monitoring by HPLC that the reaction of compound 6 is completed, quench with 5 wt% aqueous citric acid solution (30 ml), extract and separate layers, wash the organic phase with an appropriate amount of saturated brine, dry, concentrate under reduced pressure, purify by reverse HPLC (0.1 wt% aqueous TFA solution: ACN = 9:1, v / v), freeze-dry to obtain compound 8 (2.10 g, two-step yield: 51.33%, purity: 96.62%); MS: m / z 1147.60 [M + H] + 。

[0148] 4. Synthesis of compound 9

[0149]

[0150] S6: Add compound 8 (0.68 g, 0.59 mmol, 1.00 eq) into a 250 mL three-necked flask, add DCM (10 mL) to dissolve it, and then slowly add dropwise (completed within 5 min) 50 wt% aqueous TFA solution (10 mL), and stir magnetically at room temperature (25 °C) for 1 - 3 h. Concentrate under reduced pressure, purify by reverse HPLC (0.1 wt% aqueous TFA solution: ACN = 9:1, v / v), freeze-dry to obtain compound 9 (272.8 mg, yield: 42.28%, purity: 98.16%); MS: m / z 979.45 [M + H] + ; 1 1H NMR (400 MHz, DMSO-d6) spectrum is as Figure 2 shown.

[0151] 5. Synthesis of compound CR

[0152]

[0153] S7 & S8: Add the PBS solution of anti-CD16 monoclonal antibody (0.2 mL, 0.01534 μmol, 11.51 mg / mL, 1.00 eq, commercially available) and PBS (6 mL, 50 mM, 5 mM EDTA, pH = 7.2) into a 20 mL sterile test tube. Then add TCEP (0.2300 μmol, 15.00 eq), and incubate in a shaker at 37 °C for 2 - 3 h (to reduce the disulfide bonds in the monoclonal antibody and obtain anti-CD16 monoclonal antibody containing free sulfhydryl groups, or anti-CD16 monoclonal antibody-SH); after cooling to 25 °C, add the DMSO solution (0.1 mL) of compound 9 (0.2300 μmol, 15.00 eq), and carry out the coupling reaction in a shaker for 2 - 3 h; after monitoring the reaction by RP-C-HPLC and SEC-HPLC, put the reaction solution into an ultrafiltration tube (10 kd), ultrafilter with PBS, collect the product, and obtain the PBS solution (5 mL, concentration approximately 4.3 mg / ml) of compound CR (21.45 mg). According to the above feeding ratio prediction, the theoretical DAR value of compound CR should be 12, and the measured DAR value by TOF-MS is 11.13. The test results are as Figure 3 shown in Table 1.

[0154] Table 1. Detection results of DAR values by TOF-MS

[0155]

[0156] Experimental example: Activity study of immunoconjugate drug (compound CR)

[0157] 1. Experimental materials

[0158] 1.1 Cells

[0159] RM-1 cells (mouse prostate cancer cell line, source: Wuhan Punosai Life Science Co., Ltd.).

[0160] 1.2 Cytokines

[0161] Mouse IL-2 recombinant protein (brand: Abcam, catalog number: ab238291, specification: 100 μg);

[0162] Mouse IL-15 recombinant protein (brand: Abcam, catalog number: ab264456, specification: 50 μg).

[0163] 1.3 Antibodies

[0164] CD45 monoclonal antibody (30-F11), FITC, brand: Invitrogen, catalog number: MCD4501;

[0165] NK1.1 monoclonal antibody (PK136), APC, brand: Invitrogen, catalog number: A14739;

[0166] Anti-mouse CD16 / CD32 antibody, brand: BioXCell, catalog number: BE0307.

[0167] 1.4 Others

[0168] Advanced RPMI 1640 medium, brand: Gibco, catalog number: 12633012;

[0169] Poly(I:C), brand: Absin, catalog number: abs42025416;

[0170] 70μm cell strainer, brand: Corning; catalog number: 352350;

[0171] DPBS (without calcium and magnesium), brand: Hyclone, catalog number: SH30028.02;

[0172] Mouse NK cell isolation kit, brand: Miltenyi Biotec, catalog number: 130-115-818.

[0173] ACK Lysis Buffer, brand: Gibco, catalog number: A1049201.

[0174] 2. Isolation and purification of NK cells

[0175] 2.1 Experimental procedures

[0176] 2.1.1 In vivo stimulation of NK cells

[0177] (1) Prepare 3 Balb / c mice (Jicuiyaokang) (6-8 weeks old, body weight about 20 g / mouse).

[0178] (2) 18 h before euthanasia of the mice, dilute 150 μg of Poly(I:C) (TLR3 agonist) in 1xDPBS and then inject it intraperitoneally into the mice.

[0179] 2.1.2 Preparation of NK cells (all operations are ensured to be completed on ice or at 4°C)

[0180] (1) Euthanize the mice, take out the spleen and place it in a petri dish. Add 1 ml of DPBS, rinse and filter the spleen with DPBS. After filtration, transfer the spleen cells to a 50-ml centrifuge tube. Centrifuge (500 g, 4 °C, 5 min), discard the supernatant, add 2 ml of ACK Lysis Buffer to the cell pellet, gently pipette to mix well, lyse for 1 - 2 min, add 4 ml of DPBS, centrifuge (500 g, 4 °C, 5 min), and count the cells.

[0181] (2) Use an NK cell isolation kit to isolate NK cells (see the instruction manual of Miltenyi magnetic bead sorting) (leave a small amount of unsorted spleen cells as a control).

[0182] (3) Detection of sorting purity: For unsorted spleen cells and sorted NK cells, take 100,000 cells respectively, label them with CD45-FITC and NK1.1-APC respectively, and perform flow cytometry.

[0183] 2.2 Experimental results

[0184] It can be seen from Figure 4 that after sorting, the proportion of NK cells changes from 3.86% to 93.40%.

[0185] 3. Proliferation, stimulation and killing of tumor cells by NK cells

[0186] 3.1 Experimental procedures

[0187] (1) Stimulate NK cells using four protocols respectively: anti-CD16 antibody (100 μg / ml), compound 9 (100 μg / ml), a mixture of anti-CD16 antibody and compound 9, and compound CR. Add IL-2 and IL-15, with the working concentrations being IL-2 (400 unit / ml) and IL-15 (20 ng / ml) respectively.

[0188] (2) Stain RM-1 cells with CSFE. Incubate RM-1 cells with a PBS solution of 2 μM CFSE (1 - 2 ml, preheated at 37 °C) for 10 min, and then wash with complete medium (5 ml). Co-culture with NK cells at E:T (50:1 and 5:1) ratios for 72 h.

[0189] (3) Perform flow cytometry to detect the ratio of NK cells to tumor cells.

[0190] 3.2 Experimental results

[0191] 3.2.1 Compound CR promotes the killing effect of immune cells on tumor cells

[0192] As Figure 5As shown, the RM-1 cells were labeled with CSFE before co-culture with NK cells. CSFE can label cell DNA, and as the cells divide, the CSFE signal value in the daughter cells will be halved. The CSFE-labeled tumor cells were detected by flow cytometry to evaluate the proliferation ability of the tumor cells. The molecule to be detected was added during co-culture for 72 h, and the size of NK cells and tumor suppression were detected by flow cytometry. In the flow cytometry plot, two main peaks can be seen, which are the NK cell peak and the tumor cell peak respectively. Among them, the tumor cell peak can be further divided into a tumor proliferation peak and a tumor initial peak according to the CSFE fluorescence signal intensity. The number of tumor cells in the compound CR group was significantly reduced, and at the same time, the proportion of the tumor proliferation peak was significantly reduced, indicating that compound CR significantly inhibited the proliferation of tumor cells.

[0193] 3.2.2 Compound CR itself has no obvious inhibitory effect on tumor cells

[0194] As the cells proliferate, the CFSE-labeled tumor cells will show a decrease in fluorescence intensity. Therefore, the number of CFSE peaks of tumor cells can be used to indicate the number of cell proliferation times.

[0195] As Figure 6 shown, in the blank control plot, only the tumor cells labeled with CFSE have two peaks, and the proportion of the daughter peak (proliferation peak) is 35.87%. In the groups with added compounds, the proportions of the daughter peaks are 39.56% (anti-CD16 monoclonal antibody), 47.61% (compound 9), 37.96% (anti-CD16 monoclonal antibody + compound 9), and 41.58% (compound CR) respectively, all of which are not lower than the blank group. The results show that in the absence of NK cells, compound CR itself does not have a killing effect on tumor cells.

[0196] 3.2.3 Compound CR promotes the aggregation of immune cells around tumor cells and mediates the killing of tumor cells

[0197] After co-culturing NK cells and RM-1 cells for 72 h, photos were taken under a microscope. As Figure 7 shown, in the compound CR group, NK cells aggregated around the tumor cells into clusters, the number of tumor cells decreased, and the morphology was incomplete. While in the blank control group, compound 9 group, anti-CD16 monoclonal antibody group, and anti-CD16 monoclonal antibody + compound 9 group, the growth state of tumor cells was normal. Thus, it can be seen that compound CR can promote the aggregation of immune cells around tumor cells and mediate the killing of tumor cells.

[0198] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Without departing from the principles and spirit of the present invention, those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention, and these changes, modifications, substitutions, and variations are all covered within the scope of the present invention.

Claims

1. An immunoconjugate drug, or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled substance, active metabolite or prodrug thereof, characterized in that: The immunoconjugate drug comprises: A tumor-targeted fragment that targets and binds to a tumor marker; and Targeted immune cell fragments that bind to immune cell surface molecules.

2. The immunoconjugate drug according to claim 1, or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled substance, active metabolite or prodrug thereof, characterized in that: The tumor-targeting fragment targets and binds to PSMA.

3. The immunoconjugate drug according to claim 1 or 2, or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled substance, active metabolite or prodrug thereof, characterized in that: The tumor-targeting fragment is in the form of a ligand or an antibody; Preferably, the ligand is formed by a polypeptide or a small molecule compound.

4. The immunoconjugate drug according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled substance, active metabolite or prodrug thereof, characterized in that: The targeted immune cell fragment is in the form of an antibody; Preferably, the antibody is a monoclonal antibody.

5. The immunoconjugate drug according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled substance, active metabolite or prodrug thereof, characterized in that: The tumor targeting fragment and the immune cell targeting fragment are connected by a linker fragment.

6. The immunoconjugate drug according to claim 5, or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled substance, active metabolite or prodrug thereof, characterized in that: The targeted immune cell fragment is connected to a targeted tumor fragment via a linker fragment, or the targeted immune cell fragment is connected to a plurality of targeted tumor fragments via a plurality of linker fragments; When a plurality of linker fragments are included, the plurality of linker fragments are completely identical, partially identical, or completely different from each other; When a plurality of tumor-targeting fragments are included, the plurality of tumor-targeting fragments are completely identical, partially identical, or completely identical to each other.

7. An immunoconjugate drug, or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled substance, active metabolite or prodrug thereof, characterized in that: The immunoconjugate drug has a structure as shown in Formula I, in TTF indicates targeted tumor fragment; Lk represents the linker fragment; ITF stands for targeted immune cell fragment; n is 2-12.

8. The immunoconjugate drug according to claim 7, characterized in that: Each TTF independently targets a tumor marker, preferably PSMA.

9. The immunoconjugate drug according to claim 7 or 8, characterized in that: Each TTF is independently in the form of a ligand or an antibody; Preferably, the ligand is formed by a polypeptide or a small molecule compound.

10. The immunoconjugate drug according to any one of claims 1 to 9, characterized in that: The targeted tumor fragments are each independently formed by a targeting ligand compound as shown in the following formula, or each TTF is each independently formed by a targeting ligand compound as shown in the following formula, Best in PG1 is hydrogen or C1-C6 alkyl, preferably C1-C6 alkyl, more preferably tert-butyl; R Lg1 Hydrogen or Wherein R1 is a C1-C6 alkylamino group substituted with hydroxyl or amino.

11. The immunoconjugate drug according to any one of claims 1 to 10, characterized in that: The targeted tumor fragments are each independently formed by any one of the following targeting ligand compounds, or each TTF is each independently formed by any one of the following targeting ligand compounds:

12. The immunoconjugate drug according to any one of claims 5 to 11, characterized in that: The linker fragment is cleavable or non-cleavable, preferably non-cleavable, or Lk is cleavable or non-cleavable, preferably non-cleavable.

13. The immunoconjugate drug according to any one of claims 5 to 12, characterized in that: The linker fragments are each independently formed by a linker compound as shown in the following formula, or each Lk is each independently formed by a linker compound as shown in the following formula, in R Lk1 Hydroxyl or Where R2 is hydrogen or 14. The immunoconjugate drug according to any one of claims 5 to 13, characterized in that: The linker fragments are each independently formed by any one of the following linker compounds, or each Lk is each independently formed by any one of the following linker compounds:

15. The immunoconjugate drug according to any one of claims 7 to 14, characterized in that: ITF is in the form of an antibody; Preferably, the antibody is a monoclonal antibody, preferably an anti-CD16 monoclonal antibody.

16. An immunoconjugate drug, or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled substance, active metabolite or prodrug thereof, wherein: The immunoconjugate drug has a structure as shown in Formula II, Preferably, it has a structure as shown in Formula II', in ITF stands for targeted immune cell fragment; n is 2-12.

17. The immunoconjugate drug according to claim 16, characterized in that: ITF is in the form of an antibody; Preferably, the antibody is a monoclonal antibody, preferably an anti-CD16 monoclonal antibody.

18. A pharmaceutical composition comprising the immunoconjugate drug according to any one of claims 1 to 17, or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope-labeled substance, active metabolite or prodrug thereof; Preferably, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

19. Use of the immunoconjugate drug according to any one of claims 1 to 17, or a pharmaceutically acceptable salt, ester, solvate, polymorph, isotope label, active metabolite or prodrug thereof, or the pharmaceutical composition according to claim 18 in the preparation of a drug for preventing and / or treating a disease or condition; Preferably, the disease or disorder is cancer, preferably prostate cancer.

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