Pharmaceutical composition

Abstract

A pharmaceutical composition, comprising a first drug and a second drug different from the first drug, wherein the first drug is an inhibitor of topoisomerase I. A pharmaceutical composition, comprising a structure represented by formula (II): , wherein M represents a targeting polypeptide; L 1 represents a linking unit connecting M and D 1; L 2 represents a linking unit connecting M and D 2; D 1 represents the first drug, and D 2 represents the second drug; p and q are independently integers selected from 1-20.

Description

A pharmaceutical composition Technical Field

[0001] This application relates to the field of biomedicine, specifically to a pharmaceutical composition. Background Technology

[0002] Currently, antibody-drug conjugates (ADCs) can selectively deliver drugs to cancer cells and kill tumor cells, while having little impact on normal cells, ushering in a new era in cancer treatment. As ADCs, several drugs have already received FDA approval, such as DS-8201, which is composed of a Her2 antibody and a camptothecin derivative Dxd, for the treatment of HER2-positive breast cancer patients.

[0003] However, due to tumor heterogeneity and multidrug resistance (MDR) regulation, DXd or SN-38ADCs still face inherent and acquired resistance.

[0004] Therefore, there is an urgent need to develop an ADC (anti-tumor drug) that is not prone to drug resistance, is highly effective, and safe, and has a multi-drug, multi-target mechanism of action. This is of great significance for developing anti-tumor drugs with excellent anti-tumor efficacy and safety. Summary of the Invention

[0005] This application introduces two drugs with different mechanisms into an ADC. Specifically, it screens for drugs that synergistically act with camptothecin or its derivatives (e.g., exatecan, DXD, topotecan, irinotecan, belotecone, 7-ethyl-10-hydroxycamptothecin (SN38)) from different tumor growth pathway inhibitors (such as TKIs, CDKIs, ATKIs, etc.). Camptothecin or its derivatives are then sequentially coupled to the same antibody using a sequential coupling method, resulting in a dual-load ADC combining camptothecin or its derivatives with other drugs of different targets and mechanisms of action. This achieves one or more of the following advantages:

[0006] (1) The precise combination therapy at the cellular level of this application, the combination of chemical drugs with different mechanisms of action, blocks the survival channels of tumor cells on a larger scale. According to the different mechanisms of tumor development, the combination of drugs with different mechanisms of action can achieve a synergistic therapeutic effect of 1+1>2.

[0007] (2) Traditional single-load conjugates often use high cytotoxic loads and high DAR value designs due to their single mechanism of action, which inevitably leads to serious toxic side effects in clinical applications. This application uses a combination of low cytotoxic compounds with different mechanisms of action and synergistic effects, which relatively reduces the overall toxicity of the conjugate and improves patients' quality of life and treatment compliance;

[0008] (3) Dual-load ADC therapy carrying drugs with multiple mechanisms of action and multiple targets avoids acquired drug resistance caused by long-term administration of single drugs;

[0009] It extended the patient's survival time.

[0010] On the one hand, the application itself provides a pharmaceutical composition comprising a first drug and a second drug different from the first drug, wherein the first drug is an inhibitor of topoisomerase I.

[0011] In some embodiments, the inhibitor of topoisomerase I includes camptothecin or a camptothecin derivative.

[0012] In some embodiments, the camptothecin derivative includes exatecan, DXD, topotecan, irinotecan, belotecan, 7-ethyl-10-hydroxycamptothecin (SN38), or a derivative of the compound.

[0013] In some embodiments, the first drug is:

[0014] In some embodiments, the second drug comprises a nucleoside analog antimetabolite.

[0015] In some embodiments, the second drug is gemcitabine or a derivative of gemcitabine.

[0016] In some embodiments, the molecular formula of the second drug is:

[0017] In some embodiments, the second drug comprises an epidermal growth factor receptor tyrosine kinase inhibitor.

[0018] In some embodiments, the second drug is gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, or ametinib.

[0019] In some embodiments, the molecular formula of the second drug is:

[0020] In some embodiments, the second drug includes a microtubule inhibitor.

[0021] In some embodiments, the microtubule inhibitors include dolastatin or its derivatives, auristatin-like cytotoxic molecules or their derivatives, or maytansine-like cytotoxic molecules or their derivatives.

[0022] In some embodiments, the auristatin-type cytotoxic molecules include MMAE or MMAF, and the maytansine-type cytotoxic molecules include DM1, DM4, or derivatives thereof.

[0023] In some embodiments, the second drug includes a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor.

[0024] In some embodiments, the NAMPT inhibitor comprises:

[0025] In some embodiments, the pharmaceutical composition further comprises a targeting peptide, wherein the pharmaceutical composition is an antibody-drug conjugate.

[0026] In some embodiments, the targeting peptide includes an antibody, an antibody fragment, a bispecific antibody, or an antigen-binding fragment.

[0027] In some embodiments, the targeted peptide includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, or dAb.

[0028] In some embodiments, the targeting peptide targets a tumor antigen.

[0029] In some embodiments, the targeting peptide targets EGFR, VEGFR, FGFR, PDGFR, HER2, HER3, HER4, RET, cMET, Trop2, NTRK, PR / ER, CD20, PD-L1, B7-H3, Nectin-4, CLAN18.2, ROR1, MSLN, TNF-α, CD25, ENPP3, Muc1, Axl, CD20, ROR2, GPNMB, CEACAM6, and CD138. PSMA, GC-C, LIV-1, CA6, FUT3, CD56, CD37, HER3, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4, B7H3, CD228, Notch-3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, FCRL5, CEACAM5, Tissue Factor, FRα, BCMA, CD19, CD79b, CLDN18.2, or their mutants.

[0030] In some embodiments, the pharmaceutical composition comprises a structure as shown in formula (II):

[0031] in,

[0032] M represents the targeting peptide;

[0033] L1 represents the connection unit that connects M and D1;

[0034] L2 represents the connection unit that connects M and D2;

[0035] D1 represents the first drug, and D2 represents the second drug;

[0036] p and q are independent integers selected from 1 to 20.

[0037] In some embodiments, the connecting unit includes a detachable connector, a non-detachable connector, a hydrophilic connector, a hydrophobic connector, a charged connector, and an uncharged connector.

[0038] In some embodiments, the connecting unit is connected to M via a thiol, amino, carboxyl, phenolic hydroxyl, azide, or amide group on M.

[0039] In some embodiments, the M includes a first heavy chain and a second heavy chain, wherein the first heavy chain and / or the second heavy chain includes sites capable of being connected to the connecting unit.

[0040] In some embodiments, L1 and / or L2 independently have the following structures:

[0041] —La—Lb—Lc——

[0042] (Formula III)

[0043] Where La is the connecting component;

[0044] Lb is either a key or a release component;

[0045] Lc represents a bond or a drug isolation component.

[0046] In some embodiments, La includes maleimide, succinimide, or DBCO.

[0047] In some embodiments, La has the following structure:

[0048] It is linked to Lb, Lc, D1, or D2 via -C(=O)- or -O-, and can be linked to the target peptide via the 3- and / or 4-positions of maleimide / maleamide;

[0049] And R a -(CH2) n (C(=O)NH) m (CH2CH2O) s (CH2) t -,

[0050] in,

[0051] n is an integer between 0 and 10.

[0052] m is 0 or 1.

[0053] s is an integer between 0 and 10.

[0054] t is an integer between 0 and 10.

[0055] In some embodiments, La includes the following structure:

[0056] In some embodiments, Lb is an acid pyrolysis release component.

[0057] In some embodiments, Lb has the following structure:

[0058] In some embodiments, Lb is an enzyme lysis and release component.

[0059] In some embodiments, Lb is selected from one of the following: -glycine-, -alanine-, -valine-, -leucine-, -isoleucine-, -proline-, -phenylalanine-, -tryptophan-, -methionine-, -tyrosine-, -serine-, -threonine-, -cysteine-, -asparagine-, -glutamine-, -aspartic acid-, -glutamic acid-, -lysine-, -arginine-, -histidine-, -citrulline-, -lysine(triphenylmethyl)-, -lysine(monomethoxytriphenylmethyl)-, -lysine Acids (fluorenoxycarbonyl)-; -valine-citrulline-(-Val-Cit-), -valine-alanine-(-Val-Ala-), -valine-lysine-(-Val-Lys-), -valine-lysine (triphenylmethyl)-(-Val-Lys(Trt)-), -valine-lysine (monomethoxytriphenylmethyl)-(-Val-Lys(Mmt)-), -valine-lysine (fluorenoxycarbonyl)-(-Val-Lys(Fmoc)-), -valine-arginine-( -Val-Arg-), -phenylalanine-citrulline-(-Phe-Cit-), -phenylalanine-lysine-(-Phe-Lys-), -phenylalanine-lysine (triphenylmethyl)-(-Phe-Lys(Trt)-), -phenylalanine-lysine (monomethoxytriphenylmethyl)-(-Phe-Lys(Mmt)-), -phenylalanine-lysine (fluorenyloxycarbonyl)-(-Phe-Lys(Fmoc)-), -leucine-citrulline-(-Leu-Cit-), -isoleucine Amino acids -citrulline-(-Ile-Cit-), -phenylalanine-arginine-(-Phe-Arg-); -phenylalanine-arginine-arginine-(-Ala-Arg-Arg-); -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -glycine-phenylalanine-leucine-glycine-(-Gly-Phe-Leu-Gly-), -alanine-leucine-alanine-leucine-(-Ala-Leu-Ala-Leu-).

[0060] In some embodiments, the structure of Lc includes one of the following:

[0061] Where X is -O-, -NH-, or -S-;

[0062] R c This includes bonds, branches containing amide groups, or branches containing carbonyl groups.

[0063] In some embodiments, the structure of Rc is as follows:

[0064] -C(=O)NHCH2C(=O)NHCH2OCH2C(=O)-.

[0065] In some embodiments, the pharmaceutical composition comprises the following structure:

[0066] In some embodiments, the pharmaceutical composition comprises the following structure:

[0067] On the other hand, this application provides a pharmaceutical composition comprising any one of the pharmaceutical compositions described in the previous application, optionally comprising a pharmaceutically acceptable carrier.

[0068] On the other hand, this application provides a method for modulating the tumor microenvironment of a subject, comprising the steps of administering to the subject any of the aforementioned pharmaceutical compositions, or the aforementioned pharmaceutical compositions.

[0069] On the other hand, this application provides a method for modulating the immune response of a subject, which includes the following steps: administering to the subject any of the aforementioned pharmaceutical compositions or pharmaceutical preparations.

[0070] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0071] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0072] Figure 1 shows a comparison of the in vivo tumor-inhibiting effect of the ADC described in this application with other ADCs;

[0073] Figure 2 shows the HNMR (400.130MHz) of the HM-2031B described in this application;

[0074] Figure 3 shows the in vitro efficacy evaluation of the conjugate described in this application in HCC827;

[0075] Figure 4 shows the in vitro efficacy evaluation of the conjugate described in this application in NCI-H929;

[0076] Figure 5 shows the in vitro efficacy evaluation of the conjugate described in this application in JIMT1;

[0077] Figure 6 shows the in vitro efficacy evaluation of the conjugate described in this application on PC9;

[0078] Figure 7 shows the in vivo efficacy evaluation of the conjugate described in this application in HCC827;

[0079] Figure 8 shows the in vivo efficacy evaluation of the conjugate described in this application in Canpan-1;

[0080] Figure 9 shows the in vivo efficacy evaluation of the conjugate described in this application in JIMT1. Detailed Implementation

[0081] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0082] Terminology Definition

[0083] In this application, the term "antibody-drug conjugate" generally refers to an ADC, i.e., a binding protein (e.g., an antibody or its antigen-binding fragment) linked to one or more chemical drugs. The chemical drugs can be any therapeutic agent and / or cytotoxic agent. The antibody-drug conjugate can have any number of drugs conjugated to the antibody from 1 to 8, for example, it can include 2, 4, 6, or 8 drug-loaded species. In this application, the drugs can include mitotic inhibitors, antitumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemotherapeutic agents, hormones, anti-hormonal agents, corticosteroids, photosensitizing agents, oligonucleotides, radioactive isotopes, topoisomerase inhibitors, tyrosine kinase inhibitors, and / or radiosensitizers.

[0084] In this application, the term "drug / antibody ratio" or "DAR" generally refers to the number of drugs linked to an antibody in an ADC. The DAR of an ADC can range from 1 to 8, or even higher loads (e.g., 10), and the range of DAR can depend on the number of binding sites on the antibody. In this application, the DAR can be the number of drugs loaded onto a single antibody. The DAR can also be the average or mean DAR of a group of ADCs.

[0085] In this application, the term "bispecific antibody" generally refers to an antibody capable of binding to two antigens or antigenic epitopes, respectively. The bispecific antibody may include light and heavy chains of an antibody capable of specifically binding to a first antigen or antigenic epitope, and light and heavy chains of an antibody capable of specifically binding to a second antigen or antigenic epitope. In one embodiment of this application, the light chains of the antibody capable of specifically binding to the first antigen or antigenic epitope and the light chains of the antibody capable of specifically binding to the second antigen or antigenic epitope in the bispecific antibody have the same sequence. In one embodiment of this application, the heavy chains of the antibody capable of specifically binding to the first antigen or antigenic epitope and the heavy chains of the antibody capable of specifically binding to the second antigen or antigenic epitope in the bispecific antibody have different sequences.

[0086] In this application, the term "pharmaceutical composition" generally refers to a formulation in which one or more active ingredients are biologically effective. A pharmaceutical composition may include a first drug and a second drug. In some embodiments, the first drug and the second drug are present in the form of an antibody-drug conjugate. In some embodiments, the first drug and the second drug are conjugated to different targeting peptides, i.e., the pharmaceutical composition includes at least two different antibody-drug conjugates. In some embodiments, the first drug and the second drug are conjugated to the same targeting peptide, i.e., the pharmaceutical composition includes at least one antibody-drug conjugate with a dual drug load.

[0087] In this application, the term "targeting peptide" generally refers to a peptide that specifically or selectively binds to a target molecule, cell, particle, tissue, or aggregate. For example, a targeting peptide can be an antibody, antibody fragment, bispecific antibody, antigen-binding fragment, or other antibody-based molecule or compound. Other examples of targeting moieties may also be those known in the art, such as aptamers, avimers, receptor-binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides, or proteins.

[0088] In this application, the term "antibody" is generally used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). The native form of an antibody is typically a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the variable regions (VL and VH) of the light and heavy chains together are primarily responsible for binding to the antigen. The variable domains of the light and heavy chains consist of a framework region interrupted by three hypervariable regions (also known as "complementarity-determining regions" or "CDRs"). The constant regions are recognized by and interact with the immune system. (See, for example, Janeway et al., 2001, Immunol. Biology, 5th ed., Garland Publishing, New York). Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Antibodies can be derived from any suitable species. In some embodiments, antibodies are human or murine. Antibodies can be, for example, human, humanized, or chimeric antibodies.

[0089] In this application, the term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous group of antibodies (i.e., the individual antibodies constituting the group are identical except for a small amount of possible naturally occurring mutations). Monoclonal antibodies are highly specific, targeting a single antigenic site. The modifier "monoclonal" indicates that the antibody acquires this characteristic from a substantially homogeneous group of antibodies, and should not be construed as requiring the antibody to be produced by any particular method.

[0090] In this application, a “complete antibody” is generally an antibody that comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, CH3, and CH4. The constant domain can be a natural sequence constant domain (e.g., a human natural sequence constant domain) or a variant thereof.

[0091] In this application, an "antibody fragment" generally comprises a portion of a complete antibody, including its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, double-chain antibodies, triple-chain antibodies, quadruple-chain antibodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from one or more antibody fragments, one or more fragments generated from a Fab expression library, or an epitope-binding fragment of any of the above, which binds immune-specifically to a target antigen (e.g., cancer cell antigen, viral antigen, or microbial antigen).

[0092] In this application, the term "antigen" generally refers to an entity that an antibody specifically binds to.

[0093] In this application, the terms "specific binding" and "specifically binding" refer to the ability of an antibody or antibody derivative to bind to a corresponding epitope of its target antigen in a highly selective manner without binding to a variety of other antigens. Typically, the antibody or antibody derivative binds at a concentration of at least about 1 x 10-1. -7 M, preferably 10 -8 M to 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M binds with its affinity and binds to the predetermined antigen with an affinity at least twice as great as that it binds to non-specific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein).

[0094] In this application, the term "antigen-binding fragment" refers to a portion of a complete antibody and specifically to the antigen-determining variable region of a complete antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv and single-chain Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antigen-binding fragments.

[0095] In this application, the term "Fab" generally refers to a fragment containing a heavy chain variable domain and a light chain variable domain, and also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain; the term "Fab'" generally refers to a fragment different from Fab by adding a small number of residues (including one or more cysteine ​​residues from the antibody hinge region) to the carboxyl terminus of the heavy chain CH1 domain; the term "F(ab')2" generally refers to a dimer of Fab', comprising an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region. The term "Fv" generally refers to the smallest antibody fragment containing a complete antigen recognition and binding site. In some cases, this fragment may consist of a dimer of a heavy chain variable region and a light chain variable region bound tightly non-covalently; the term "dsFv" generally refers to a disulfide-bonded Fv fragment, wherein the bond between a single light chain variable region and a single heavy chain variable region is a disulfide bond. The term "dAb fragment" generally refers to an antibody fragment composed of VH domains. In this application, the term "scFv" generally refers to a monovalent molecule formed by the covalent pairing of a heavy chain variable domain and a light chain variable domain of an antibody via a flexible peptide linker; such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0096] In this application, the term "fully human antibody" generally refers to an antibody expressed by transferring a human antibody-encoding gene into a genetically engineered animal lacking an antibody gene. All parts of the antibody (including the variable and constant regions) are encoded by a human-derived gene. Fully human antibodies can significantly reduce the immune side effects caused by heterologous antibodies in humans. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology, among others.

[0097] In this application, the term "specific binding" generally refers to binding to a specific target without cross-reactivity to other targets. For example, when an antibody binds to an epitope via its antigen-binding domain, and this binding requires some complementarity between the antigen-binding domain and the epitope, according to this definition, an antibody is said to "specifically bind" to the antigen when it is more likely to bind to an epitope via its antigen-binding domain than to a random, unrelated epitope. Selective reactivity, on the other hand, generally refers to preferential binding to a specific target.

[0098] In this application, the term "tumor-specific antigen" generally refers to a protein that is primarily or exclusively expressed on tumor cells. "Primary expression" means that the protein is expressed at a relatively higher level on tumor cells than on normal somatic cells, while "exclusively expressed" means that the protein is expressed on tumor cells but is not detectable on normal somatic cells by standard methods known in the art.

[0099] In this application, the term "conjugate" refers to a polypeptide (e.g., peptide, nucleic acid, protein, enzyme, sugar, polysaccharide, lipid, glycoprotein, and lipoprotein) and its chemical portion that are covalently or non-covalently (e.g., via a linker) linked into a larger molecule. The chemical portion that may be included in a conjugate may be a therapeutic agent or a cytotoxic agent, and non-limiting examples include: mitotic inhibitors, antitumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, anti-hormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radioactive isotopes, topoisomerase inhibitors, kinase inhibitors, and radiosensitizers.

[0100] In this application, the term "Her2" generally refers to a type I transmembrane protein belonging to the epidermal growth factor receptor family, also known as c-erbB2, ErbB2, or Neu (Slamon, et al., Science 235(1987) 177-182; Swiss-Prot P04626). In this application, the term "Her2" may also encompass homologs, variants, and isoforms of Her2, including splice isoforms. The term "Her2" also includes proteins having one or more sequences from Her2 homologs, variants, and isoforms, as well as fragments of such sequences, provided that the variant protein (including isoforms), homolog, and / or fragment can be recognized by one or more Her-specific antibodies (such as pertuzumab or trastuzumab). Her2 is associated with tumor transformation in human breast cancer cells, and overexpression of Her2 protein has been detected in patients with breast cancer, gastric cancer, pancreatic cancer, ovarian cancer, peritoneal cancer, or colorectal cancer.

[0101] In this application, the terms "Trop2" and "TROP2" generally refer to a single-pass transmembrane type I cell membrane protein, also known as tumor-associated calcium signal transducer 2 (TACSTD2), GA733-1, EGP-1, or MIS1. In this application, the term "Trop2" may also encompass homologs, variants, and isoforms of Trop 2, including splice isoforms. The term "Trop" also includes proteins having one or more sequences of Trop 2 homologs, variants, and isoforms, as well as fragments of such sequences, provided that the variant protein (including isoforms), homologous protein, and / or fragment can be recognized by one or more Trop-specific antibodies (such as hRS7 or hTINA1). Trop2 may be human Trop2, the DNA sequence and amino acid sequence of which are available in public databases, such as NCBI accessions NM_002353 and NP_002344.

[0102] In this application, the term "EGFR" generally belongs to the ErbB family of receptor tyrosine kinases and plays a key role in the physiological mechanisms of epithelial cells. The amino acid sequence of human EGFR protein can be found in UniProt / Swiss-Prot accession number P00533. In this application, the targeting peptide can bind to EGFR protein. In this application, the terms "EGFR protein," "EGFR antigen," and "EGFR-Fc recombinant protein" are used interchangeably and include any variant or isotype thereof naturally expressed by cells.

[0103] In this application, the terms "c-met," "cmet," or "Met" refer to any natural or variant (whether natural or synthetic) c-met polypeptide. c-MET, short for c-mesenchymal-epithelial transition factor (c-Met), is a receptor tyrosine kinase that, upon binding to its ligand hepatocyte growth factor, activates various cell signaling pathways, including those involved in proliferation, motility, migration, and invasion. c-Met proteins include polypeptides encoded by the nucleotide sequence identified with GenBank accession number NM_000245, proteins encoded by the polypeptide sequence identified with GenBank accession number NM_000236, or their extracellular domains. The term "wild-type c-met" generally refers to a polypeptide containing the amino acid sequence of a naturally occurring c-met protein. The term "wild-type c-met sequence" generally refers to the amino acid sequence found in naturally occurring c-met.

[0104] In this application, the term "topoisomerase inhibitor" includes both topoisomerase I inhibitors and topoisomerase II inhibitors. Examples of topoisomerase I inhibitors include, but are not limited to, topotecan, gimatecan, irinotecan, camptothecin and its analogues, 9-nitrocamptothecin and macrocamptothecin conjugate PNU-166148 (compound A1 in WO 99 / 17804); 10-hydroxycamptothecin acetate; etoposide; idarubicin hydrochloride; irinotecan hydrochloride; teniposide; topotecan, topotecan hydrochloride; doxorubicin; epirubicin, epirubicin hydrochloride; 4'-epirarubicin, mitoxantrone, mitoxantrone hydrochloride; daunorubicin, daunorubicin hydrochloride, pentorubicin, and dasatinib (BMS-354825). In this application, examples of the term "topoisomerase I inhibitor" include, but are not limited to, topotecan, gimatecan, irinotecan, camptothecin and its analogues, 9-nitrocamptothecin and macrocamptothecin conjugate PNU-166148 (compound A1 in WO 99 / 17804); 10-hydroxycamptothecin acetate; etoposide; idarubicin hydrochloride; irinotecan hydrochloride; teniposide; topotecan, topotecan hydrochloride; doxorubicin; epirubicin, epirubicin hydrochloride; 4'-epirarubicin, mitoxantrone, mitoxantrone hydrochloride; daunorubicin, daunorubicin hydrochloride, pentorubicin and dasatinib (BMS-354825).

[0105] In this application, the term "nucleoside analog antimetabolite" refers to molecules that act in a nucleoside-like manner during DNA synthesis. These include a range of antiviral products used to prevent viral replication in infected cells. Nucleoside analogs can be used to combat hepatitis B virus, hepatitis C virus, herpes simplex virus, and HIV. Once phosphorylated, nucleoside analogs bind to the growing DNA strand with sufficient similarity to nucleotides to exert an antimetabolite effect. Examples of antimetabolites include, but are not limited to, 6-mercaptopurine; cytarabine; fludarabine; flexuridine; fluorouracil; capecitabine; raltitrexed; methotrexate; cladribine; gemcitabine; gemcitabine hydrochloride; thioguanine; hydroxyurea; DNA demethylating agents such as 5-azacytidine and decitabine; edaraxal; and folic acid antagonists, such as, but not limited to, pemetrexed.

[0106] In this application, the term "tyrosine kinase inhibitor" includes any of a variety of therapeutic agents or drugs that are selective or non-selective inhibitors of receptor tyrosine kinases and / or non-receptor tyrosine kinases. Theoretically speaking, tyrosine kinase inhibitors typically inhibit target tyrosine kinases by binding to ATP-binding sites in enzymes.

[0107] In this application, the term "epidermal growth factor receptor tyrosine kinase inhibitor" or EGFR-TKI can refer to a compound that selectively and effectively inhibits epidermal growth factor receptor tyrosine kinase. Examples of the epidermal growth factor receptor tyrosine kinase inhibitors include, but are not limited to, gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or pharmaceutically acceptable salts thereof. In some embodiments, the epidermal growth factor receptor tyrosine kinase inhibitor is Osimertinib, Almonertinib, Alflutinib, or a pharmaceutically acceptable salt thereof.

[0108] In this application, "microtubules" or "tubulin" are components of the cytoskeleton, distributed throughout the cytoplasm. These tubular polymers composed of tubulin can be up to 50 micrometers long, with an average length of 25 micrometers, and are highly dynamic. The outer diameter of a microtubule is approximately 24 nanometers, and the inner diameter is approximately 12 nanometers. Microtubules are found in eukaryotic cells and are composed of dimers of two globular proteins (α- and β-tubulin). Tubulin is one of several members in a small family of globular proteins. The tubulin superfamily includes five distinct families: α-, β-, γ-, δ-, and ε-tubulin, as well as a sixth family found only in protozoa. The most common members of the tubulin family are α-tubulin and β-tubulin, which are the proteins that make up microtubules. Microtubules are crucial in many cellular processes. They are involved in maintaining cellular structure.

[0109] In this application, the term "tubulin regulator" is intended to mean a drug that stabilizes or destabilizes tubulin synthesis and / or polymerization.

[0110] In this application, the term "microtubule inhibitor" includes any drug that can disrupt the normal organization and dynamics of microtubules.

[0111] In this application, the term "auritstatins" refers to a class of tubulin inhibitors that block the binding of tubulin to GTP and the binding of microtubules to vincristine binding sites, thereby inducing apoptosis and inhibiting tumor growth. Currently, MMAE (US6884869) and MMAF (US7498298) are the most commonly used ADC warheads, both of which are pentapeptides derived from the modified dolastatin 10.

[0112] In this application, the term "NAMPT" or nicotinamide phosphoribosyltransferase (NAMPT) refers to a regulator of the intracellular nicotinamide adenine dinucleotide (NAD) pool, regulating the activity of NAD-dependent enzymes. NAMPT acts as a catalyst in the condensation reaction between nicotinamide and 5-phosphoribosyl-1-pyrophosphate, producing a key intermediate in the biosynthesis of nicotinamide mononucleotide—NAD. As the rate-limiting factor in the mammalian NAD biosynthesis pathway, NAMPT plays a central role in cellular processes. In addition to its enzymatic function, the secreted form of NAMPT has a dual nature, acting as both an immunomodulatory cytokine and an adipokin with anti-diabetic properties. NAMPT plays a crucial role in regulating circadian rhythm function by coordinating NAD oscillations through the release of CLOCK-BMAL1 heterodimers from NAD-dependent SIRT1-mediated inhibition, thereby regulating the expression of clock target genes. The amino acid sequence of the human NAMPT protein can be found in UniProt / Swiss-Prot accession number P43490.

[0113] In this application, the term "NAMPT inhibitor" can refer to a compound that reduces the physiological function of NAMPT.

[0114] In this application, the term "suppression" generally refers to reducing the detectable amount or completely preventing it.

[0115] In this application, the term "tumor" generally refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. In this application, the tumor may include solid tumors and / or hematomas. The terms "cancer," "cancerous," "cellular proliferative disorder," "proliferative lesion," and "tumor" are not mutually exclusive when used herein. In some embodiments, a tumor may refer to a mass containing a majority of cancer cells, such as a mass of flesh exhibiting the characteristic cells of any cancer described herein. Examples of tumors may include primary tumors of any of the above-described types of cancer or metastatic tumors at a second site derived from any of the above-described types of cancer.

[0116] In this application, the term "tumor antigen" generally includes the meaning known in the art, encompassing any molecule expressed on (or associated with) tumor cells, known or believed to play a role in the tumorigenic properties of tumor cells. Many tumor antigens are known in the art. Whether a molecule is a tumor antigen can also be determined using techniques and assays well known to those skilled in the art, such as clonogenic assays, transformation assays, in vitro or in vivo tumorigenesis assays, gel migration assays, gene knockout analyses, etc. The term "tumor antigen" can refer to human transmembrane proteins, i.e., cell membrane proteins anchored in the cellular lipid bilayer. Human transmembrane proteins as used herein typically contain an "extracellular domain" that can bind ligands, a lipophilic transmembrane domain, a conserved intracellular domain, such as a tyrosine kinase domain, and a carboxyl-terminal signaling domain having several phosphorylated tyrosine residues. Tumor antigens include molecules such as EGFR, HER2, HER3, HER4, EpCAM, CEA, TRAIL, TRAIL receptor 1, TRAIL receptor 2, lymphotoxin β receptor, CCR4, CD19, CD20, CD22, CD28, CD33, CD40, CD80, CSF-1R, CTLA-4, fibroblast activation protein (FAP), hepsin, melanoma-associated chondroitin sulfate proteoglycan (MCSP), prostate-specific membrane antigen (PSMA), VEGF receptor 1, VEGF receptor 2, IGF1-R, TSLP-R, TIE-1, TIE-2, TNF-α, weak TNF-like apoptosis inducer (TWEAK), or IL-1R.

[0117] In this application, the terms “trastuzumab”, “Pertuzumab”, “Inetetamab”, “Cetuximab”, “panitumumab”, “Necitumumab”, “Matuzumab” and “Nimotuzumab” are used according to their general and common meaning as understood in the art.

[0118] In this application, the term "cytotoxic agent" or "cytotoxic agent" generally refers to a substance that has cytotoxic activity and causes cell damage. This term is intended to include radioactive isotopes, chemotherapeutic agents, and toxins (e.g., small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin), including their synthetic analogues and derivatives. The term "cytotoxic activity" generally refers to the cell-killing effect of intracellular metabolites of a drug (such as camptothecin conjugates). Cytotoxic activity can be expressed as an IC50 value, which is the concentration (moles or mass) of half the cells that survive.

[0119] In this application, the term "cell growth inhibitor" or "cell inhibitor" generally refers to a substance with cell growth inhibitory activity, including substances that inhibit cell growth or proliferation. Cell inhibitors include inhibitors such as protein inhibitors, for example, enzyme inhibitors. Cell inhibitors have cell inhibitory activity. The term "cell inhibitory activity" generally refers to the antiproliferative effect of intracellular metabolites of drugs (such as camptothecin conjugates).

[0120] In this application, the term "camptothecin" generally refers to a pyrroloquinoline cytotoxic alkaloid composed of a quinoline ring AB, a pyrrole ring C, a pyridone ring D, and an α-hydroxylactone ring E, wherein the 20-position is S-configured, and its structure is as follows:

[0121] The lactone ring (E ring), pyridone ring (D ring), and hydroxyl group at C20 are considered essential groups for CPT to exert its topoisomerase I inhibitory effect and achieve antitumor effects.

[0122] In this application, the terms “camptothecin,” “ixotecan,” “topotecan,” “irinotecan,” “belototecan,” “letopotecan,” “CKD-602,” “gematotecan,” “karenitecin,” “BN-80915,” “hydroxycamptothecin,” “9-aminocamptothecin,” “9-nitrocamptothecin,” “7-ethyl-10-hydroxycamptothecin,” and “7-ethyl-10-difluoromethylcamptothecin” are used according to their general and common meaning as understood in the art.

[0123] In this application, the term "pharmaceuticalally acceptable" ingredient generally refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0124] In this application, the term "solvent" generally refers to an association or complex of one or more solvent molecules with a compound of this application. Non-limiting examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" generally refers to a complex in which the solvent molecule is water.

[0125] In this application, the terms "optical isomer" or "stereoisomer" generally refer to compounds having the same chemical composition but different spatial arrangements of atoms or groups, including any variety of stereoisomers that may exist, including geometric isomers. It should be understood that substituents can be attached to the chiral center of a carbon atom. The term "chiral" refers to a molecule that has a non-overlapping characteristic with its mirror-image pair, while the term "chiral" refers to a molecule that can overlap with its mirror-image pair. Therefore, the present invention includes enantiomers, diastereomers, or racemates of compounds. An "enantiomer" is a pair of stereoisomers that are mirror images of each other and do not overlap. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. Where appropriate, this term is used to name racemic mixtures. A "diastereomer" is a stereoisomer having at least two asymmetric atoms, but which are not mirror images of each other. Absolute stereochemistry is described according to the Cahn-Lngold-Prelog RS system. When the compound is a pure enantiomer, the stereochemistry of each chiral carbon can be designated as R or S. Resolved compounds with unknown absolute configurations can be designated as (+) or (-) depending on their direction of rotation (right-handed or left-handed) at the sodium D-line wavelength when rotating plane-polarized light. Some compounds described herein contain one or more asymmetric centers or axes, thus producing enantiomers, diastereomers, and other stereoisomers, which can be designated as (R)- or (S)- based on their absolute stereochemistry.

[0126] In this application, the term "tautomer" generally refers to structural isomers with different energies that can interconvert through low energy barriers. For example, proton tautomers (also known as proton-interconvertible isomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerization. Valence tautomers include interconversions via rearranging some bonding electrons.

[0127] In this application, the term "isotope" generally includes atoms having the same number of atoms but different mass numbers. Examples of isotopes that can be incorporated into the compounds described in this application and their pharmaceutically acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, for example, 2 H, 3 H, 11 C 13 C 14 C 15 N、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 36 Cl、 123 I and 125I. The subject matter disclosed in this application may also include compounds described in this application in isotopically labeled form.

[0128] In this application, the term "metabolite" generally refers to a product produced by the metabolism of a specific compound or its salt in the body. Metabolites of compounds can be identified using conventional techniques known in the art, and their activity can be determined using tests as described in this application. Such products can be generated by, for example, oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc., of the compound to which the application is administered. Therefore, this application may also include metabolites of the compounds of this application, including compounds produced by methods comprising exposing the compounds of this application to mammals for a period sufficient to produce their metabolites.

[0129] In this application, the terms "prodrug" or "prodrug precursor" generally refer to a drug precursor compound that, when administered to a subject, undergoes a chemical transformation through metabolism or a chemical process to yield the compound of this application or its salt. Information regarding prodrugs is well known in the art (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19).

[0130] In this application, the term "pharmaceutically acceptable salt" means a salt that retains the biological effects and properties of the compound of this application, and that generally has no biological or other disadvantages. It includes pharmaceutically acceptable organic or inorganic salts, and exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, hydrogen sulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannins, pantothenates, hydrogen tartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, glycosides, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, bis(hydroxynaphthyl)ate (i.e., 1,1′-methylene-bis(2-hydroxy-3-naphthyl)ate), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. This counterion can be any organic or inorganic component that stabilizes the charge on the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. In instances where multiple charged atoms are part of a pharmaceutically acceptable salt, the salt may have multiple counterions. Therefore, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more counterions.

[0131] In this application, the term "linker" or "linking unit" generally refers to the bifunctional portion in a drug-ligand conjugate that links a drug (such as camptothecin) to a ligand unit. The linker unit of this application has several components, such as a linking component La, a release component Lb, and a drug isolation component Lc.

[0132] In this application, the term "optionally substituted" means that the group in question may be substituted or unsubstituted. When substituted, the substituents of the "optionally substituted" group may include, but are not limited to, one or more substituents independently selected from, alone or in combination, the following groups or a specially designated group of groups: alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxyl, alkoxy, mercapto, cyano, halogen, carbonyl, thiocarbonyl, isocyanate, thiocyanate, isothiocyanate, nitro, perhaloalkyl, and amino groups including monosubstituted and disubstituted amino groups, and their protected derivatives. Non-limiting examples of optional substituents include halogens, -CN, =O, =N-OH, =N-OR, =NR, -OR, -C(O)R, -C(O)OR, -OC(O)R, -OC(O)OR, -C(O)NHR, -C(O)NR2, -OC(O)NHR, -OC(O)NR2, -SR-, -S(O)R, -S(O)2R, -NHR, -N(R)2, -NHC(O)R, -NRC(O)R, -NHC(O)OR, -NRC(O)OR, S(O)2NHR, -S(O)2N(R)2, -NHS(O)2NR2, -NRS(O)2NR2, -NHS(O)2R, -NRS(O)2R, C 1-6 Alkyl, C 1-6 Alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C 1-6 Alkyl and halogen-substituted C 1-6 Alkoxy groups, wherein each R is independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C 1-6 Alkyl and halogen-substituted C 1-6 Alkoxy groups. The position and number of such substituents are determined by well-known valence states of each group; for example, =O is a suitable substituent for alkyl groups but not for aryl groups. Two substituents can be linked together to form a five-, six-, or seven-membered aromatic or non-aromatic carbocyclic or heterocyclic ring containing one to three heteroatoms, such as forming methylenedioxy or ethylenedioxy.

[0133] When it comes to specific naming, the substituent is usually placed before the group it replaces, for example, "C". 1-3 Alkoxy C 3-8 cycloalkyl C 1-6 "alkyl" refers to C1-6 Alkyl groups, which are C 3-8 Cycloalkyl substitution, and the C 3-8 Cycloalkyl groups are also C 1-3 Alkoxy substitution, for example: the structural formula of methoxycyclobutylmethyl is:

[0134] In this application, the number of carbon atoms is typically indicated by the prefix "C". x -C y "or "C x-y The term "C1-C6 alkyl" indicates the minimum number of carbon atoms in a substituent, where x is the minimum number and y is the maximum number. For example, "C1-C6 alkyl" or "C 1-6 "Alkyl" refers to an alkyl substituent containing 1 to 6 carbon atoms. Further examples include C3-C6 cycloalkyl groups or C... 3-6 Cycloalkyl refers to saturated cycloalkyl groups containing 3 to 6 carbon ring atoms.

[0135] In this application, the term "alkyl" generally refers to a straight-chain, branched, or cyclic saturated substituent consisting of carbon and hydrogen. Non-limiting examples of alkyl groups include: methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl, and tert-butyl), pentyl, isopentyl, hexyl, etc. Where appropriate, the alkyl group may optionally be substituted at each carbon as defined in the claims. Typical substituents include, but are not limited to: fluorine, chlorine, OH, cyano, alkyl (optionally substituted), cycloalkyl, etc.

[0136] In this application, the term "cycloalkyl" generally refers to a saturated monocyclic cycloalkyl group. A monocycle typically comprises 3 to 10 carbon atoms. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, etc.

[0137] In this application, the term "heterocyclic group" or "heterocycle" generally refers to a ring structure containing at least one heteroatom, specifically, for example, meaning one or more non-aromatic heterocyclic groups, bicyclic heterocyclic groups, and polycyclic heterocyclic groups containing the same or different heteroatoms arbitrarily selected from O, S, and N.

[0138] The term "aryl" generally refers to a monocyclic or bicyclic aromatic ring system of hydrocarbons, wherein such rings can be fused. If the rings are fused, one of the rings must be a completely unsaturated ring, and the fused ring can be a completely saturated, partially unsaturated, or completely unsaturated ring. The term "fused" means that the second ring exists (i.e., shares) two adjacent atoms with the first ring. The term "aryl" includes aromatic groups, such as phenyl, naphthyl, tetrahydronaphthyl, indenyl, biphenyl, 4-(pyridin-3-yl)phenyl, 2,3-dihydro-1H indenyl, and 1,2,3,4-tetrahydronaphthyl.

[0139] The term "heteroaryl" generally refers to an aromatic group (e.g., pyrrole, pyridinyl, pyrazolyl, indole, indazole, thiophene, furanyl, benzofuranyl, oxazolyl, imidazoleyl, tetrazolyl, triazine, pyrimidinyl, pyrazinyl, thiazolyl, purine, benzimidazolyl, quinolinyl, isoquinolinyl, benzothiophene, benzoxazolyl, 1H-benzo[d][1,2,3]triazolyl, etc.) containing at least one heteroatom (e.g., oxygen, sulfur, nitrogen, or combinations thereof) in a 5- to 10-membered aromatic ring system. Heteroaryl groups can consist of monocyclic or fused-ring systems. Typical monocyclic heteroaryl rings are 5- to 6-membered rings containing 1 to 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, while typical fused-ring heteroaryl ring systems are 9- to 10-membered ring systems containing 1 to 4 heteroatoms independently selected from oxygen, sulfur, and nitrogen. A fused heteroaryl ring system may consist of two fused heteroaryl rings or a heteroaryl ring fused to an aryl group (e.g., phenyl).

[0140] In this application, wedge-shaped solid line keys are typically used. and wedge-shaped dashed key The absolute configuration of the center of a solid is represented by a straight solid line key. and straight dashed key The relative configurations of the stereocenters are indicated. Unless otherwise specified, all compounds appearing in this invention are intended to include all possible optical isomers, such as compounds with a single chirality, or mixtures of various chiral compounds (i.e., racemates). In all compounds of this invention, each chiral carbon atom may optionally be in the R configuration or S configuration, or a mixture of R and S configurations.

[0141] In this application, the term "carrier" can include pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals at the doses and concentrations used. Physiologically acceptable carriers are typically pH-buffered aqueous solutions. Non-limiting examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICS™. In some embodiments, the pharmaceutically acceptable carrier is a non-naturally occurring pharmaceutically acceptable carrier.

[0142] In this application, the term "abnormal" generally refers to a deviation from a standard, such as a normal healthy subject or cell and / or a group of normal healthy subjects or cells. As used herein, the term "abnormal expression" refers to the abnormal expression of a cell or subject's gene product (e.g., RNA, protein, polypeptide, or peptide) compared to a normal healthy cell or subject and / or a group of normal healthy cells or subjects. This abnormal expression can be due to gene amplification or gene expression inhibition. In some embodiments, "abnormal expression" related to DNase topoisomerase I refers to elevated, decreased, or inappropriate expression of DNase topoisomerase I. In specific embodiments, the term "abnormal activity" refers to DNase topoisomerase I deviating from its normal activity in healthy cells or subjects and / or a group of normal healthy cells or subjects.

[0143] In this application, the term "expression upregulation" generally refers to an increase in the expression of nucleic acid mRNA or peptide. The term may also refer to post-translational modifications required for increased peptide activity and / or function, such as the addition of a sugar moiety, phosphorylation, etc.

[0144] In this application, the term "inhibitor" generally refers to compounds / substances known in the art that are capable of completely or partially preventing or reducing the physiological function (i.e., activity) of one or more specific proteins (e.g., topoisomerase I). Inhibitors are also known as "antagonists".

[0145] In this application, the term "treat" or "treatment" generally refers to therapeutic and preventative treatment aimed at inhibiting or slowing (alleviating) undesirable physiological changes or conditions, such as the development or spread of cancer. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, attenuation of disease severity, stabilization of disease state (i.e., non-deterioration), delay or slowing of disease progression, improvement or mitigation of disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean extended survival compared to expected survival without treatment. Those requiring treatment include those who already have the disease or condition and those who are susceptible to it. Within the scope of cancer, the term "treatment" may also include any or all of the following: killing tumor cells; inhibiting the growth of tumor cells, cancer cells, or tumors; inhibiting the replication of tumor cells or cancer cells; reducing the overall tumor burden or the number of cancer cells; and improving one or more symptoms of accompanying diseases.

[0146] The terms “administration” or “application” include the route by which the compound is introduced into a subject to achieve its intended function. Non-limiting examples of possible routes of administration include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal), topical, oral, inhalation, rectal, and transdermal.

[0147] The term "contact" generally refers to two or more substances of different types coming into contact with each other in any order, in any manner, and for any duration. When applied to cells, "contact" means a method of delivering the compound of this application to or placing it in direct proximity to a target cell, whether the delivery is in vitro or in vivo.

[0148] In this application, the term "effective amount" generally includes the amount that effectively achieves the desired result within the necessary dose and time period. The effective amount of a compound can vary depending on factors such as the subject's disease state, age, and weight, as well as the compound's ability to elicit the desired response in that subject. Dosing regimens can be adjusted to provide the optimal therapeutic response.

[0149] In this application, the term "therapeuticly effective amount" generally refers to the amount of a conjugate that is effective in treating a disease or disorder in mammals. In the case of cancer, a therapeutically effective amount of the conjugate can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., delay to a certain extent, preferably terminate) cancer cell infiltration into surrounding organs; inhibit (i.e., delay to a certain extent, preferably terminate) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with said cancer to a certain extent. In terms of the extent to which the drug can inhibit cancer cell growth and / or kill existing cancer cells, it may be growth-inhibiting and / or cytotoxic. In the context of cancer treatment, efficacy can be detected, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0150] In this application, the terms "subject" or "patient" refer to an animal, such as a mammal, including but not limited to primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In some embodiments, the subject is a human.

[0151] Invention Details

[0152] Pharmaceutical Composition

[0153] On one hand, this application provides a pharmaceutical composition comprising a first drug and a second drug different from the first drug, wherein the first drug is an inhibitor of topoisomerase I.

[0154] In some embodiments, the inhibitor of topoisomerase I may include camptothecin or a camptothecin derivative. For example, the camptothecin derivative may include exatecan, Dxd, topotecan, irinotecan, belotecan, 7-ethyl-10-hydroxycamptothecin (SN38), or derivatives of the compound (e.g., derivatives formed by the above-mentioned camptothecin or camptothecin derivative with a linker).

[0155] In some embodiments, the first drug may include the following structures, or derivatives of the following structures (e.g., connecting the following structures with linkers):

[0156] In some implementations, the first drug may be

[0157] In some embodiments, the second drug may include a nucleoside analog antimetabolite. For example, the second drug is gemcitabine or a derivative of gemcitabine.

[0158] In some embodiments, the molecular formula of the second drug may be:

[0159] In some embodiments, the second drug may include an epidermal growth factor receptor tyrosine kinase inhibitor. For example, the second drug may be one of gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, avitinib (Abivertinib), or alflutinib (also known as furmonertinib). In some embodiments, the second drug may include the following structures, or derivatives of the following structures (e.g., the following structures linked to a linker):

[0160] In some embodiments, the second drug may include a tubulin inhibitor. This tubulin inhibitor may include dolastatin or a derivative thereof, auristatin-like cytotoxic molecules or derivatives thereof, or maytansine-like cytotoxic molecules or derivatives thereof. For example, dolastatin may include dolastatin 10 or dolastatin 15, auristatin-like cytotoxic molecules may include MMAE or MMAF, and maytansine-like cytotoxic molecules may include DM1 (Mertansine), DM4, or derivatives thereof.

[0161] In some embodiments, the second drug may include the following structures, or derivatives of the following structures (e.g., linking the following structures with linkers):

[0162] In some embodiments, the second drug may be a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor. For example, the NAMPT inhibitor includes:

[0163] Antibody-drug conjugates

[0164] In this application, the pharmaceutical composition is in the form of an antibody-drug conjugate. In some embodiments, the first drug and the second drug are conjugated to different targeting peptides, i.e., the pharmaceutical composition includes at least two different antibody-drug conjugates. In some embodiments, the antibody-drug conjugate may include a first conjugate and a second conjugate. Specifically, the antibody-drug conjugate may be M-(L1-D1). u and M-(L2-D2) v The mixture is defined as follows: M represents the targeting peptide; L1 represents the linker unit connecting M and D1; L2 represents the linker unit connecting M and D2; D1 represents the first drug; and D2 represents the second drug. u and v are independently integers selected from 1 to 20, for example, u can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and v can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0165] In some embodiments, the first and second drugs are conjugated to the same targeting peptide, meaning the pharmaceutical composition includes at least one antibody-drug conjugate with dual drug loading. In some embodiments, the antibody-drug conjugate has the structure of Formula II:

[0166] in,

[0167] M represents the targeting peptide;

[0168] L1 represents the connection unit that connects M and D1;

[0169] L2 represents the connection unit that connects M and D2;

[0170] D1 represents the first drug, and D2 represents the second drug;

[0171] p and q are independently integers selected from 1 to 20. In some embodiments, p and q are independently integers selected from 1 to 10. In some embodiments, p can be an integer from 1 to 8, for example, p can be 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, q can be an integer from 1 to 8, for example, q can be 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the sum of p and q can be 8.

[0172] In some implementations, M can be an antibody, an antibody fragment, or an antigen-binding fragment. The antibody can be multispecific or single-specific.

[0173] In some embodiments, M may be a monoclonal antibody. In more specific cases, the targeting peptide may be a multivalent and / or multispecific monoclonal antibody. The targeting peptide may be a mouse, chimeric, humanized, or fully human monoclonal antibody, which may be in whole, fragment (Fab', Fab, F(ab)2, F(ab')2, Fv, or scFv) or subfragment (single-chain construct) form, or be an isotype of IgG1, IgG2, IgG3, IgG4, IgA, or a submolecule thereof. Those skilled in the art will recognize that the conjugates, methods, compositions, and uses described herein can utilize any of the many antibodies known in the art. The antibodies used are commercially available from a variety of known sources.

[0174] In some embodiments, M may be a fully human antibody. Methods for producing fully human antibodies using combinatorial methods or transgenic animals transformed with human immunoglobulin loci are known in the art. The fully human antibody is expected to exhibit fewer side effects than chimeric or humanized antibodies and function as a substantially endogenous human antibody in vivo.

[0175] In some embodiments, M can be an antibody fragment. Antibody fragments can be obtained by conventional methods, such as digesting a full-length antibody with pepsin or papain. For example, antibody fragments can be produced by digesting an antibody with pepsin to provide a 5S fragment denoted as F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally using a blocking group of thiol groups resulting from disulfide bond cleavage, to produce a 3.5S Fab' monovalent fragment. For example, pepsin digestion produces two monovalent Fab fragments and one Fc fragment. Other methods of antibody cleavage can also be used, such as separating the heavy chain to form a monovalent light-heavy chain fragment, further cleaving the fragment, or other enzymatic, chemical, or genetic techniques, as long as the fragment binds to an antigen recognized by the intact antibody. In some cases, the Mab can be a single-chain antibody. These single-chain antibodies can be prepared by constructing a structural gene comprising a DNA sequence encoding a VH domain and a VL domain linked by an oligonucleotide linker sequence. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as *Escherichia coli*. The recombinant host cell synthesizes a single polypeptide chain, wherein a linker peptide bridges two variable domains. Methods for generating scFv are well known in the art.

[0176] In some implementations, M can recognize or bind to markers or tumor-associated antigens, and can be expressed primarily or exclusively on cells that are diseased in relatively normal tissues, and can be internalized by cells.

[0177] In some embodiments, the targets or antigens that M can recognize or bind to may include: EGFR, VEGFR, FGFR, PDGFR, HER2, HER3, HER4, RET, cMET, Trop2, NTRK, PR / ER, CD20, PD-L1, B7-H3, Nectin-4, CLAN18.2, ROR1, MSLN, TNF-α, CD25, ENPP3, Muc1, Axl, CD20, ROR2, GPNMB, CEACAM6, CD138, PSMA, GC-C, LIV-1, CA6, FUT3, CD56, CD37, HER3, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4, B7H3, CD228, Notch-3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, FCRL5, CEACAM5, Tissue Factor, FRα, BCMA, CD19, CD79b, CLDN18.2, or their mutants.

[0178] In some embodiments, M can specifically bind to one or more tumor-specific antigens. In some cases, the targeting peptide can be an antibody or an antigen-binding fragment thereof that reacts with an antigen or epitope expressed on tumor cells. Available tumor-specific antigens include one or more combinations of EGFR, cMET, HER2, and TROP2. For example, M can target EGFR, cMET, HER2, or TROP2 alone, or M can target EGFR and cMET simultaneously.

[0179] In some implementations, M may include an anti-HER2 antibody.

[0180] In some embodiments, the anti-HER2 antibody includes trastuzumab, pertuzumab, inetetamab, and margetuximab.

[0181] In some implementations, M may include an anti-EGFR antibody.

[0182] In some embodiments, the anti-EGFR antibody may include cetuximab, panitumumab, necitumumab, or nimotuzumab.

[0183] In some implementations, M may include an anti-TROP2 antibody.

[0184] In some embodiments, the anti-TROP2 antibody may include hRS7.

[0185] In some implementations, M can be an anti-EGFR / c-MET bispecific antibody.

[0186] In some embodiments, the anti-EGFR / c-MET bispecific antibody may include PM1080 or amivantamab.

[0187] The linking units (L1 and / or L2) include cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, and uncharged linkers. In some embodiments, the linking unit is linked to M via a thiol, amino, carboxyl, phenolic hydroxyl, azide, or amide group on M. M comprises a first heavy chain and a second heavy chain, the first heavy chain and / or the second heavy chain containing sites capable of linking to the linking unit.

[0188] L1 and / or L2 can independently have the following structures:

[0189] ——La-Lb-Lc——

[0190] (Formula III)

[0191] Where La is the connection component; Lb is the key or release component; and Lc is the key or drug isolation component.

[0192] The primary function of the linker component La is to connect the first drug and / or the second drug to M. The connection occurs through a thiol, amino, carboxyl, phenolic hydroxyl, azide, or amide group on M. Therefore, La can include maleimide, succinimide, or DBCO groups. For example, La has the following structure:

[0193] It is linked to Lb, Lc, D1, or D2 via -C(=O)- or -O-, and can be linked to the target peptide (M) via the 3- and / or 4-positions of maleimide / maleamide; and R a -(CH2) n (C(=O)NH) m (CH2CH2O) s (CH2) t -,in,

[0194] n is an integer from 0 to 10. For example, n can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0195] m is 0 or 1.

[0196] s is an integer from 0 to 10. For example, s can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0197] t is an integer from 0 to 10. For example, t can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0198] Specifically, La can include

[0199] In some embodiments, Lb is a bond or a release component. The main function of Lb is that when the antibody-drug conjugate reaches tumor cells under the action of M, Lb can cleave under certain conditions, releasing drugs D1 and / or D2 into the tumor cells. Therefore, based on its mechanism, Lb can be a pH-controlled release component; for example, Lb is an acid cleavage release component with the following structure:

[0200] For example, Lb can be an enzyme cleavage and release component. Specifically, Lb can include -glycine-, -alanine-, -valine-, -leucine-, -isoleucine-, -proline-, -phenylalanine-, -tryptophan-, -methionine-, -tyrosine-, -serine-, -threonine-, -cysteine-, -asparagine-, -glutamine-, -aspartic acid-, -glutamic acid-, -lysine-, -arginine-, -histidine-, -citrulline-, -lysine (triphenylmethyl)-, -lysine (monomethoxytriphenylmethyl)- -Lysine (fluorenoxycarbonyl)-; -valine-citrulline-(-Val-Cit-), -valine-alanine-(-Val-Ala-), -valine-lysine-(-Val-Lys-), -valine-lysine (triphenylmethyl)-(-Val-Lys(Trt)-), -valine-lysine (monomethoxytriphenylmethyl)-(-Val-Lys(Mmt)-), -valine-lysine (fluorenoxycarbonyl)-(-Val-Lys(Fmoc)-), -valine-arginine- (-Val-Arg-), -phenylalanine-citrulline-(-Phe-Cit-), -phenylalanine-lysine-(-Phe-Lys-), -phenylalanine-lysine (triphenylmethyl)-(-Phe-Lys(Trt)-), -phenylalanine-lysine (monomethoxytriphenylmethyl)-(-Phe-Lys(Mmt)-), -phenylalanine-lysine (fluorenyloxycarbonyl)-(-Phe-Lys(Fmoc)-), -leucine-citrulline-(-Leu-Cit-), -isoleucine One of the following: -citrulline-(-Ile-Cit-), -phenylalanine-arginine-(-Phe-Arg-), -phenylalanine-arginine-arginine-(-Ala-Arg-Arg-), -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -glycine-phenylalanine-leucine-glycine-(-Gly-Phe-Leu-Gly-), or -alanine-leucine-alanine-leucine-(-Ala-Leu-Ala-Leu-).

[0201] In some implementations, the structure of Lc can be Where X is -O-, -NH-, or -S-. R c This includes either bonds that are branched with amide groups or branched with carbonyl groups. For example, R c The structure can be a bond, -(C=O)- or -(C(=O)NH)CH2(C(=O)NH)(CH2OCH2)C(=O)-.

[0202] In some implementations, L1 and / or L2 may have the following structures:

[0203] In some implementations, L1-D1 can be one of the following structures:

[0204] Table 1

[0205] In some implementations, L2-D2 can be one of the following structures:

[0206] Table 2

[0207] In some embodiments, the antibody-drug conjugate has the following structure:

[0208] In some embodiments, the antibody-drug conjugate has the following structure:

[0209] Pharmaceutical preparations and applications

[0210] On the other hand, this application provides a pharmaceutical formulation comprising the pharmaceutical composition described in this application, or a pharmaceutically acceptable carrier.

[0211] In this application, the pharmaceutical formulation may include the pharmaceutical composition described in this application at a "therapeutic effective dose" or a "preventive effective dose." The "therapeutic effective dose" can be the amount that effectively achieves the desired therapeutic outcome at the necessary dose and duration. The therapeutic effective dose can be determined by those skilled in the art, and for example, can vary depending on factors such as the disease state, the subject's age, sex, weight, and the ability of the antibody-drug conjugate to elicit the desired response in the subject. The "preventive effective dose" can be the amount that effectively achieves the desired preventive outcome at the necessary dose and duration. For example, the preventive effective dose may be lower than the therapeutic effective dose.

[0212] In this application, the pharmaceutical formulation may be configured in a form suitable for administration (e.g., suitable for parenteral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intratumoral, and / or mucosal administration). In this application, the pharmaceutical formulation may include other active pharmaceutical ingredients.

[0213] In this application, the pharmaceutically acceptable carrier may include any or all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. The pharmaceutically acceptable carrier may include one or more of water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc., and combinations thereof. The pharmaceutically acceptable carrier may also include isotonic agents, wetting agents, emulsifiers, preservatives, and / or buffers.

[0214] On the other hand, this application provides a method for modulating the tumor microenvironment of a subject, comprising the steps of administering the pharmaceutical composition or the pharmaceutical preparation described in this application to the subject.

[0215] On the other hand, this application provides a method for modulating the immune response of a subject, which includes the following steps: administering the pharmaceutical composition or the pharmaceutical preparation described in this application to the subject.

[0216] This application provides for the use of the antibody-drug conjugate or the pharmaceutical composition described in this application in the preparation of a drug, wherein the drug can modulate the tumor microenvironment of a subject and / or modulate the subject's immune response.

[0217] This application provides the pharmaceutical composition or pharmaceutical formulation described herein, which is used to modulate the tumor microenvironment of a subject and / or modulate the subject's immune response.

[0218] On the other hand, this application provides for the use of the pharmaceutical composition or pharmaceutical preparation described in this application in the preparation of a drug, wherein the drug can prevent and / or treat tumors.

[0219] This application provides a method for preventing and / or treating tumors, comprising the steps of: administering the pharmaceutical composition or pharmaceutical preparation described in this application to a subject to prepare a drug.

[0220] This application provides the pharmaceutical composition or pharmaceutical preparation described herein for the prevention and / or treatment of tumors.

[0221] In this application, the tumor may include solid tumors and / or non-solid tumors.

[0222] For example, the tumor may include gastric cancer, breast cancer (e.g., ductal carcinoma of the breast), and / or pancreatic cancer. For example, the tumor in this application may include HER2 weakly positive tumors and / or HER2 negative tumors; for example, the technical solution of this application may have a bystander killing effect.

[0223] Without being limited by any theory, the embodiments described below are only for illustrating the various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0224] Example

[0225] Example 1 Antibody-Drug Conjugation

[0226] Example 1.1 Antibody Reduction

[0227] Prepare 20mM TCEP reducing agent (Tris-2-carboxyethy1-phosphine) with purified water, and prepare 20mM EDTA (Ethylene Diamine Tetraacetic Acid) protecting agent with purified water. Add a certain molar ratio (e.g., 1:2) of reducing agent and a certain volume ratio (e.g., 1:1) of protecting agent to a certain concentration of 3-30 mg / mL monoclonal / bispecific antibody according to different conjugation efficiencies and conjugation requirements. Stir the reaction system at 25℃-37℃ for 1-3 hours. The reduced antibody can be directly conjugated.

[0228] Example 1.2 Conjugation of antibody with -L1-D1

[0229] The linker-active drug loading D1 (i.e., the structure of -L1-D1) was dissolved in 10%-25% DMSO (dimethyl sulfoxide) to prepare a stock solution of a certain concentration of 5mM-10mM. According to different coupling efficiencies and coupling requirements, a certain molar ratio of linker-active drug loading -D1 was added. After slow addition of the drug, the mixture was stirred at 25℃ for 1-14h to obtain antibody-drug conjugate-1 (i.e., M-L1-D1).

[0230] After the reaction, the antibody-drug conjugate-1 was purified by centrifugation and ultrafiltration 3-5 times with PBS (pH: 7.4) buffer, or purified by a 40KD desalting column to remove excess unreacted drug and free small molecules such as DMSO. The conjugation status of antibody-drug conjugate-1 was detected by hydrophobic high-performance liquid chromatography (HIC-HPLC) or reversed-phase high-performance liquid chromatography (RP-HPLC), and the residual status of linker-active drug loading-D1 was detected by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0231] Example 1.3 Coupling of M-L1-D1 and -L2-D2

[0232] Adjust the concentration of antibody-drug conjugate-1 to 3-30 mg / mL. Add a reducing agent at a certain molar ratio (e.g., 1:2) and a protective agent at a certain volume ratio (e.g., 1:1) according to different conjugation efficiencies and conjugation requirements. Stir the reaction system at 25℃-37℃ for 1-3 hours.

[0233] The linker-active drug loading-D2 (i.e., the structure-L2-D2) is dissolved in a certain proportion (10%-25%) of organic solvent, such as DMSO (dimethyl sulfoxide), to prepare a stock solution of a certain concentration (5mM-10mM). According to different coupling efficiencies and coupling requirements, a certain molar ratio of active drug-D2 is added. After slow addition, the mixture is stirred at 25°C for 1-14 hours to obtain D2-L2-M-L1-D1, which is the final form of the drug composition.

[0234] After the reaction, D2-L2-M-L1-D1 was purified by centrifugation and ultrafiltration 3-5 times with PBS (pH: 7.4) buffer, or purified by a 40KD desalting column to remove excess unreacted drug and free small molecules such as DMSO. The coupling status of D2-L2-M-L1-D1 was detected by hydrophobic high performance liquid chromatography (HIC-HPLC) or reversed-phase high performance liquid chromatography (RP-HPLC), and the residual status of linker-active drug loading-D2 was detected by reversed-phase high performance liquid chromatography (RP-HPLC).

[0235] Example 2 Preparation of pharmaceutical formulation

[0236] Example 2.1 Preparation of dual-drug combination ADC

[0237] Following the steps in Example 1, 16 different drugs 1-16 were prepared. The specific types of antibodies, -L1-D1, and -L2-D2 are shown in Table 3 below.

[0238] Table 3 Raw materials for the synthesis of drugs 1-16

[0239] 2.2 Preparation of Single-Loaded Drugs

[0240] Following the steps in Examples 1.1 and 1.2, single-loaded antibody-drug conjugates were prepared, drugs 17-36, and the specific antibodies and LD types are shown in Table 4 below.

[0241] Table 4. Raw materials for the synthesis of drugs 17-40

[0242] Table 5 shows the structures involved in Example 2.

[0243] In this application, CP refers to the aforementioned drugs. For example, CP2 refers to drug 2, CP3 refers to drug 3, and CP22 refers to drug 22.

[0244] Example 3: In vitro experiment of EGFR / cMET dual-drug combination ADC

[0245] The in vitro efficacy of drugs 2 and 3 (CP2 and CP3) targeting EGFR*cMET was tested against other tumor cells expressing EGFR and cMET targets, including human malignant melanoma cells A-375 (low EGFR and low cMET expression), non-small cell lung cancer cells H1975 (medium EGFR and medium cMET expression), human colon cancer cells LS180 (medium EGFR and medium cMET expression), and human colon cancer cells HT-29 (high EGFR and high cMET expression). Drugs 17-22 (CP17, CP18, CP19, CP20, CP21, and CP22) used as controls, either alone or in combination. Cell lines were cultured in DMEM medium containing 10% placenta or 1640 medium containing 10% placenta, and seeded at 30,000 cells / well in 96-well plates. The cells were treated with antibody-drug conjugates 24 hours after seeding. The test sample was serially diluted with basal medium and transferred to cell culture plates containing cells at 100 μL / well. Incubation was performed at 37°C, 5% CO2, for 70-74 h. CCK-8 was diluted 10-fold with medium. The old medium was removed from the 96-well plate, and 100 μL of the diluted CCK-8 solution was added to each well. Incubation was performed at 37°C, 5% CO2, for 2-4 h. Readings were taken using a microplate reader at wavelengths of 450 nm / 655 nm. The percentage of inhibition and the maximum percentage of inhibition were calculated.

[0246] The percentage of inhibition was calculated. The results (Figures 3 and 4) show that bispecific antibodies targeting EGFR / cMET, coupled with drug 2 (an inhibitor of topoisomerase I + Gefitinib chloride) and drug 3 (icherotecan + a NAMPT inhibitor), exhibited significant cytotoxicity in cell lines expressing EGFR and cMET. Furthermore, the results indicate that bispecific antibodies targeting EGFR / cMET coupled with the dual-loaded drugs of this invention, compared to a single-loaded drug 22 (an inhibitor of topoisomerase I), demonstrated superior cytotoxic efficacy against cell lines expressing EGFR and cMET.

[0247] The results showed that the synergistic killing effect produced by the dual-antibody binding to two toxin molecules with different mechanisms of action (such as an inhibitor of topoisomerase I + Gefitinib chloride, or an inhibitor of topoisomerase I + NAMPT inhibitor) enabled the dual-loaded drugs to achieve a higher inhibition percentage at the same concentration. In the in vitro efficacy evaluation experiment of HCC827, at a drug concentration of 100 nM, the dual-loaded drug 2 (an inhibitor of topoisomerase I + Gefitinib chloride) showed an approximately 13% higher inhibition percentage compared to the single-loaded drug 22 (an inhibitor of topoisomerase I), and drug 3 (an inhibitor of topoisomerase I + NAMPT inhibitor) showed an approximately 38% higher inhibition percentage compared to the single-loaded drug 22 (an inhibitor of topoisomerase I). In the in vitro efficacy evaluation experiment of NCI-H292, at a drug concentration of 10 nM, the dual-loaded drug 2 (an inhibitor of topoisomerase I + Gefitinib chloride) showed an approximately 10% higher inhibition percentage compared to the single-loaded drug 22 (an inhibitor of topoisomerase I), and drug 3 (an inhibitor of topoisomerase I + NAMPT inhibitor) showed an 18% higher inhibition percentage compared to the single-loaded drug 22 (an inhibitor of topoisomerase I). The dual-antibody conjugate dual-drug combination effectively improves therapeutic efficacy and avoids the drug resistance problems caused by monotherapy.

[0248] Example 4: In vitro experiment of anti-Her2 dual-load ADC

[0249] The in vitro efficacy of Her2-targeting drugs 5, 6, and 7 on other Her2-targeting tumor cells, including human breast cancer cells SK-BR-3 (Her2-highly expressed), human gastric cancer cells NCI-N87 (Her2-highly expressed), human endometrial cancer cells HEC-1-A (Her2-highly expressed), and ovarian cancer cells SKOV-3 (Her2-highly expressed), was tested. Drugs 23-28, administered alone or in combination, served as controls. Cell lines were cultured in DMEM medium containing 10% placenta or 1640 medium containing 10% placenta, and seeded at 30,000 cells / well in 96-well plates. The cells were treated with the antibody-drug conjugate 24 hours after seeding. The test samples were serially diluted with basal medium and transferred to cell culture plates at 100 μL / well; incubated at 37°C, 5% CO2 for 70-74 hours. Dilute CCK-8 10 times with culture medium, remove the old culture medium from the 96-well plate, add 100 μL of diluted CCK-8 solution to each well, and develop the color at 37℃ and 5% CO2 for 2-4 hours. Use a microplate reader with a detection wavelength of 450 nm / 655 nm for reading.

[0250] The percentage of inhibition and the maximum percentage of inhibition were calculated. The results (Figure 5) show that the Her2-targeting drug of this invention has high cytotoxic efficacy against Her2-expressing cell lines.

[0251] The percentage of inhibition and the maximum percentage of inhibition were calculated. The results (Figure 5) show that the Her2-targeting drug of this invention has higher cytotoxic efficacy against Her2-expressing cell lines compared to single-loaded drugs and / or combinations of single-loaded drugs.

[0252] The percentage of inhibition was calculated. The results (Figure 5) show that the monoclonal antibody targeting Her2, conjugated with the dual-loaded drugs of this invention—drug 5 (an inhibitor of topoisomerase I + Gemcitabine), drug 6 (an inhibitor of topoisomerase I + Gefitinib chloride), and drug 7 (an inhibitor of topoisomerase I + NAMPT inhibitor)—exhibited significant cytotoxicity in the Her2-expressing JIMT1 cell line. Simultaneously, the results (Figure 5) show that the Her2 monoclonal antibody conjugated with the dual-loaded drugs of this invention exhibits superior cytotoxic efficacy against Her2-expressing cell lines compared to the single-loaded drug 28 (an inhibitor of topoisomerase I).

[0253] The results showed that the synergistic killing effect produced by the monoclonal antibody binding to two toxin molecules with different mechanisms of action (such as ixenocarbine + gemcitabine, ixenocarbine + gefitinib chloride, and ixenocarbine + NAMPT inhibitor) enabled higher inhibition percentages at the same concentration of dual-loaded drugs. In the in vitro pharmacodynamic evaluation experiment of JIMT1, at a drug concentration of 10 nM, the dual-loaded drug 5 (anti-topoisomerase I + gemcitabine) showed an approximately 40% higher inhibition percentage than the single-loaded drug 28 (anti-topoisomerase I), drug 6 (anti-topoisomerase I + gefitinib chloride) showed an approximately 11% higher inhibition percentage than the single-loaded drug 28 (anti-topoisomerase I), and drug 7 (anti-topoisomerase I + NAMPT inhibitor) showed an approximately 24% higher inhibition percentage than the single-loaded drug 28 (anti-topoisomerase I). Her2 monoclonal antibody combined with dual-drug combination therapy effectively improves treatment efficacy and avoids drug resistance problems caused by monotherapy.

[0254] Example 5: In vitro experiment of anti-EGFR dual-load ADC

[0255] The in vitro efficacy of EGFR-targeting drugs 13, 14, and 15 against other EGFR-targeting tumor cells, including human non-small cell lung cancer H1975 (EGFR-expressing), human breast cancer HCC1806 (EGFR-highly-expressing), and human orthotopic pancreatic adenocarcinoma BxPC-3 (EGFR-highly-expressing), was tested. Drugs 35-40, administered alone or in combination, served as controls. Four cell lines were cultured in DMEM medium containing 10% placental plasma or 1640 medium containing 10% placental plasma. Cells were seeded at 30,000 cells / well in 96-well plates and treated with the antibody-drug conjugate 24 hours after seeding. The test samples were serially diluted with basal medium and transferred to cell culture plates at 100 μL / well. Incubation was performed at 37°C, 5% CO2 for 70-74 hours. Dilute CCK-8 10-fold with culture medium, remove the old culture medium from the 96-well plate, add 100 μL of diluted CCK-8 solution to each well, and incubate at 37°C with 5% CO2 for 2-4 hours. Use a microplate reader with a detection wavelength of 450 nm / 655 nm for readings. Calculate the percentage of inhibition and the maximum percentage of inhibition.

[0256] The percentage of inhibition and the maximum percentage of inhibition were calculated. The results (Figure 6) show that the EGFR-targeting drug of this invention has high cytotoxic efficacy against EGFR-expressing cell lines.

[0257] The percentage of inhibition and the maximum percentage of inhibition were calculated. The results (Figure 6) show that the EGFR-targeting drug of this invention has higher cytotoxic efficacy against EGFR-expressing cell lines compared to single-loaded drugs and / or combinations of single-loaded drugs.

[0258] The percentage of inhibition was calculated. The results (Figure 6) showed that monoclonal antibodies targeting EGFR, conjugated with the dual-loaded drugs 13 (an inhibitor of topoisomerase I + Gemcitabine) and 14 (an inhibitor of topoisomerase I + Gefitinib chloride) of this invention, exhibited significant cytotoxicity in EGFR-expressing cell lines. In in vitro pharmacodynamic evaluation experiments on PC9 cells, the dual-loaded drug 13 (an inhibitor of topoisomerase I + Gemcitabine) achieved the highest inhibition rate (96.5%) at approximately 10 nM, and the dual-loaded drug 14 (an inhibitor of topoisomerase I + Gemcitabine) also achieved the highest inhibition rate (97.4%) at approximately 10 nM, while the single-loaded drug 40 (an inhibitor of topoisomerase I) showed an inhibition rate of only about 50% at the same concentration. The dual-loaded drugs 13 and 14 showed significantly lower inhibition rates at low concentrations (approximately 10 nM). -1At approximately 1 nM, the drug showed activity and exhibited inhibitory effects on tumor cells, achieving a 50% inhibition rate. In contrast, the single-loaded drug 40 showed zero inhibition at this concentration, only achieving killing efficiency at approximately 10 nM. The dual-loaded drugs 13 and 14 had an effective dose approximately two orders of magnitude lower than the single-loaded drug 40, and an IC50 approximately one order of magnitude lower. The EGFR monoclonal antibody-drug combination still showed effective therapeutic efficacy at low doses, suggesting that even lower doses could be achieved in clinical applications, further optimizing treatment dosage and reducing off-target risks.

[0259] Example 6: In vivo experiment of EGFR / cMET dual antibody dual load ADC

[0260] The efficacy of a dual-load drug targeting EGFR*cMET was tested in mice: Human malignant melanoma cells A-375 or other cell lines expressing EGFR and cMET were selected, maintained in vitro, and routinely passaged twice a week. Cells in the exponential growth phase were harvested and counted for tumor inoculation. Female Ba1b / c nude mice, 6-8 weeks old and weighing approximately 18-22g, were subcutaneously inoculated with 0.2ml of PBS and Matrige1 (1:1) containing tumor cells (10x10⁻¹²). 6 Allow the tumor to grow. When the average visually estimated tumor volume reaches approximately 150-250 mm... 3 At that time, all animals were weighed and the volume of each tumor was measured. Then, all mice were randomly divided into groups and injected with equal doses of dual-loaded drugs, with one or both groups receiving drug 17-22 serving as controls. Body weight and tumor size were recorded every 3-4 days.

[0261] The results (Figure 7) showed that the dual-loaded drugs targeting EGFR*cMET of this invention, namely, drug 1 (an inhibitor of topoisomerase I + Gemcitabine) and drug 2 (an inhibitor of topoisomerase I + Gefitinib chloride), effectively inhibited tumor growth in an HCC827 tumor CDX model expressing EGFR and cMET. The final tumor volume was 34±7 mm3 in the dual-loaded drug group, 38±22 mm3 in the dual-loaded drug group, and 2075±894 mm3 in the Vehicle group. The inhibitory effect of the dual-drug combination remained stable throughout the treatment period.

[0262] The results (Figure 7) showed that the tumor suppression effects of the EGFR*cMET dual-loaded drugs 1 and 2 of this invention were superior to those of the combination of single-loaded drugs 18 and 19. Compared with the combined administration of two single-loaded ADC drugs (10 mg / kg), the dual-loaded drugs at the same dose (5 mg / kg) had better therapeutic effects. Furthermore, the mice in the dual-loaded drug treatment group maintained stable body weight, indicating that the drugs did not cause significant systemic toxicity at effective doses. This suggests that the dual-loaded drugs have a wider therapeutic window and better safety profile.

[0263] Example 7: In vivo experiment of anti-Her2 dual-load ADC

[0264] The efficacy of a dual-load drug targeting Her2 was tested in mice: Human gastric cancer cells NCI-N87 expressing Her2 or other Her2-expressing cell lines were selected and maintained in vitro and routinely passaged twice a week. Cells in the exponential growth phase were harvested and counted for tumor inoculation. Female Ba1b / c nude mice, 6-8 weeks old and weighing approximately 18-22g, were subcutaneously inoculated with tumor cells (10x10⁻¹) in 0.2 ml PBS and Matrige1 (1:1) in the right flank. 6 Allow the tumor to grow. When the average visually estimated tumor volume reaches approximately 150-250 mm... 3 At that time, all animals were weighed and the volume of each tumor was measured. Then, all mice were randomly divided into groups and injected with equal doses of the dual-load drug, with one or both groups receiving the drug (drug 23-28) serving as controls. Body weight and tumor size were recorded every 3-4 days.

[0265] The results showed that the Her2-targeting dual-load drug 5 (an inhibitor of topoisomerase I + Gemcitabine) of the present invention effectively inhibited tumor growth in the Her2-expressing Canpan-1 tumor CDX model and JIMT-1 tumor CDX model (Figures 8 and 9). In the Canpan-1 tumor CDX model: the average final tumor volume in the dual-load drug 5 group was 61 mm3, while that in the Vehicle group was 859 mm3; in the JIMT-1 tumor CDX model: the average final tumor volume in the dual-load drug 5 group was 3 mm3, with the tumor almost completely disappearing, while that in the Vehicle group was 1091 mm3. The inhibitory effect of the dual-drug combination remained stable throughout the treatment period, demonstrating a significant tumor-suppressing effect.

[0266] The results (Figures 8 and 9) showed that the Her2 dual-loaded drug groups 5 and 6 exhibited superior tumor suppression effects compared to the combination of single-loaded drugs 24 and 25. Compared to the combination of two single-loaded ADC drugs (at a dose of 10 mg / kg), the dual-loaded drug demonstrated better therapeutic efficacy at the same dose (5 mg / kg). Furthermore, the mice in the dual-loaded drug treatment group maintained stable body weight, indicating that the drugs did not cause significant systemic toxicity at effective doses. This finding suggests that the dual-loaded drug has a wider therapeutic window and higher safety profile.

[0267] Example 8: Synthesis of a novel HM-2031B

[0268] HM-2031B_1 (1.20 g, 6.49 mmol) was added to DCM (15 mL), followed by TEA (1.31 g, 12.9 mmol). The mixture was cooled to 0 °C, and HY-696_1 (1.20 g, 6.49 mmol) was added. The mixture was reacted at room temperature for 1 hour. The mixture was then cooled to 0 °C again, and HM-313_7F (791.8 mg, 6.49 mmol) was dissolved in DMF (3 mL) and added dropwise to the above solution. The mixture was reacted at room temperature for 1 hour. After low-temperature rotary evaporation, the mixture was purified by reverse phase (phase A: NH4OAc, phase B: CAN, gradient B: 65-95, 40 min). HM-2031B_3 (240 mg, 0.572 mmol) was added to THF (7.2 mL), and the mixture was cooled to -50 °C. NaBH4 (8.7 mg, 0.229 mmol) was added, and the mixture was reacted at -50 °C for 5 hours. The reaction solution was added dropwise to water at 0°C, and extracted with 50 mL of EA. The organic phases were combined and evaporated to dryness, then directly purified by reverse-phase chromatography (phase A: NH4OAc, phase B: CAN, gradient B: 65-95, 40 min). HM-2031B_4 (46 mg, 0.11 mmol) and phenyl p-nitrochloroformate (33 mg, 0.16 mmol) were dissolved in DCM (2 mL), and DIPEA (28 mg, 0.22 mmol) was added. The reaction was carried out at room temperature for 16 hours. After the reaction, the solution was purified by column chromatography (60% EA in PE). HM-2031B_5 (31 mg, 0.05 mmol) and HM-2031B_6 (31 mg, 0.05 mmol) were dissolved in DMF (1 mL), and DIPEA (6.8 mg, 0.05 mmol) and HOBT (7 mg, 0.05 mmol) were added. The reaction was carried out at room temperature for 1 hour. The final product HM-2031B was obtained by reverse-phase purification (ammonium acetate system) (50-80% 10mM ammonium acetate aqueous solution / ACN).

[0269] Example 9: Comparison Experiment of Different Types of Load Combinations

[0270] Following the steps in Examples 1.1 and 1.2, single-loaded antibody-drug conjugates targeting EGFR, namely Nimotuzumab-Mc-GGFG-Dxd (DAR8), Nimotuzumab-HM-2031B (DAR8), Nimotuzumab-Mc-Val-Cit-PAB-Gefitinib (DAR8), and Nimotuzumab-Mc-O-Si(di-iso)-O-Gemcitabine (DAR8), were prepared. Following the steps in Example 1, dual-loaded antibody-drug conjugates, Nimotuzumab-HM-2031B & MAL-di-EG-Val-Cit-PAB-MMAF (DAR4+DAR4), were prepared. The specific types of antibodies, -L1-D1, and -L2-D2 are shown in Table 6 below.

[0271] Table 6. Reagents and raw materials used in Example 12

[0272] The efficacy of the aforementioned EGFR-targeting drugs was tested in mice. Non-small cell lung cancer cells HC4006 were maintained in vitro and routinely passaged twice weekly. Cells in the exponential growth phase were harvested and counted for tumor inoculation. CB17 SCID mice, 6-8 weeks old and weighing approximately 18-22g, were subcutaneously inoculated with HC4006 tumor cells (10x10⁻¹) in 0.2ml PBS and Matrigel1 (1:1) in the right flank. 6 Tumor development was monitored. Body weight and tumor volume were recorded for all mice on day 15, with an average tumor volume of 176.43 mm. 3 All mice were randomly assigned to groups and administered the medication via tail vein injection at a dose of 5 mg / kg, as shown in Table 7 below. Body weight and tumor size were recorded every 3–4 days.

[0273] Table 7 Tail vein injection methods

[0274] The results showed that, within a 21-day treatment period, the combined use of Nimotuzumab-Mc-O-Si(di-iso)-O-Gemcitabine (DAR8) and Nimotuzumab-HM-2031B (DAR8), which have lower cytotoxicity, could achieve the same therapeutic effect as Nimotuzumab-Mc-GGFG-Dxd (DAR8), which has higher cytotoxicity.

[0275] The dual-loaded Nimotuzumab-HM-2031B & MAL-di-EG-Val-Cit-PAB-MMAF (DAR4+DAR4) prepared by sequential coupling method has a therapeutic effect comparable to Nimotuzumab-Mc-GGFG-Dxd (DAR8). However, the dual-loaded Nimotuzumab-HM-2031B & MAL-di-EG-Val-Cit-PAB-MMAF uses a combination of a topoisomerase I (TOP1) inhibitor and a tubulin inhibitor, which effectively avoids acquired resistance caused by single-mechanism drugs, thereby avoiding or significantly delaying cancer recurrence / disease progression.

[0276] The results showed that the dual-load drug formulations with different combinations of chemical drugs of the present invention could effectively inhibit tumor growth in the HC4006 CDX model. Furthermore, from the perspective of drug action mechanism and the pathogenesis of tumor occurrence, survival, and metastasis, the dual-load ADC with multiple targets and mechanisms of action has significant advantages in avoiding and alleviating drug resistance in patients during clinical treatment.

Claims

1. A pharmaceutical composition comprising a first drug and a second drug different from the first drug, wherein the first drug is an inhibitor of topoisomerase I.

2. The pharmaceutical composition according to claim 1, wherein the inhibitor of topoisomerase I comprises camptothecin or a camptothecin derivative.

3. The pharmaceutical composition according to claim 2, wherein the camptothecin derivative comprises exatecan, DXD, topotecan, irinotecan, belotecan, 7-ethyl-10-hydroxycamptothecin (SN38), or a derivative of the compound.

4. The pharmaceutical composition according to any one of claims 1-3, wherein the first drug is:

5. The pharmaceutical composition according to any one of claims 1-4, wherein the second drug comprises a nucleoside analog antimetabolite.

6. The pharmaceutical composition according to claim 5, wherein the second drug is gemcitabine or a derivative of gemcitabine.

7. The pharmaceutical composition according to claim 6, wherein the molecular formula of the second drug is:

8. The pharmaceutical composition according to any one of claims 1-4, wherein the second drug comprises an epidermal growth factor receptor tyrosine kinase inhibitor.

9. The pharmaceutical composition of claim 8, wherein the second drug is gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, or ametinib.

10. The pharmaceutical composition according to claim 9, wherein the molecular formula of the second drug is:

11. The pharmaceutical composition according to any one of claims 1-4, wherein, The second drug includes a microtubule inhibitor.

12. The pharmaceutical composition according to claim 11, wherein, The microtubule inhibitors include dolastatin or its derivatives, auristatin-like cytotoxic molecules or their derivatives, or maytansine-like cytotoxic molecules or their derivatives.

13. The pharmaceutical composition according to claim 12, wherein, The auristatin-type cytotoxic molecules include MMAE or MMAF, and the maytansine-type cytotoxic molecules include DM1, DM4, or their derivatives.

14. The pharmaceutical composition according to any one of claims 1-4, wherein, The second drug includes a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor.

15. The pharmaceutical composition of claim 14, wherein the NAMPT inhibitor comprises:

16. The pharmaceutical composition according to claims 1-15, further comprising a targeting peptide, wherein the pharmaceutical composition is an antibody-drug conjugate.

17. The pharmaceutical composition of claim 16, wherein the targeting polypeptide comprises an antibody, an antibody fragment, or an antigen-binding fragment.

18. The pharmaceutical composition of claim 16, wherein the targeting peptide comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, or dAb.

19. The pharmaceutical composition according to any one of claims 16-18, wherein the targeting polypeptide targets a tumor antigen.

20. The pharmaceutical composition of claim 19, wherein the targeting peptide targets EGFR, VEGFR, FGFR, PDGFR, HER2, HER3, HER4, RET, cMET, Trop2, NTRK, PR / ER, CD20, PD- L1, B7-H3, Nectin-4, CLAN18.2, ROR1, MSLN, TNF-α, CD25, ENPP3, Muc1, Axl, CD20, ROR2, GPNMB, CEACAM6, CD138, PSMA, GC-C, LIV-1, CA6, FUT3, CD56, CD37, HER3, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4, B7H3, CD228, Notch-3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, FCRL5, CEACAM5, Tissue Factor, FRα, BCMA, CD19, CD79b, CLDN18.2, or their mutants.

21. The pharmaceutical composition according to any one of claims 16-20, wherein the pharmaceutical composition comprises a structure as shown in formula (II): in, M represents the targeting peptide; L1 represents the connection unit that connects M and D1; L2 represents the connection unit that connects M and D2; D1 represents the first drug, and D2 represents the second drug; p and q are independent integers selected from 1 to 20.

22. The pharmaceutical composition of claim 21, wherein the connecting unit comprises a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, and an uncharged linker.

23. The pharmaceutical composition according to any one of claims 21-22, wherein the linking unit is linked to M via a thiol, amino, carboxyl, phenolic hydroxyl, azide, or amide group on M.

24. The pharmaceutical composition according to any one of claims 21-23, wherein the M comprises a first heavy chain and a second heavy chain, the first heavy chain and / or the second heavy chain comprising sites capable of being connected to a linker unit.

25. The pharmaceutical composition according to any one of claims 21-24, wherein L1 and / or L2 independently have the following structure: ——La-Lb-Lc—— (Formula III) Where La is the connecting component; Lb is either a key or a release component; Lc represents a bond or a drug isolation component.

26. The pharmaceutical composition according to any one of claims 21-25, wherein La comprises maleimide, succinimide or DBCO.

27. The pharmaceutical composition according to claims 21-26, wherein, La has the following structure: It is linked to Lb, Lc, D1, or D2 via -C(=O)- or -O-, and to the target peptide via maleimide / maleamide at the 3- and / or 4-positions; And R a -(CH2) n (C(=O)NH) m (CH2CH2O) s (CH2) t -, in, n is an integer between 0 and 10. m is 0 or 1. s is an integer between 0 and 10. t is an integer between 0 and 10.

28. The pharmaceutical composition according to any one of claims 21-27, wherein, La includes the following structures:

29. The pharmaceutical composition according to any one of claims 21-28, wherein, Lb is the acid pyrolysis release component.

30. The pharmaceutical composition according to claim 29, wherein Lb has the following structure:

31. The pharmaceutical composition according to claims 21-28, wherein Lb is an enzyme cleavage and release component.

32. The pharmaceutical composition according to claim 31, wherein Lb is selected from one of the following: -glycine-, -alanine-, -valine-, -leucine-, -isoleucine-, -proline-, -phenylalanine-, -tryptophan-, -methionine-, -tyrosine-, -serine-, -threonine-, -cysteine-, -asparagine-, -glutamine-, -aspartic acid-, -glutamic acid-, -lysine-, -arginine-, -histidine-, -citrulline-, -lysine (triphenylmethyl)-, -lysine (monomethoxytrimethyl)- -phenylmethyl)-, -lysine (fluorenoxycarbonyl)-; -valine-citrulline-(-Val-Cit-), -valine-alanine-(-Val-Ala-), -valine-lysine-(-Val-Lys-), -valine-lysine (triphenylmethyl)-(-Val-Lys(Trt)-), -valine-lysine (monomethoxytriphenylmethyl)-(-Val-Lys(Mmt)-), -valine-lysine (fluorenoxycarbonyl)-(-Val-Lys(Fmoc)-), -valine -Arginine-(-Val-Arg-), -Phenylalanine-citrulline-(-Phe-Cit-), -Phenylalanine-lysine-(-Phe-Lys-), -Phenylalanine-lysine (triphenylmethyl)-(-Phe-Lys(Trt)-), -Phenylalanine-lysine (monomethoxytriphenylmethyl)-(-Phe-Lys(Mmt)-), -Phenylalanine-lysine (fluorenyloxycarbonyl)-(-Phe-Lys(Fmoc)-), -Leucine-citrulline-(-Leu-Cit-) -Isoleucine-citrulline-(-Ile-Cit-), -Phenylalanine-arginine-(-Phe-Arg-); -Phenylalanine-arginine-arginine-(-Ala-Arg-Arg-); -Glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -Glycine-phenylalanine-leucine-glycine-(-Gly-Phe-Leu-Gly-), -Alanine-leucine-alanine-leucine-(-Ala-Leu-Ala-Leu-).

33. The pharmaceutical composition according to claims 21-32, wherein Lc comprises the following structure: Where X is -O-, -NH-, or -S-; R c It consists of a bond, a branch containing an amide group, or a branch containing a carbonyl group.

34. The pharmaceutical composition according to claim 33, wherein the structure of Rc is -(C=O)- or -C(=O)NHCH2C(=O)NHCH2OCH2C(=O)-.

35. The pharmaceutical composition according to any one of claims 1-34, wherein the pharmaceutical composition comprises the following structure:

36. The pharmaceutical composition according to any one of claims 1-31, wherein the pharmaceutical composition comprises the following structure:

37. A pharmaceutical formulation comprising the pharmaceutical composition according to any one of claims 1-36, optionally comprising a pharmaceutically acceptable carrier.

38. A method for modulating the tumor microenvironment of a subject, comprising the following steps: The pharmaceutical composition of any one of claims 1-36 or the pharmaceutical preparation of claim 38 is administered to the subject.

39. A method for modulating the immune response of a subject, comprising the following steps: The pharmaceutical composition of any one of claims 1-36 or the pharmaceutical preparation of claim 38 is administered to the subject.

40. The use of the pharmaceutical composition according to any one of claims 1-36, or the pharmaceutical preparation according to claim 38, in the preparation of a medicament, wherein the medicament can prevent and / or treat tumors.

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