Antibody drug conjugate and its application

Abstract

The present invention provides a method for simultaneously coupling one or more drugs to a limited number of connection sites of an antibody by using one or more cysteine ​​residues or cysteine ​​derivative residues as drug connection carriers, thereby preparing an ADC product with a higher drug load or selecting a drug with lower toxicity to prepare an ADC product, thereby obtaining an ADC product with a larger therapeutic window. In addition, since multiple drug molecules can be coupled to one connection site, when preparing antibody-drug conjugates with the same DAR value, the ADC product obtained by the method of the present invention has better uniformity. In addition, the amount of antibody required for production can also be greatly reduced, thereby effectively reducing production costs. Compared with antibody-drug conjugates to which only one drug molecule can be connected to the same site, the antibody-drug conjugate prepared by the method of the present invention can still have the same inhibitory or killing effect on tumor cells while the total amount of coupled drug molecules is greatly reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly to an antibody-drug conjugate formed based on one or more cysteine ​​residues or cysteine ​​derivative residues as drug linker carriers and its application. The drug linker carrier can simultaneously couple one or more drugs to limited linking sites of an antibody as needed. Background Art

[0002] Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that link a biologically active drug (drug) and an antibody (antibody) via a chemical linker. ADCs are like precision-guided weapon systems, where the biologically active drug acts as a lethal munition, guided by the antibody to precisely strike diseased cells. Thus, ADCs combine the high potency of cytotoxic drug molecules with the highly targeted properties of antibodies. As of July 2019, only six antibody-drug conjugates had been approved globally (Table 1).

[0003] Table 1 Marketed Antibody Drug Conjugates

[0004] Generic name of the drug company Time to market Indications Brentuximabvedotin Seattle, Takeda 2011 Hodgkin lymphoma Trastuzumabemtansine Genentech 2013 Breast cancer Inotuzumabozogamicin Pfizer 2017 Acute lymphoblastic leukemia Gemtuzumabozogamicin Pfizer 2017 Acute myeloid leukemia Moxetumomabpasudotox AstraZeneca 2018 Hairy cell leukemia Polatuzumabvedotin-piiq Genentech 2019 Diffuse large B-cell lymphoma

[0005] Due to the non-uniqueness of the attachment sites selected on antibodies and the complexity of the attachment reaction, the final ADC product is a mixture of ADCs with varying numbers of drug-linked antibodies at varying sites. This heterogeneity in ADC drug preparation poses significant challenges to drug production and quality control. A key metric for product homogeneity is the drug-antibody ratio (DAR), which measures the number of drug molecules that can be attached to a unit amount of antibody. A low average DAR value indicates that too few drug molecules are attached to the antibody, compromising overall efficacy. A high average DAR value indicates that too many drug molecules are attached to the antibody, leading to overall ADC instability, altered pharmacokinetic parameters, and potentially increased plasma clearance, shortened half-life, and increased systemic toxicity.

[0006] Currently, there are three classic coupling methods for ADCs: amino coupling, thiol coupling, and cross-link coupling.

[0007] Amino-coupling involves coupling drugs to lysine (Lys) residues on antibodies via linkers. The first-generation ADC drug, Mylotarg, employed amino-coupling. However, IgG contains nearly 100 lysine residues, and coupling may occur at nearly 40 exposed lysine residues on the antibody's light and heavy chains. Furthermore, through lysine coupling, each antibody can be coupled to multiple small-molecule drugs in varying numbers. Therefore, the ADC drugs obtained through amino-coupling are highly heterogeneous and have extremely poor product uniformity, which seriously affects the drug's PK / PD and therapeutic window (see http: / / www.sohu.com / a / 277791166_464404).

[0008] Thiol conjugation involves first opening the interchain disulfide bonds in the antibody to form free cysteine ​​(Cys) residues, and then conjugating the linker-drug complex that can pair with the cysteine ​​residues. Since there are only four pairs of interchain disulfide bonds on IgG, eight free cysteine ​​residues will be formed after all the interchain disulfide bonds are opened. Therefore, the average DAR value of the ADC formed by single thiol conjugation is 0-8. Although thiol conjugation can better control the number of connections on each antibody, the severance of the interchain disulfide bonds will greatly reduce the stability of the antibody.

[0009] Bridging coupling is a new coupling method evolved on the basis of thiol coupling. Like thiol coupling, it first opens the interchain disulfide bonds in the antibody to form two free cysteine ​​residues, and then pairs with these two free cysteine ​​residues at the same time. Since there are only 4 pairs of interchain disulfide bonds on an antibody, 8 free cysteine ​​residues will be formed after all the interchain disulfide bonds are opened. Therefore, the ADC formed by bridging coupling has an average DAR value of 0-4. Compared with thiol coupling, bridging coupling can better control the uniformity of the product and greatly improve the stability of the coupled antibody. However, the maximum DAR value of the bridging coupling method is 4, that is, a maximum of 4 drugs can be coupled to an antibody. Due to the limited number of antigens on the surface of tumor cells, the antigen expression level required for effective ADC activity varies according to the characteristics of different antigens. ADC requires at least 10 4 Ideally, the antigen targeted by the antibody portion of the ADC should be uniformly expressed on the surface of tumor cells and have a high copy number (>10 5 Tumor cells usually have only a limited number of antigens on their surface (approximately 5,000 to 10 6antigens / cell), while the average DAR of ADC is 3.5-4 (such as the average DAR of Brentuximab vedotin is 4, and the average DAR of Trastuzumab emtansine is 3.5). The amount of drug delivered to tumor cells is very low, so the efficacy on tumor cells is low, which is also considered to be one of the main reasons for the clinical failure of ADC.

[0010] Since there are only a limited number of antigens on the surface of tumor cells, in order to ensure that the limited number of small molecule drugs carried by antibodies can effectively kill tumor cells, small molecule drugs with particularly strong toxicity are often used clinically. The small molecule drugs currently used in ADCs mainly include auristatin, maytansine, calicheamicin, doxorubicin and other categories. Compared with traditional chemotherapy drugs, these small molecule drugs have stronger killing effects on cancer cells. Usually, an average dose of four to six molecules can achieve target cell killing. However, once these highly toxic small molecule drugs appear off-target in the body, the damage to the patient is fatal. In addition, due to the particularly strong toxicity of these small molecule drugs (such as DM1 on the IC of many cells), 50 About 10 -11 mol / L, IC of DM4 50 About 10 -12 mol / L (Document 1: Widdison WC, Wilhelm SD, Cavangh EE, et al. Semisynthetic maytansine analogues for the targeted treatment of cancer [J]. J Med Chem, 2006, 49: 4392-4408; Document 2: Lambert JM. Antibody-maytansinoid conjugates: a new strategy for the treatment of cancer [J]. Drugs Future,2010,35:471-480.), MMAE’s IC 50 About 10 -11 -10 -9mol / L), so if off-target and premature drug release occur, there will be great risks to patients. The FDA conducted a summary analysis of 20 ADC investigational new drugs (INDs) received and found that the toxicity exhibited by ADC drugs in animals was mainly hematopoietic system toxicity, liver toxicity and reproductive toxicity, and some also had skin toxicity and kidney toxicity. Among them, the toxicity of the hematopoietic system, liver and reproductive system was directly related to small molecule cytotoxic drugs (Reference 3: Saber H, Leighton JK. An FDA oncology analysis of antibody-drug conjugates [J]. Regul Toxicol Pharmacol, 2015, 71 (3): 444-452). If a small molecule drug with low toxicity is selected, the amount of small molecule drug with effective loading is often too low, resulting in low efficacy and thus clinical failure. Summary of the Invention

[0011] In order to solve the above problems, the present invention provides a novel antibody-drug conjugate capable of coupling more active units (Drug). The technical solution of the present invention is as follows:

[0012] The present invention provides an antibody-drug conjugate as shown in formula (I)-(IV),

[0013]

[0014]

[0015] in:

[0016] A is any antibody or its functional binding fragment;

[0017] B1, B2, B, ..., B n is a cysteine ​​residue or a cysteine ​​derivative residue, which may be the same or different, B1 and B2, B2 and B3, ..., B n-1 With B n Linked by peptide bonds formed by dehydration condensation;

[0018] L1, L2, L3, L4,…,L n+1 are any linking units and are independent of each other and can be the same or different. The L1 is covalently connected to the amino end of B1, the L2 is covalently connected to B1, L3 is covalently connected to B2, L4 is covalently connected to B3, ..., L n+1 With B n The L2 is covalently linked to D1, L3 to D2, L4 to D3, ..., L n+1 With D n covalently linked;

[0019] D1, D2, D3, ..., D n are any active drug units and are independent of each other and may be the same or different;

[0020] Z is any group covalently linked to the carbonyl group of B1 in formula (I) or the carbonyl group of B2 in formula (II) or the carbonyl group of B3 in formula (III) or the carbonyl group of B in formula (IV) n On the carbonyl group;

[0021] n is an integer greater than or equal to 4, representing the number of branches connecting the active drug unit;

[0022] m is selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0023] Furthermore, the B1, B2, B3, ..., B n The structures are shown in formula (V):

[0024]

[0025] Wherein, p is selected from 1, 2, 3, 4, 5, 6, 7, 8.

[0026] Furthermore, the Z is selected from:

[0027] OH, SH, NH2,

[0028] Furthermore, the antibody-drug conjugate has the structure shown in formula (VI-1)-(VI-5):

[0029]

[0030]

[0031] in:

[0032] A is any antibody or its functional binding fragment;

[0033] L1, L2, L3, L4, and L5 are any connecting units and are independent of each other and can be the same or different;

[0034] D1, D2, D3, and D4 are any active drug units and are independent of each other and may be the same or different;

[0035] m is selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0036] Furthermore, the L1 is covalently linked to an amino residue or a thiol residue on the antibody; preferably, the L1 is covalently linked to a thiol residue on the antibody; more preferably, the L1 is covalently linked to a thiol residue formed after the opening of the interchain disulfide bond on the antibody.

[0037] Furthermore, the L1, L2, L3, L4, ..., L n+1 It is a cleavable linker, a combination of cleavable linkers, or a non-cleavable linker.

[0038] Furthermore, the cleavable linker comprises a peptide unit and a polysulfide bond, wherein the peptide unit comprises 2-20 amino acids, preferably the peptide unit is selected from -valine-citrulline-(-Val-Cit-), -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -valine-alanine-(-Val-Ala-), -valine-lysine-(-Val-Lys-), -valine-arginine-(-Val-Arg-), -phenylalanine-citrulline-(-Phe-Cit-), -phenylalanine-lysine-(-Phe-Lys-), -phenylalanine-arginine-(-Phe-Arg-) and combinations thereof; the polysulfide bond comprises 2-8 sulfur atoms, preferably the polysulfide bond is selected from a disulfide bond (-SS-), a trisulfide bond (-SSS-), and a tetrasulfide bond (-SSSS-).

[0039] In certain specific embodiments, the L1 can be selected from the following structures:

[0040]

[0041]

[0042]

[0043]

[0044] In certain specific embodiments, the L2, L3, L4, ..., L n+1 You can choose from the following structures:

[0045]

[0046]

[0047]

[0048] Furthermore, the antibody or its functional binding fragment includes a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, a single-chain Fv ("scFv"), a diabody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a fusion protein comprising the antigen-binding portion of an antibody; preferably, the antibody is a humanized monoclonal antibody or a fully human antibody.

[0049] Furthermore, the antibody is an IgG antibody or a functional binding fragment thereof, and more preferably, the antibody is IgG1, IgG2, IgG3, or IgG4.

[0050] Furthermore, the active drug unit is a cytotoxic molecule, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating autoimmune diseases, an anti-inflammatory drug, or a drug for treating infectious diseases.

[0051] Furthermore, the cytotoxic molecules include but are not limited to tubulin inhibitors or DNA damaging agents; further preferably, the tubulin inhibitors include but are not limited to dolastatin and auristatin cytotoxic molecules, maytansine cytotoxic molecules; the DNA damaging agents include but are not limited to calicheamicin, duocarmycin, anthramycin derivatives PBD, camptothecins and camptothecin derivatives, SN-38; further preferably, the auristatin cytokine molecules include but are not limited to MMAE or MMAF or their derivatives, the maytansine cytotoxic molecules include but are not limited to DM1, DM4 or their derivatives; further preferably, the cytotoxic molecules include the following molecules:

[0052]

[0053]

[0054]

[0055] Furthermore, the structure of the antibody drug conjugate is shown below:

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] in:

[0065] A is any antibody or its functional binding fragment;

[0066] m is selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0067] The present invention also provides a pharmaceutical composition comprising an effective amount of any one of the above-mentioned antibody-drug conjugates or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.

[0068] The present invention also provides use of any of the above-mentioned antibody-drug conjugates in the preparation of drugs for treating cancer.

[0069] The antibody-drug conjugates of the present invention use one or more cysteine ​​residues or cysteine ​​derivative residues as drug linker carriers to simultaneously couple one or more drugs to the limited linking sites of the antibody, thereby easily preparing antibody-drug conjugates with higher drug loads. In theory, since ADCs with higher drug loads can be prepared, the range of drugs that can be selected for coupling is also wider, so that drugs with lower toxicity can be selected to prepare ADC products, thereby obtaining ADC products with a larger therapeutic window. In addition, since multiple drug molecules can be coupled to one linking site, the ADC product obtained by the method of the present invention has better uniformity when preparing antibody-drug conjugates with the same DAR value. And the amount of antibody required for production can also be greatly reduced, thereby effectively reducing production costs. In addition, in experiments, we also surprisingly found that the antibody-drug conjugates prepared by the method of the present invention, compared with antibody-drug conjugates in which only one drug molecule can be connected to the same site, can still have the same inhibitory or killing effect on tumor cells when the total amount of coupled drug molecules is greatly reduced. DETAILED DESCRIPTION

[0070] [definition]

[0071] Various terms related to various aspects of the specification are used throughout the specification and claims. Unless otherwise indicated, such terms are given their ordinary meaning in the art. Other specifically defined terms should be understood in a manner consistent with the definitions provided herein.

[0072] As used herein, the terms "a," "an," and "the" are used according to standard convention and mean one or more unless the context indicates otherwise. Thus, for example, reference to "an antibody drug conjugate" includes a combination of two or more antibody drug conjugates, and so forth.

[0073] It should be understood that wherever aspects are described herein with the language "comprising," similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0074] Although the numerical ranges and parameter approximations shown in the broad scope of the present invention, the numerical values ​​shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" should be considered to include any and all sub-ranges between a minimum value of 1 and a maximum value of 10 (including endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0075] The present invention uses Refers to containing The groups are connected to other groups via chemical bonds.

[0076] The linking units and linkers in the present invention can be used interchangeably; the active units, drugs and poisons in the present invention can be used interchangeably.

[0077] The term "antigen" in the present invention refers to any molecule that triggers an immune response or is capable of being bound by an antibody or antigen binding molecule. The immune response may involve the production of antibodies or the activation of specific immunocompetent cells or both. Those skilled in the art will readily appreciate that any macromolecule, including nearly all proteins or peptides, can serve as an antigen. Typically, antigens can be endogenously expressed, i.e., expressed from genomic DNA, or they can be recombinantly expressed, or they can be chemically synthesized. The "antigens" referred to in the present invention specifically refer to those tumor-associated antigens, which are well known in the art and can be prepared using antibody preparation methods and information well known in the art. In order to develop effective cellular targets for cancer diagnosis and treatment, researchers have sought to identify transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells, while being little or not expressed on the surface of one or more non-cancerous cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells than on the surface of non-cancerous cells. Identifying such tumor-associated factors can greatly improve the specific targeting properties of antibody-based cancer treatments. Tumor-associated antigens include, but are not limited to, the tumor-associated antigens listed below (1)-(36). For convenience, information related to antigens known in the art is indicated below, including name, other names, and gene bank accession number. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases such as Genbank. Antibodies targeting tumor-associated antigens include all amino acid sequence variants and isotypes that have at least 70%, 80%, 85%, 90%, or 95% homology to the actual confirmed sequence, or have biological properties and characteristics that are completely consistent with the referenced tumor-associated antigen sequence. Tumor-associated antigens (1)-(37):

[0078] (1) BMPR1B (bone morphogenetic protein receptor type IB, Genbank accession number NM_001203);

[0079] (2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486);

[0080] (3) STEAP1 (six-transmembrane prostate epithelial antigen, Genbank accession number NM_012449);

[0081] (4) 0772P (CA125, MUC16, Genbank accession number AF361486);

[0082] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM_005823);

[0083] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate) member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM_006424);

[0084] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878);

[0085] (8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628);

[0086] (9) ETBR (endothelin type B receptor, Genbank accession number AY275463);

[0087] (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM_017763);

[0088] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six-transmembrane prostate epithelial antigen 2, six-transmembrane prostate protein, Genbank accession number AF455138);

[0089] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM_017636);

[0090] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession number NP_003203 or NM_003212);

[0091] (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792, Genbank accession number M26004);

[0092] (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-related β), B29, Genbank accession number NM_000626);

[0093] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchoring protein 1a), SPAP1B, SPAP1C, Genbank accession number NM_030764);

[0094] (17) HER2 (ErbB2, Genbank accession number M11730);

[0095] (18) NCA (CEACAM6, Genbank accession number M18728);

[0096] (19) MDP (DPEP1, Genbank accession number BC017023);

[0097] (20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession number AF184971);

[0098] (21) Brevican (BCAN, BEHAB, Genbank accession number AF229053);

[0099] (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession number NM_004442);

[0100] (23) ASLG659 (B7h, Genbank accession number AX092328);

[0101] (24) PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436);

[0102] (25) GEDA (Genbank accession number AY260763);

[0103] (26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF116456);

[0104] (27) CD22 (B cell receptor CD22-B isoform, Genbank accession number AK026467);

[0105] (28) CD79a (CD79A, CD79α, immunoglobulin-associated α, can covalently interact with Igβ (CD79B) and form a complex with Ig M molecules on the surface, transducing B cell-specific proteins involved in B cell differentiation signals, Genbank accession number NP_001774.1);

[0106] (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, plays a role in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and possibly in AIDS, lymphoma, myeloma, and leukemia, Genbank accession number NP_001701.1);

[0107] (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen), which binds peptides and presents them to CD4 + T lymphocytes, Genbank accession number NP_002111.1);

[0108] (31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, its defect may lead to the pathophysiological condition of idiopathic detrusor instability, Genbank accession number NP_002552.2);

[0109] (32) CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_001773.1);

[0110] (33) LY64 (lymphocyte antigen 64 (RP105), a member of the leucine-rich repeat-rich type I membrane protein (LRR) family, regulates B cell activation and apoptosis, and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, Genbank accession number NP_005573.1);

[0111] (34) FcRH1 (Fc receptor-like protein 1, a putative immunoglobulin Fc domain receptor containing C2-type Ig-like and ITAM domains, which may play a role in B lymphocyte differentiation, Genbank accession number NP_443170.1);

[0112] (35) IRTA2 (translocation-associated immunoglobulin superfamily receptor 2, a putative immune receptor that may play a role in B cell development and lymphomagenesis; gene dysregulation caused by translocation occurs in some B cell malignancies, Genbank accession number NP_112571.1);

[0113] (36) TENB2 (putative transmembrane proteoglycan related to the EGF / heregulin family of growth factors and follistatin, Genbank accession number AF179274);

[0114] (37) Other related antigens.

[0115] The term "antibody" in the present invention is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. Generally, an antibody can comprise at least two heavy chains and two light chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each heavy chain comprises a heavy chain variable region and a heavy chain constant region. The heavy chain constant region comprises three constant domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region and a light chain constant region. The light chain constant region comprises one constant domain: CL. The heavy chain variable region and the light chain variable region can be further subdivided into highly variable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each heavy chain variable region and light chain variable region comprises three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of Ab can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Unless otherwise indicated, the term "antibody" in the present invention also encompasses complete immunoglobulins or antigen-binding portions thereof that compete with complete antibodies for specific binding. The antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. Antigen-binding portions particularly include Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), fragments including complementary determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, bivalent antibodies, trivalent antibodies, tetravalent antibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigens that bind to the polypeptide.The term "antibody" as used herein also includes both naturally occurring and non-naturally occurring (recombinantly produced) antibodies, human and non-human antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies (see, e.g., Stocks, (2004) Drug Discovery Today 9(22):960-66), antibody fusions (which term encompasses antibody-drug conjugates and which are sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affybodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics") and antigen-binding fragments thereof.

[0116] The term "functional fragment" in the present invention refers to an antibody fragment consisting of or comprising a partial sequence of the heavy or light variable chain of the antibody from which it is derived, wherein the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1 / 100, and more preferably at least 1 / 10, of the affinity of the antibody from which it is derived. Such a functional fragment will contain a minimum of 5 amino acids, preferably 10, 15, 25, 50, or 100 consecutive amino acids of the antibody sequence from which it is derived.

[0117] The term "humanized antibody" refers to an antibody that comprises a CDR region derived from a non-human antibody and the rest of the antibody molecule is derived from one (or several) human antibodies. In addition, some residues in the framework (called FR) segment may be modified to retain binding affinity (Jones et al., Nature, 321: 522-525, 1986; Verhoeyen et al., Science, 239: 1534-1536, 1988; Riechmann et al., Nature, 332: 323-327, 1988). The humanized antibodies or fragments thereof according to the present invention can be prepared by techniques known to those skilled in the art (for example, as described in the documents Singer et al., J. Immun. 150: 2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10: 1-142, 1992; or Bebbington et al., Bio / Technology, 10: 169-175, 1992).

[0118] The term "chimeric antibody" refers to an antibody in which the variable region sequence is from one species and the constant region sequence is from another species, for example, an antibody in which the variable region sequence is from a mouse antibody and the constant region sequence is from a human antibody. Chimeric antibodies or fragments thereof according to the present invention can be prepared by using genetic recombination technology. For example, the chimeric antibody can be produced by cloning recombinant DNA, which comprises a promoter and a sequence encoding the variable region of a non-human, especially mouse, monoclonal antibody according to the present invention, and a sequence encoding the constant region of a human antibody. The chimeric antibody of the present invention encoded by such a recombinant gene will be, for example, a mouse-human chimera, the specificity of the antibody being determined by the variable region derived from the mouse DNA, and its isotype being determined by the constant region derived from the human DNA. For methods of preparing chimeric antibodies, for example, reference can be made to the document Verhoeyn et al. (BioEssays, 8:74, 1988).

[0119] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0120] In certain specific embodiments, the present invention relates to antibodies including but not limited to the following: muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab, etanercept, basiliximab, gemtuzumab, alemtuzumab, ibritumomab tiuxetan, adalimumab, afacept, omalizumab, efalizumab, tositumomab, cetuximab, ABT-806, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, linacept, certolizumab pegol, romiplostim, AMG-531, golimumab, ustekinumab, ABT-874, belatacept, belimumab, atacicept, anti-CD20 antibodies, canakinumab, tocilizumab, atezolizumab, mepolizumab, pertuzumab, HuMax CD20, tremelimumab, tesimumab, ipilimumab, IDEC-114, intuzumab, HuMax EGFR, aflibercept, HuMax-CD4, teplizumab, ortecilizumab, catumaxomab, anti-EpCAM antibody IGN101, adelimumab, ogavuzumab, dexmecitinib, giretuximab, denosumab, bapinezumab, motavizumab, efavirenz, rixibac, LY2469298, and veltuzumab.

[0121] The term "linker" in this invention refers to a bifunctional or multifunctional molecule that can react with both a protein / antibody molecule and a drug molecule, thereby serving as a "bridge" to connect the protein / antibody and drug molecule. Based on the mechanism of intracellular drug release, "linkers" or "antibody-drug conjugate linkers" can be divided into two categories: non-cleavable linkers and cleavable linkers.

[0122] A non-cleavable linker is a relatively stable linker whose structure is difficult to degrade and break in the in vivo environment. For antibody-drug conjugates containing non-cleavable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is internalized by the cell, the antibody is enzymatically hydrolyzed in the lysosome, releasing the active molecule composed of the small molecule drug, the linker, and the antibody amino acid residues. The resulting change in the drug molecular structure does not weaken its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into neighboring cells. Therefore, this type of active drug cannot kill neighboring tumor cells that do not express the target antigen (antigen-negative cells) (bystander effect) (Bioconjugate Chem. 2010, 21, 5-13). Common non-cleavable linkers include MC linkers and MCC linkers:

[0123]

[0124] Cleavable linkers, as the name suggests, can be cleaved within target cells to release the active drug (the small molecule drug itself). Cleavable linkers can be divided into two main categories: chemically labile linkers and enzyme-labile linkers.

[0125] Chemically labile linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties, such as pH and glutathione concentration.

[0126] pH-sensitive linkers, also commonly referred to as acid-cleavable linkers, are relatively stable in the neutral environment of blood (pH 7.3-7.5), but are hydrolyzed in the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). First-generation antibody-drug conjugates (ADCs) primarily utilize these linkers, such as hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid-cleavable linkers, ADCs based on these linkers typically have a short half-life (2-3 days). This short half-life has, to a certain extent, limited the application of pH-sensitive linkers in newer generation ADCs.

[0127] Glutathione-sensitive linkers, also known as disulfide linkers, trigger drug release based on the difference between the high intracellular glutathione concentration (millimolar range) and the relatively low glutathione concentration (micromolar range) in blood. This is particularly true for tumor cells, where low oxygen levels lead to increased reductase activity, resulting in higher glutathione concentrations. Disulfide bonds are thermodynamically stable, resulting in good stability in plasma.

[0128] Enzyme-labile linkers, such as peptide linkers, enable better control of drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as cathepsin B or plasmin (the levels of such enzymes are increased in some tumor tissues). This peptide linkage is considered very stable in the plasma circulation because proteases are generally inactive outside the cell due to the unfavorable extracellular pH and serum protease inhibitors. Due to their high plasma stability and good intracellular cleavage selectivity and efficiency, enzyme-labile linkers are widely used as cleavable linkers in antibody-drug conjugates. Typical enzyme-labile linkers include the VC linker.

[0129]

[0130] Suicide linkers are typically embedded between a cleavable linker and an active drug, or they themselves are part of the cleavable linker. The mechanism of action of a suicide linker is that upon cleavage of the cleavable linker under appropriate conditions, the suicide linker undergoes spontaneous structural rearrangement, releasing the attached active drug. Common suicide linkers include p-aminobenzyl alcohol (PAB).

[0131]

[0132] The term "active unit" in the present invention generally refers to any compound having the desired biological activity and a reactive functional group for preparing the conjugate of the present invention. The desired biological activity includes the diagnosis, cure, relief, treatment, and prevention of diseases in humans or other animals. As new drugs are continuously discovered and developed, these new drugs should also be included in the drugs described in the present invention. Specifically, the drugs include but are not limited to cytotoxic drugs, cell differentiation factors, stem cell trophic factors, steroid drugs, drugs for treating autoimmune diseases, anti-inflammatory drugs or drugs for infectious diseases. More specifically, the drugs include but are not limited to microtubule inhibitors or DNA, RNA damaging agents. Preferably, the active units involved in the present invention include but are not limited to the following:

[0133] (a) Erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787 / ZK222584, oxaliplatin, 5-fluorouracil, folinic acid, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carbaquinone, metodepa, uredepa, ethyleneimine, hexamethylmelamine, triethylene melamine, triethylene phosphoramide, triethylenethiophosphoramide, trihydroxymethylmelamine, sucralose, sucralose octanone, camptothecin, topotecan, bryostatin, calistin, CC-1065, adolesin, carzelesin, bisezole, sucralose 1, Candida albicans 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, acanthopanax, hyoscyamine, stoloniferol, sponge inhibitor, chlorambucil, naphthalene nitrogen mustard, clofosamide, estramustine, ifosfamide, dichloromethyl diethylamine, melphalan, nembixin, phenylmethane cholesterol, prednimustine, trofosamide, uracil mustard, carmustine, chlorozonomycin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, danemycin, danemycin A, clodronate, esperamicin, neocarcinogen, aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carbicycline, carmomycin, carmosin, Chromomycin, dactinomycin, daunorubicin, detoxrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, mexicomycin, mitomycin C, mycophenolic acid, nogamycin, olivomycin, pyromycin, porphyromycin, puromycin, triferon-doxorubicin, rhodorubicin, streptomycin, streptozocin, tuberculin, ubenimex, zotocin, daunorubicin, 5-fluorouracil, dimethylfolate, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiopurine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine , doxifluridine, enocitabine, floxuridine, kalutestosterone, drostanolone propionate, cyclothiocarbamate, melastane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglucuronolactone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, basbutane, bisantrene, edatrexate, defosfamide, colcemid, diazocine, eflornithine, elixir Ammonium, etoglu, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansine, ansamitocin, mitoguanidine, mitoxantrone, mopidarol, diamine nitrazepam, pentostatin, methaminomustine, pirarubicin, losoxantrone, 2-ethylhydrazide, methylbenzylhydrazine, polysaccharide-k, razoxane, rhizoxin, sizosone, germanium spiroamine, fine cross-sporic acid, triaminoquinone, 2,2',2"-Trichlorotriethylamine, T-2 toxin, fusobactin A, baculosporin A, and serpentin, urethane, vindesine, dacarbazine, mannitol mustard, dibromomannitol, dibromodulanol, piperoman, dry cytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, mitoxantrone, teniposide, edatrexate, daunorubicin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine, or any pharmaceutically acceptable salt, solvate or acid thereof;

[0134] (b) Monokines, lymphokines, traditional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, liver growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α, tumor necrosis factor-β, Mullerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinductive factor, interferon, interferon-α, interferon-β, interferon-γ, colony stimulating factor ("CSF"), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin (IL), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factor, LIF, kit ligand, or any combination of the foregoing;

[0135] (c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, amanitin derivatives, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, tubulin, geldanamycin, maytansine, calicheamicin, daunorubicin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analogs, auristatin, candidatin, camptothecin, camptothecin derivatives and metabolites, rhizoctonia, rhizoctonia derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycin, enediyne antibiotics, esperamicins, epothilones, azafil, aplidine, toxoids, or any combination of the foregoing;

[0136] (d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, a stimulator, a neurotransmitter, a radioisotope, or a combination of any of the foregoing;

[0137] (e) a radioactive label, 32P, 35S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, digoxigenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a combination of any of the foregoing;

[0138] (f) immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or combinations of any of the foregoing;

[0139] (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, troloxifene, naloxifene, LY117018, onapristone, or toremifene;

[0140] (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole;

[0141] (i) Flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine;

[0142] (j) aromatase inhibitors;

[0143] (k) protein kinase inhibitors;

[0144] (1) lipid kinase inhibitors;

[0145] (m) antisense oligonucleotides;

[0146] (n) ribozymes;

[0147] (o) vaccines; and

[0148] (p) Anti-angiogenic agents.

[0149] In some embodiments of the present invention, the "active unit" is selected from: maytansinoids, V-ATPase inhibitors, pro-apoptotic agents, Be12 inhibitors, McL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTOr inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, dolastatin, MetAP (methionine aminopeptidase), nuclear export inhibitors of protein CRM1, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA minor groove binders, DHFR inhibitors, and dolastatin peptides, vitamin A precursors, and folic acid.

[0150] In some embodiments of the present invention, the "active unit" is a cytotoxic drug (e.g., antimetabolite, antitumor antibiotic, alkaloid), an immunopotentiator, or a radioisotope. Preferably, the drug can be selected from amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicines, colcimids, combretastatins, cryptophycins, discodermolides, docetaxel, doxorubicin, n), echinomycins, eleutherobins, epothilones, estramustines, lexitropsins, maytansines, methotrexate, netropsins, puromycins, rhizoxins, taxanes, tubulysins, or vinca alkaloids. More preferably, the “drug” can be selected from MMAD (Monomethyl auristatin D) and its derivatives, MMAE (Monomethyl auristatin E) and its derivatives, MMAF (Monomethyl auristatin F) and its derivatives, maytansine derivative DM1 (Mertansine derivative M1), maytansine derivative DM4 (Mertansine derivative M4), Duocarmycine and its derivatives, Calichemicin and its derivatives, PBDA (Pyrrolobenzodiazepines), Doxorubicin, Vinca Alkaloids, Metrotrexate, Vinblastine, Daunorubicin and its derivatives, tubulysins and its derivatives.

[0151] In certain specific embodiments, the "active unit" is maytansine or maytansinoids. Maytansine compounds inhibit cell proliferation by inhibiting the microtubule formation of tubulin (Science 1975, 189, 1002-1005; US 5208020). Maytansinoids are derivatives of maytansine. Both maytansine and maytansinoids have efficient cytotoxicity, but they have great limitations in the clinical application of cancer treatment, which is mainly due to the low selectivity of such molecules to tumors. However, this high cytotoxicity prompts them to become the preferred drug part of antibody drug conjugates. Listed below are maytansine, maytansinoids, and three maytansinoid molecular structures that are frequently used in antibody drug conjugate applications.

[0152]

[0153] The main raw material for synthetic maytansinoids is maytansinol, which is primarily obtained by hydrolysis of ansamitocins. Ansamitocins can be produced by fermentation. Ansamitocin derivatives (WO 2012 / 061590) and alanyl maytansinol (US 2012 / 0121615) have also been reported as drug "warheads" for antibody-drug conjugates.

[0154]

[0155] In certain specific embodiments, the "active unit" is an auristatin peptide drug. Auristatin peptide drugs are analogs of dolastatin 10, a biologically active polypeptide isolated from the marine mollusk Aplysia (US 7498298). Dolastatin 10 inhibits tubulin polymerization by binding to tubulin (the same binding region as vincristine). Dolastatin 10, auristatin peptide PE, and auristatin peptide E are all linear polypeptides containing four amino acids (three of which are unique to dolastatin compounds) and a C-terminal amide group. Two representative auristatin peptide compounds, monomethyl auristatin peptide E (MMAE) and monomethyl auristatin peptide F (MMAF), are both preferred drug moieties for antibody drug conjugates.

[0156]

[0157] In certain specific embodiments, the "active unit" is a Tubulysin drug. Tubulysins are a class of natural products extracted from myxobacteria that can effectively inhibit the polymerization of tubulin and therefore have anti-mitotic activity. Tubulysin D has the best activity. Tubulysin D is a complex tetrapeptide compound that contains O-acyl / N,O-acetal functional groups in its structure and is therefore unstable under both acidic and alkaline conditions. US2011 / 0021568 and US2013 / 0224228 respectively disclose a series of tubulysin analogs that have the above unstable functional groups removed from their structures while having high cellular activity.

[0158]

[0159] In certain specific embodiments, the "active unit" is a calichemicin. Calcheamicins are antitumor antibiotics that bind to the minor groove of DNA and induce double-helix DNA breaks at specific sites, leading to cell apoptosis. While calichemicins exhibit sub-picomolar activity in vitro, their low therapeutic index precludes clinical application. However, this high activity makes them ideal candidates for antibody-drug conjugates (e.g., gemtuzumab ozogamicin and inotuzumab ozogamicin).

[0160]

[0161] In certain specific embodiments, the "active unit" is doxorubicin. Doxorubicin can be embedded in the DNA double helix structure to block DNA replication and is therefore used as a chemotherapeutic drug. However, due to the low cytotoxicity of doxorubicin (for human cancer cell lines, the half-inhibitory concentration is 0.1-0.2 micromolar, while the cytotoxic drug activity for antibody-drug conjugates is generally sub-nanomolar), its application in antibody-drug conjugates is not common.

[0162]

[0163] In certain specific embodiments, the "active unit" is a benzodipyrrole antibiotic (duocarmycins, CC-1065, etc.) and other cyclopropapyrroloind-4-one (CPI) derivatives. These compounds are effective DNA minor groove binding-alkylating agents. Cyclopropabenzindol-4-one (CBI) analogs have more stable chemical structures, higher biological activity, and are easier to synthesize than their parent compounds containing natural CPI alkylating subunits. A representative CBI derivative is the phenolic hydroxyl-protected derivative CBI (see the figure below), which has weakened prodrug toxicity and enhanced water solubility.

[0164]

[0165] In certain specific embodiments, the "active unit" is a pyrrolo[2,1-c][1,4]benzodi-azepine (PBDs) or PBD dimers. PBD is a natural product produced by Streptomyces, and its unique property is that it can form non-twisted covalent adducts in the minor groove of DNA, specifically at the purine-guanine-purine sequence. The use of PBD as part of a small molecule strategy to target DNA sequences and as a new type of anticancer and antibacterial drug has attracted increasing interest (Biochemistry 2008, 47, 11818-11829). A flexible carbon chain is used to connect the C8 / C8' hydroxyl groups of two PBD units, and the resulting dimer has enhanced biological activity (WO 2011 / 130616). PBD dimers are believed to be able to produce sequence-selective DNA damage, such as inverted 5'-Pu-GATC-Py-3' interchain crosslinks, thereby leading to their biological activity. These compounds have been shown to be highly potent cytotoxic drugs and may serve as candidates for antibody-drug conjugates.

[0166]

[0167] In other specific embodiments, the "active unit" is not limited to the above-mentioned categories, but also includes all drugs that can be used in antibody-drug conjugates.

[0168] The term "pharmaceutical composition" as used herein refers to a combination of at least one drug and, optionally, a pharmaceutically acceptable carrier or excipient, combined to achieve a specific purpose. In certain embodiments, the pharmaceutical composition includes temporally and / or spatially separated combinations, provided they function together to achieve the purpose of the present invention. For example, the components of the pharmaceutical composition may be administered to a subject as a whole or separately. When the components of the pharmaceutical composition are administered to a subject separately, the components may be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or a polyalkylene glycol such as polypropylene glycol, triglycerides, or the like. The type of pharmaceutically acceptable carrier used depends, inter alia, on whether the composition according to the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The composition according to the present invention may contain a wetting agent, emulsifier, or buffer as an additive.

[0169] The pharmaceutical composition, vaccine or pharmaceutical preparation of the present invention can be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.

[0170] The term "effective amount" as used herein encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. [Specific embodiment]

[0172] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Reagents for which specific sources are not specified are conventional reagents purchased from the market. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0173] The units of weight-volume percentage in the present invention are well known to those skilled in the art, for example, refer to the weight of the solute in 100 ml of solution.

[0174] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0175] Example 1 Preparation of Compound 1

[0176]

[0177] Dissolve 145.4 mg of maleimidocaproic acid in 10 mL of DMF, add 244.7 mg of HATU and 226 μL of DIPEA, and stir at room temperature. Dissolve 201.9 mg of MMAF in 5 mL of DMF and slowly add dropwise to the reaction system, stirring at room temperature for 16 h. Distill off the solvent under reduced pressure, and purify by preparative liquid chromatography to obtain 90.3 mg of the product (maleimidocaproyl-MMAF, Mc-MMAF) in a yield of 36%. LC-MS: (M+H) + 924.8, (MH) - 923.2.

[0178] Dissolve 36.1 mg of maleimidocaproyl-MMAF in 1.5 mL of DMF, add 5.6 mg of Cys-Cys and 2.1 μL of DIPEA, and stir at room temperature for 3 hours. Then add 1.1 mg of Cys-Cys and stir at room temperature for 2 hours. Add 17.7 mg of 6-(maleimido)hexanoic acid succinimidyl ester and 25 μL of DIPEA, and stir at room temperature for 15 hours. Distill off the solvent under reduced pressure, and purify by preparative liquid chromatography to obtain 120.0 mg of the compound in a 45% yield. LC-MS: (M+2H) 2+ 1134.1, (M-2H) 2- 1132.2.

[0179] Example 2 Preparation of Compound 2

[0180]

[0181] Maleimidocaproyl-MMAF was prepared according to the preparation method of Example 1. 40.1 mg of maleimidocaproyl-MMAF was dissolved in 1.5 mL of DMF, 4.4 mg of Cys-Cys-Cys and 2.1 uLDIPEA were added, and the mixture was stirred at room temperature for 2 h. 1.8 mg of Cys-Cys-Cys was added, and the mixture was reacted at room temperature for 2 h. 0.9 mg of Cys-Cys-Cys was added, and the mixture was reacted at room temperature for 2 h. 13.5 mg of 6-(maleimido)hexanoic acid succinimidyl ester and 21 uLDIPEA were added, and the mixture was stirred at room temperature for 15 h. The solvent was distilled off under reduced pressure, and the mixture was purified by preparative liquid phase to obtain 223.1 mg of compound with a yield of 49%. LC-MS: (M+3H) 3+ 1648.4, (M-3H) 3- 1646.3.

[0182] Example 3 Preparation of Compound 3

[0183]

[0184] Maleimidocaproyl-MMAF was prepared according to the preparation method of Example 1. 31.6 mg of maleimidocaproyl-MMAF was dissolved in 2 mL of DMF. 3.9 mg of Cys-Cys and 2.9 uLDIPEA were added, stirred at room temperature for 4 h, then 1.2 mg of Cys-Cys was added, stirred at room temperature for 3 h, then 0.8 mg of Cys-Cys was added, and stirred at room temperature for 16 h. Separately, 12.1 mg of 1,3,5-triacryloylhexahydro-1,3,5-triazine-thioglycolic acid and 10.4 mg of TSTU were dissolved in 1.5 mL of DMF, 17 uL of DIPEA was added, stirred at room temperature for 2 h, and then added to the above reaction system, and stirred at room temperature for 5 h. The solvent was distilled off under reduced pressure, and the product was purified by preparative liquid phase to obtain 318.4 mg of compound with a yield of 29%. LC-MS: (M+2H) 2+ 1199.3, (M-2H) 2- 1197.4.

[0185] Example 4 Preparation of Compound 4

[0186]

[0187] Maleimidocaproyl-MMAF was prepared according to the preparation method of Example 1. 55.0 mg of maleimidocaproyl-MMAF was dissolved in 1.5 mL of N,N-dimethylformamide, 11.6 mg of Cys-Cys was added, 4.3 uL of N,N-diisopropylethylamine was added, and the mixture was stirred at room temperature for 3 h. 36.1 mg of MP2-PNP (2-(2-Maleimidoethoxy)ethyl(4-nitrophenyl)carbonate, maleimidoethoxyethyl-p-nitrophenyl carbonate) was added to the reaction system, and 51 uL of N,N-diisopropylethylamine was added, and the mixture was stirred at room temperature for 15 h. The solvent was distilled off under reduced pressure, and the mixture was purified by preparative liquid chromatography to obtain 418.0 mg of compound 4 with a yield of 27%. LC-MS: (M+2H) 2+ 1142.3, (M-2H) 2- 1141.1.

[0188] Example 5 Preparation of Compound 5

[0189]

[0190] Maleimidocaproyl-MMAF was prepared according to the preparation method of Example 1. 55.0 mg of maleimidocaproyl-MMAF was dissolved in 1.5 mL of N, N-dimethylformamide, 11.6 mg of Cys-Cys was added, 4.3 uL of N, N-diisopropylethylamine was added, and the mixture was stirred at room temperature for 3 h. 71.0 mg of maleimidopropionamide-octapolyethylene glycol propionate succinimide ester (Mal-Propionamide-PEG8-propionate-OSu) was added to the reaction system, and 51 uL of N, N-diisopropylethylamine was added, and the mixture was stirred at room temperature for 15 h. The solvent was distilled off under reduced pressure, and the mixture was purified by preparative liquid phase to obtain 519.3 mg of compound. Yield 28%, LC-MS: (M+2H) 2+ 1324.1, (M-2H) 2- 1324.0.

[0191] Example 6 Preparation of Compound 6

[0192]

[0193] Maleimidocaproyl-MMAF was prepared according to the preparation method of Example 1. 63.0 mg of maleimidocaproyl-MMAF was dissolved in 2.0 mL of N,N-dimethylformamide, 13.3 mg of Cys-Cys was added, 4.8 uL of N,N-diisopropylethylamine was added, and the mixture was stirred at room temperature for 3 h. 86.6 mg of Mc-VC-PAB-PNP was added to the reaction system, followed by 58 uL of N,N-diisopropylethylamine, and the mixture was stirred at room temperature for 15 h. The solvent was distilled off under reduced pressure, and the mixture was purified by preparative liquid chromatography to obtain 647.4 mg of the compound with a yield of 52%. LC-MS: (M+2H) 2+ 1336.0, (M-2H) 2- 1334.6.

[0194] Example 7 Preparation of Compound 7

[0195]

[0196] Maleimidocaproyl-MMAF was prepared according to the preparation method of Example 1. 46.6 mg of maleimidocaproyl-MMAF was dissolved in 2.0 mL of N,N-dimethylformamide, 10.9 mg of Cys-Cys was added, 3.6 uL of N,N-diisopropylethylamine was added, and the mixture was stirred at room temperature for 3 h. 84.4 mg of PY-MAA-VC-PAB-PNP was added to the reaction system, followed by 48 uL of N,N-diisopropylethylamine, and the mixture was stirred at room temperature for 15 h. The solvent was distilled off under reduced pressure, and the mixture was purified by preparative liquid chromatography to obtain 731.0 mg of the compound with a yield of 43%. LC-MS: (M+2H) 2+ 1401.2, (M-2H) 2- 1399.4.

[0197] Example 8 Preparation of Antibody Drug Conjugate (ADC)

[0198] The general method for the synthesis of antibody drug conjugates is:

[0199] Method A: Anti-Her-2 antibody was prepared into a 10 mg / mL solution using PBS buffer at pH 7.4. 2.4 molar equivalents of TCEP were added and the solution was shaken and mixed for 1 hour. 5.0 molar equivalents of linker-toxin were then added and shaken and mixed. The solution was reacted for 1 hour. After the reaction, residual small molecules were removed by ultrafiltration and the solution was loaded onto hydrophobic chromatography (HIC-HPLC) for DAR, drug distribution, and naked antibody ratio detection.

[0200] Method B: Anti-Her-2 antibody was prepared into a 10 mg / mL solution using boric acid-borax buffer at pH 9. 5 molar equivalents of TCEP were added and the solution was shaken for 1 hour. 6.0 molar equivalents of linker-toxin were then added and shaken for 3 hours. After the reaction, residual small molecules were removed by ultrafiltration and the solution was loaded onto hydrophobic chromatography (HIC-HPLC) for DAR, drug distribution, and naked antibody ratio detection.

[0201] The following compounds were prepared using the general ADC preparation method (A is any antibody or its functional binding fragment, m is 1, 2, 3, 4, 5, 6, 7, 8), the following compounds:

[0202]

[0203]

[0204] Example 9 Inhibitory Effect of ADC on SK-BR-3 Tumor Cell Line

[0205] SK-BR-3 tumor cells (human breast cancer cells) were digested, collected by centrifugation, counted, and the cell suspension was diluted to 0.5-1.5×10 5 / mL, 100μL of the cell suspension was added to each well of a 96-well plate. Incubate overnight in a 37°C, 5% CO2 incubator; on the next day, add 9 corresponding concentration gradients of ADC drugs, plus a group of cell controls with zero concentration of ADC drugs. After 72 hours of culture, use Cell Counting Kit-8 (CCK-8 kit for short) for activity color development, and the OD value at 450nm of the 96-well plate after color development is detected by a microplate reader. Prism software calculates the IC50 value based on the OD value. When the fitted curve is an "S-shaped curve" and R 2 When the IC50 value is ≥0.95, it is valid and can be reported.

[0206] We selected ADC-0 with an average DAR value of 3.87 (Comparative Example 1), ADC-0 with an average DAR value of 7.18 (Comparative Example 2), ADC-10 with an average DAR value of 4.3 (Comparative Example 3), and ADC-34 with an average DAR value of 4.29 (Comparative Example 4) to conduct inhibition experiments on SK-BR-3 tumor cell lines. Their IC50 values ​​are shown in Table 2:

[0207] Table 2 Inhibitory evaluation of SK-BR-3 tumor cell line

[0208]

[0209] * Taking Comparative Example 1 as the base, the change in antibody dosage = (Comparative Example X - Comparative Example 1) / Comparative Example 1; X represents the corresponding comparative example;

[0210] *Based on Comparative Example 1, change in MMAF dosage = (Comparative Example X - Comparative Example 1) / Comparative Example 1; X represents the corresponding comparative example;

[0211] Comparative Example 2 increased the DAR value to 7.18 by increasing the number of MMAF sites attached to the antibody. Compared with Comparative Example 1, while achieving the same effect, Comparative Example 2 can effectively reduce the antibody usage (i.e., the amount of antibody in the ADC, excluding antibody loss during the synthesis process, the antibody usage is equal to the ADC concentration), but the MMAF usage is increased by 19.3%.

[0212] Comparative Example 3 has two MMAFs connected to one linker, and the DAR value (4.3) is comparable to that of Comparative Example 1 (3.87). Compared with Comparative Example 1, while achieving the same effect, Comparative Example 3 effectively reduces the amount of antibody used by 65.9% and the amount of MMAF used by 24.3%.

[0213] Comparative Example 4 has three MMAFs connected to one linker, and the DAR value is comparable to that of Comparative Example 1. Compared with Comparative Example 1, while achieving the same effect, Comparative Example 4 effectively reduces the amount of antibody used by 85.5% and the amount of MMAF used by 51.8%.

[0214] The above comparative data demonstrates that ADC-10 and ADC-34 exhibit more efficient cellular activity. This effect goes beyond simply increasing the amount of MMAF in the antibody to enhance ADC efficacy. Furthermore, while carrying the same amount of MMAF, the single-point multi-warhead (cytotoxic drug) attachment method effectively reduces both antibody and toxin usage, achieving an unexpected technical benefit. Furthermore, the single-point multi-warhead approach significantly improves ADC product uniformity, significantly facilitating pharmaceutical production and quality control.

[0215] The present invention has been illustrated through various specific embodiments. However, those skilled in the art will appreciate that the present invention is not limited to these specific embodiments. Those skilled in the art may make various modifications and variations within the scope of the present invention, and the various technical features described throughout this specification may be combined without departing from the spirit and scope of the present invention. Such modifications and variations are within the scope of the present invention.

Claims

1. An antibody-drug conjugate, characterized in that: The structure of the antibody drug conjugate is shown below: in: A is any antibody or a functional binding fragment thereof, wherein the sulfhydryl residue on A is covalently linked to the linker; m is selected from 1, 2, 3, 4, 5, 6, 7, and 8.

2. The antibody-drug conjugate according to claim 1, wherein: The antibodies or functional binding fragments thereof include monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv ("scFv"), diabodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or fusion proteins comprising the antigen-binding portion of an antibody.

3. The antibody-drug conjugate according to claim 2, wherein: The antibody is a humanized monoclonal antibody or a fully human antibody.

4. The antibody-drug conjugate according to claim 1, wherein: The antibody is an IgG antibody or a functional binding fragment thereof, 5. The antibody-drug conjugate according to claim 4, wherein: The antibodies are IgG1, IgG2, IgG3, and IgG4.

6. A pharmaceutical composition comprising an effective amount of the antibody-drug conjugate according to any one of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.

7. Use of the antibody-drug conjugate according to any one of claims 1 to 5 in the preparation of a drug for treating cancer.

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