RU2787617C2 - Antibody-drug conjugates and their use - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to an antibody-drug conjugate (hereinafter – ADC) having increased drug loading, which is obtained using one or more cysteine residues or cysteine derivative residue as carriers bound to a drug and due to conjugation of one or more drugs at the same time by a limited number of antibody binding sites, or it can be obtained with a drug with low toxicity in such a way that ADC product with a wider therapeutic window is obtained. In addition, since a set of drug molecules can be conjugated by one binding site, when ADC is obtained having the same DAR value, the resulting ADC product has better homogeneity. The used amount of an antibody required for preparation can also be significantly reduced.

EFFECT: resulting ADC still can achieve the same inhibitory or destructive effect on tumor cells, while the total number of conjugated drug molecules is significantly reduced compared to ADC capable of binding only one drug molecule by the same site.

14 cl, 2 tbl, 9 ex

Description

Field of invention

The description of the present invention relates to the field of biomedicine, and in particular to an antibody-drug conjugate in which one or more cysteine residues or derivatives thereof are used as a linker, and their uses, and the linker can be loaded as desired with one or more than one drug at a limited antibody binding site.

Prior Art

An antibody-drug conjugate (ADC) refers to a class of biological drugs in which the active drug is linked to an antibody through a chemical linker. ADCs are like a precision guided weapon system that uses an active drug as a projectile to precisely kill diseased cells, directed by an antibody. Thus, ADC combines the dual advantages of high potency of cytotoxic drugs and a high degree of targeting of the antibody. At the end of July 2019, only six antibody-conjugated drugs had been approved worldwide (Table 1).

Due to the non-uniqueness of the selected binding sites on an antibody and the complexity of the ligation reaction, the actual resulting ADC product is a mixture of ADCs containing varying amounts of antibodies bound to different amounts of drugs at different sites. The heterogeneity of the resulting ADC drug creates significant difficulties for the production and quality control of drugs. When analyzing product homogeneity, there is an important indicator, namely the DAR (Drug to Antibody Ratio), which is the average amount of drugs that can be carried by one unit of antibody. If the DAR value is too small, then there are too few drug molecules carried by the antibody, which can affect the overall efficacy of the drug. However, if the average DAR is too high, then the antibody is loaded with too many drug molecules, which leads to general instability of the ADC, changes in its pharmacokinetic parameters, and can increase plasma clearance, decrease half-life, and increase systemic drug toxicity. .

There are currently three classical coupling methods for ADC, one of which is amide coupling, the other is thiol coupling, and the third is cross-linking.

Amide coupling consists in linking a drug to a lysine residue (Lys) of an antibody using a linker. The first generation of the ADC drug mylotarg utilized this amide coupling route. There are about one hundred lysines in IgG (Immunoglobulin G), binding can occur at about 40 exposed lysine residues on the light chain and heavy chain of an antibody, and each antibody can be linked to a variety of drug molecules in varying amounts by binding through lysine. Thus, ADCs produced by amide coupling are highly heterogeneous and the product has extremely poor uniformity, which seriously affects the PK/PD and the therapeutic range of the drug (see http://www.sohu.com/a/277791166_464404).

In thiol binding, the interchain disulfide bond of an antibody is first broken down to form a free cysteine residue (Cys), which is further bound to a linker-drug complex capable of combining with the cysteine residue. Since there are only 4 pairs of interchain disulfide bonds in IgG, after breaking all interchain disulfide bonds, 8 free cysteine residues can be formed, thus the average DAR value for ADCs obtained by monothiol binding is 0-8. While thiol binding may provide better control for multiple drugs on each antibody, disruption of the interchain disulfide bond greatly reduces the stability of the antibody.

Cross-linking is a new type of linkage that has been developed from thiol linkage. Like thiol binding, the interchain disulfide bond of an antibody is first broken down to form two free residues, which then bind simultaneously. Since there are only 4 pairs of interchain disulfide bonds on one antibody, there are 8 free cysteine residues after all interchain disulfide bonds are broken, thus the bridged ADCs have an average DAR of 0-4. Compared to thiol binding, bridging can provide better control of product homogeneity and can greatly contribute to the stability of the antibody after binding. However, the DAR for bridging is up to 4, meaning up to 4 drugs can be linked to a single antibody. The number of antigens on the surface of tumor cells is limited, and the level of antigen expression required for effective ADC activity varies depending on the characteristics of the various antigens. ADC requires at least 1×10 ⁴ antigens/cell to deliver a lethal amount of a cytotoxic drug. Ideally, the antigen targeted by the antibody in the ADC should be uniformly expressed on the surface of tumor cells with a high copy number (greater than 10 ⁵ /cell). Tumor cells usually have a limited number of antigens on their surface (approximately 5000 to 10 ⁶ antigens/cell), although ADCs have a DAR of 3.5-4 (e.g., brentuximab vedotin has a mean DAR of 4 and trastuzumab emtansine has a mean DAR, equal to 3.5), which results in the amount of drug delivered to the tumor cells being very low, resulting in less effect on the tumor cells. The latter is believed to be one of the main causes of clinical failure in relation to ADC.

Due to the limited number of antigens on the surface of tumor cells, in order to allow effective killing of tumor cells by a limited number of drugs that carry antibodies, small molecules with particularly strong toxicity are often used in the clinic. At present, the small molecules used in ADC mainly include the classes of auristatin, maytansine, calicheamicin, doxorubicin, and the like. These small molecules are more powerful in killing cancer cells than conventional chemotherapy drugs. Typically, killing of target cells can be achieved with an average dose of four to six molecules. However, if these highly toxic small drug molecules lose their target in the body, they are fatal to the patient. In addition, because these small molecules are particularly toxic (e.g. DM1 has an IC ₅₀ (mean inhibitory concentration) of approximately 10 ^-11 mol/L for a variety of cells, DM4 has an IC ₅₀ of approximately 10 ^-12 mol/L (ref. 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 Ref 2: Lambert JM Antibody-maytansinoid conjugates: a new strategy for the treatment of Cancer [J]. Drugs Future, 2010, 35: 471-480.), and MMAE has an IC ₅₀ of approximately 10 ^-11 -10 ^-9 mol/l). If conditions exist, such as loss of target and early release of the drug, there are very high risks for patients. The FDA (Food and Drug Administration) conducted a pooled analysis for 20 Investigational New Drugs (IND) and found that the toxicity of ADC drugs in animals is mainly determined by hematopoietic toxicity, hepatotoxicity and reproductive toxicity, and some of them also have dermal toxicity and nephrotoxicity, where hematopoietic toxicity, hepatotoxicity, and reproductive toxicity are directly related to small molecule cytotoxic drugs (ref. 3: Saber H, Leighton JK. An FDA oncology analysis of antibody-drug conjugates [J]. Regul Toxicol Pharmacol, 2015 , 71(3): 444-452). However, if a small molecule drug having low toxicity is used, then the loading amount of the small molecule drug is often too low, such that the effect obtained from the drug is too low, leading to clinical failure.

Brief summary of the invention

In order to solve the above problems, the description of the present invention proposes a new antibody-drug conjugate having the ability to bind to a large number of active drugs, and the technical solutions according to the description of the present invention are as follows.

In the description of the present invention, there is provided an antibody-drug conjugate represented by formula (I)-(IV):

where,

A is an antibody or functional binding fragment thereof;

B ₁ , B ₂ , B ₃ , ... and B _n are cysteine or a derivative thereof, and they may be the same or different; B ₁ and B ₂ , B ₂ and B ₃ , ..., B _n-1 and B _n are linked via a peptide bond in a dehydrogenation reaction with condensation;

each of L ₁ , L ₂ , L ₃ , L ₄ , ... and L _n+1 independently represents a linker, and they may be the same or different; L ₁ is covalently bonded to the N-terminus of B ₁ , L ₂ and B ₁ , L ₃ and B ₂ , L ₄ and B ₃ , ... L _n+1 and B _n are covalently bonded via a thiol group, and L ₂ and D ₁ , L ₃ and D ₂ , L ₄ and D ₃ , ... L _n+1 and D _n are covalently bonded;

each of D ₁ , D ₂ , D ₃ , ... and D _n independently represents an active drug, and they may be the same or different;

Z represents a group covalently bonded to a carbonyl group in B ₁ in formula (I), a carbonyl group in B ₂ in formula (II), a carbonyl group in B ₃ in formula (III), or a carbonyl group in B _n in formula (IV );

n is an integer equal to or greater than 4, which is the number of branches that bind to active drugs; and

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

In addition, the structures B ₁ , B ₂ , B ₃ , ..., B _n are represented by formula (V), respectively:

where p is selected from 1, 2, 3, 4, 5, 6, 7 and 8.

In addition, Z is selected from the group consisting of:

OH, SH, NH ₂ ,

In addition, the antibody-drug conjugate has the structure represented by the formula (VI-1) to (VI-5):

where,

A is an antibody or functional binding fragment thereof;

each of L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ are independently linkers, which may be the same or different;

each of D ₁ , D ₂ , D ₃ and D ₄ independently represent active drugs, which may be the same or different; and

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

In addition, L ₁ is covalently linked to the amino or thiol residue of the antibody; preferably, L ₁ is covalently linked to the thiol group of the antibody; more preferably, L ₁ is covalently linked to a thiol residue formed by breaking the interchain disulfide bond of the antibody.

In addition, L ₁ , L ₂ , L ₃ , L ₄ , ... and L _n+1 represent a cleavable linker, a combination of cleavable linkers, or a non-cleavable linker.

In addition, the cleavable linker includes a peptide linker and a polysulfide bond, and where the peptide linker contains 2-20 amino acids, preferably the peptide linker is selected from the group consisting of -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; and the polysulfide bond contains 2-8 sulfur atoms, preferably the polysulfide bond is selected from the group consisting of a disulfide bond (-S-S-), a trisulfide bond (-S-S-S-) and a tetrasulfide bond (-S-S-S-S-).

In some specific embodiments, L ₁ may be selected from the following structures:

In some specific embodiments, L ₂ , L ₃ , L ₄ , ... L _n+1 may be selected from the following structures:

In addition, an antibody or functional binding fragment thereof includes a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, single chain Fv ("scFv"), diabody, linear antibody , a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a fusion protein containing an antigen-binding antibody fragment; preferably, the antibody is a humanized monoclonal antibody or a fully human antibody.

In addition, the antibody is an IgG antibody or a functional binding fragment thereof, preferably, the antibody is an IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ antibody.

In addition, the active drug is a cytotoxic molecule, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease.

In addition, the cytotoxic molecule includes, without limitation, a tubulin inhibitor or a DNA damaging agent; preferably, the tubulin inhibitor comprises a cytotoxic molecule of dolastatins and auristatins, a cytotoxic molecule of maytansines; the DNA damaging agent includes calicheamicins, duocarmycins, pyrrolobenzodiazepine (PBD), an anthramycin derivative, camptothecins and derivatives thereof, and SN-38; moreover, preferably, the auristatin cytokine molecule includes MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), and a derivative thereof; and the maytansine cytotoxic molecule includes DM1, DM4 and a derivative thereof; and further preferably, the cytotoxic molecule comprises the following molecules:

In addition, the structures of the antibody-drug conjugate are as follows:

where,

A is an antibody or functional binding fragment thereof; and

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

The disclosure of the present invention also provides a pharmaceutical composition comprising an effective amount of any of the above antibody-drug conjugates, or a pharmaceutically acceptable salt or solvate thereof, together with a pharmaceutically acceptable excipient.

The description of the present invention also provides the use of any of the above antibody-drug conjugates in the preparation of a medicament for the treatment of cancer.

The disclosure of the present invention provides an antibody-drug conjugate (ADC) wherein one or more cysteine or derivatives thereof are used as linkers for binding to one or more drugs at limited antibody binding sites, facilitating the production of ADCs with more high drug loading. Theoretically, since higher loading ADCs can be produced, binding drugs can be selected from a larger range such that drugs with less toxicity can be used to produce ADC products, thereby providing ADC products having a wide therapeutic window. . In addition, since multiple drugs can be associated with one binding site, the ADC products obtained using the method according to the description of the present invention have better uniformity in the case of the same DAR value. In addition, the amount of antibody required for production can be significantly reduced, thus reducing the cost. In addition, it has been surprisingly found that the amount of antibody required for production can be substantially reduced, thus reducing cost. Compared to antibody-drug conjugates associated with only one drug, antibody-drug conjugates obtained using the method in accordance with the description of the present invention have the same inhibitory or killing effect on tumor cells using fewer drugs. means for linking to the same site.

Detailed description of the invention

Definitions

Various terms associated with various parts of the description are used in the description and claims. Unless otherwise indicated, such terms are given in their usual meaning in the art. Other specifically defined terms should be understood in accordance with the definitions given here.

The singular terms used herein are used in accordance with standard practice and, unless the context otherwise indicates, denote one or more than one. Thus, for example, an "antibody-drug conjugate" includes a combination of two or more antibody-drug conjugates, and the like.

It is understood that the description of the invention uses the phrase “comprise/include/include”, in addition, similar terms such as “consisting of…” and/or “essentially consisting of…” are also suggested.

Although the numerical ranges and approximate values of the parameters are presented in the broad scope of the description of the present invention, the numerical values presented in the specific examples have been given as accurately as possible. However, any numerical value may inherently contain some errors due to the standard deviations presented in the respective measurements. In addition, all ranges disclosed herein should be understood to include any and all subranges. For example, a given range of "1 to 10" should be considered to encompass any and all sub-ranges between a minimum of 1 and a maximum of 10 (including extremes); that is, all subranges starting at a minimum of 1 or greater, such as 1 to 6.1, and subranges ending at a maximum of 10 or less, such as 5.5 to 10, are included. " is to be understood as being included in its entirety.

указывает на то, что группа, содержащая

связана с другой группой химической связью.Used in the description of the present invention

indicates that the group containing

linked to another group by a chemical bond.

The binding unit and the linker in the description of the present invention can be used interchangeably; the active unit, drug and poisonous substance in the description of the present invention can be used interchangeably.

The term "antigen" in the description of the present invention refers to any molecule that elicits an immune response or has the ability to be associated with an antibody or antigen-binding molecule. The immune response may involve the production of antibodies or the activation of specific immunocompetent cells, or both. One of ordinary skill in the art will readily appreciate that any macromolecule, including almost all proteins or peptides, can act as an antigen. Typically, the antigen may be expressed endogenously, ie expressed by genomic DNA, or it may be expressed recombinantly, or it may be chemically synthesized. "Antigen", to which the description of the present invention relates, specifically refers to those tumor-associated antigens that are well known in the art, and can be obtained using methods well known in the art for preparing antibodies. In order to develop effective targets at the cellular level for the diagnosis and treatment of cancer, researchers are trying to find transmembrane or other tumor-associated polypeptides. These targets have the ability to be specifically expressed on the surface of one or more cancer cells with little or no expression on the surface of one or more non-cancerous cells. Typically, such tumor-associated polypeptides are overexpressed on the surface of cancer cells compared to the surface of non-cancerous cells. The identification of such tumor-associated factors can greatly enhance the specific targeting characteristics of antibody-based cancer treatment. Tumor-associated antigens include, without limitation, tumor-associated antigens (1)-(36) listed below. For convenience, antigen-related information well known in the art is provided below, including the name, other names, and Genbank accession number. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases such as Genbank. Tumor-associated antigens targeted by the respective antibodies include all amino acid sequence variants and homologues having at least 70%, 80%, 85%, 90%, or 95% homology with the actual verified sequence, or having completely identical biological properties and characteristics with the sequences. the aforementioned tumor-associated antigen. Tumor-associated antigens (1)-(37) are as follows:

(1) BMPR1B (bone morphogenetic protein receptor 1B, Genbank accession number NM_001203);

(2) E16 (LAT1, SLC7A5, Genbank access number NM_003486);

(3) STEAP1 (six transmembrane domain prostate epithelial antigen 1, Genbank accession number NM_012449);

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

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

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

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, brain signaling protein 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and similar to type 1), transmembrane domain (TM) and short cytoplasmic domain, (brain signaling protein) 5B, Genbank accession number AB040878);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA gene 2700050C12, Genbank accession number AY358628);

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

(10) MSG783 (RNF124, putative protein FLJ20315, Genbank accession number NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, having six trans-membrane domains prostate epithelial antigen 2, having six trans-membrane domains prostate protein, Genbank accession number AF455138);

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

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

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

(15) CD79b (CD79B, CD79β, IGb (Igb-associated beta), B29, Genbank accession number NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession number NM_030764);

(17) HER2 (ErbB2, Genbank accession number M11730);

(18) NCA (CEACAM6, Genbank access number M18728);

(19) MDP (DPEP1, Genbank accession number BC017023);

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

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

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

(23) ASLG659 (B7h, Genbank accession number AX092328);

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

(25) GEDA (Genbank accession number AY260763);

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

(27) CD22 (B-cell receptor isotype CD22-B, Genbank accession number AK026467);

(28) CD79a (CD79A, CD79α, immunoglobulin associated alpha, able to covalently interact with Igβ (CD79B) and complex with IgM molecules on the surface, transduce signals to B-cell-specific proteins involved in B-cell differentiation, accession number Genbank NP_001774.1);

(29) CXCR5 (Burkitt's Lymphoma Receptor 1, a G protein-coupled receptor activated by the chemokine CXCL13, which plays a role in lymphocyte migration and humoral defense, in infection with HIV-2 (human immunodeficiency virus 2) and possibly in AIDS ( acquired immunodeficiency syndrome), lymphoma, myeloma, and leukemia, Genbank accession number NP_001707.1);

(30) HLA-DOB (beta subunit of the MHC class II molecule (antigen Ia) that binds to the peptide and presents it to CD4+ T-lymphoid cells, Genbank accession number NP_002111.1);

(31) P2X5 (P2X purine receptor ligand-dependent ion channel 5, an extracellular ATP (adenosine triphosphate)-gated ion channel that may be involved in synaptic transmission and neuronal regeneration and whose deficiency can lead to the pathophysiological states of idiopathic detrusor instability, accession number Genbank NP_002552.2);

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

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

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

(35) IRTA2 (translocation-associated immunoglobulin superfamily receptor 2, a putative immunoreceptor that may play a role in B-cell development and lymphoma formation; translocation-induced genetic disorders occur in some B-cell malignancies, Genbank accession number NP_112571.1 );

(36) TENB2 (putative transmembrane proteoglycan associated with the EGF/heregulin family of growth factors and follistatin, Genbank accession number AF179274);

(37) Other related antigens.

The term "antibody" in the description of the present invention is used in the broadest scope and covers various antibody structures, including, without limitation, a monoclonal antibody, a polyclonal antibody, a multispecific antibody (eg, a bispecific antibody), and an antibody fragment. Typically, an antibody may contain at least two heavy chains and two light chains linked to each other by disulfide bonds, or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. The heavy chain constant region contains three constant domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region and a light chain constant region. The light chain constant region contains a constant domain: CL. In addition, the heavy chain variable region and the light chain variable region can be divided into hypervariable 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 contains three CDRs and four FRs, arranged from the N-terminus to the C-terminus in the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain and light chain variable regions contain a binding domain that interacts with an antigen. The Ab (antibody) constant region can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system. Unless otherwise indicated, the term "antibody" in the description of the present invention encompasses an intact immunoglobulin or antigen-binding fragment that competes with an intact antibody for specific binding. The antigen-binding fragment can be obtained using recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. Antigen binding fragments include Fab, Fab', F(ab') ₂ , Fv, domain antibody (dAb), complementarity determining region (CDR) fragment, single chain antibody (scFv), chimeric antibody, divalent antibody, trivalent antibody, tetravalent an antibody; and a polypeptide that contains at least an immunoglobulin fragment that is sufficient to confer specific antigen binding to the polypeptide. The term "antibodies" in the description of the present invention also includes naturally occurring and non-naturally occurring (recombinantly produced) antibodies, human and non-human antibodies, monospecific antibody, multispecific antibody (including bispecific antibody), immunoglobulin, synthetic antibody, tetrameric antibody , containing two heavy chain molecules and two light chain molecules, antibody light chain monomer, antibody heavy chain monomer, antibody light chain dimer, antibody heavy chain dimer, antibody light chain-antibody heavy chain pairs, intracellular antibody (see e.g. Stocks, ( 2004) Drug Discovery Today 9(22): 960-66), antibody fusion (this term encompasses an antibody-drug conjugate and sometimes refers to "antibody conjugate" herein), heteroconjugate antibody, single domain antibody, monovalent antibody, single chain antibody, or single chain Fv (scFv), camelized antibody, affitelo, Fab fragment, F(ab') ₂ fragment, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibody (including, for example, anti-anti-Id antibody), minibody, domain antibody, synthetic antibody (sometimes referred to herein "mimic antibody") and its antigen-binding fragment.

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

The term "humanized antibody" refers to an antibody containing a CDR region derived from a non-human antibody and another antibody fragment derived from one (or more) human(s) antibody(s). In addition, certain backbone residues (called FR) can be modified to preserve binding affinity (Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al. al., Nature, 332: 323-327, 1988). A humanized antibody or fragment thereof as described herein may be prepared using techniques known to those skilled in the art (eg, as described in: 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).

The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody having a variable region sequence derived from a mouse antibody and a constant region sequence having origin from a human antibody. Chimeric antibody or its fragment in accordance with the description of the present invention can be obtained using techniques of genetic recombination. For example, a chimeric antibody can be obtained by cloning a recombinant DNA containing a promoter and a sequence encoding the variable region of a non-human, in particular murine, monoclonal antibody in accordance with the description of the present invention, as well as a sequence encoding the constant region of a human antibody. A chimeric antibody according to the present disclosure encoded by such a recombinant gene would be, for example, a mouse-human chimera whose specificity is determined by a variable region derived from mouse DNA and whose isotype is determined by a constant region derived from human DNA. With respect to methods for producing chimeric antibodies, for example, reference may be made to Verhoeyn et al. (BioEssays, 8:74, 1988).

The term "monoclonal antibody" refers to the preparation of an antibody molecule having a single molecule composition. A monoclonal antibody exhibits single binding specificity and affinity for a particular epitope.

In some specific embodiments, antibodies as described herein include, but are not limited to: muromomab-CD3, abciximab, rituximab, daclizumab, palivizumab, inflizumab, trastuzumab, etanercept, basiliximab, gemtuzumab, alemtuzumab, ibritumomab, adalimumab, alefacept, omalizumab, efalizumab, tositumomab, cetuximab, ABT-806, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab, romiplostim, AMG-531, golimumab, ustekinumab, ABT-874, belatacept, belimumab, atacicept, anti-CD20 antibody, canakinumab, tocykinumab atezolizumab, mepolizumab, pertuzumab, HuMax CD20, tremelimumab, ticilimumab, ipilimumab, IDEC-114, intuzumab, HuMax EGFR, aflibercept, HuMax-CD4, teplizumab, telixuzumab, catumaxomab, anti-EpCAM IGN101, adakimumab, denutuksimumab, oregsimumab , bapineuzumab, motavizumab, efumgumab, raxibacumab, LY2469298, and veltuzumab.

The term "linker" in the description of the present invention refers to a molecule having a bifunctional group or a polyfunctional group that can interact with a protein/antibody molecule and a drug molecule, respectively, and thus serves as a "bridge" for binding the protein/antibody to drug molecule. According to the mechanism of drug release in cells, the "linker" or "linker of the antibody-drug conjugate" can be divided into two classes: non-cleavable linker and cleavable linker.

A non-cleavable linker is a relatively stable linker whose structure is difficult to degrade or break down in vivo. For an antibody-drug conjugate containing a non-cleavable linker, the drug release mechanism is as follows: the conjugate binds to the antigen and is then taken up by endocytosis; the antibody is hydrolyzed in the lysosome and the active molecule is released, consisting of a small drug molecule, a linker, and amino acid residues of the antibody. The resulting structural change does not reduce the cytotoxicity of the drug. However, since the active molecule is charged (via amino acid residues), it cannot enter neighboring cells. Thus, such active drugs cannot kill adjacent tumor cells that do not express the target antigen (antigen negative cells) (bystander effect) (Bioconjugate Chem. 2010, 21, 5-13). Conventional non-cleavable linkers are MC linkers and MCC linkers and the like:

A cleavable linker, as the name implies, can be cleaved in target cells and release the active drug (the smallest drug molecule itself). Cleavable linker can be divided into two main classes: chemically unstable linker and enzymatically unstable linker.

A chemically unstable linker can be selectively cleaved due to different plasmatic and cytoplasmic properties. Such properties include pH, glutathione concentration, and the like.

A pH sensitive linker, often referred to as an acid labile linker. Such a linker is relatively stable in a neutral blood environment (pH 7.3-7.5), but hydrolyzes in slightly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). Most first generation antibody-drug conjugates use this type of linker such as hydrazone, carbonate, acetal, ketals. Antibody-drug conjugates using this type of linker typically have a shorter half-life (2-3 days) due to the limited stability of the acid-labile linker in plasma. This short half-life somewhat limits the use of the pH sensitive linker in new generation antibody-drug conjugates.

A glutathione sensitive linker is also known as a disulfide linker. The release of the drug is caused by the difference between a high concentration (in the millimolar range) of intracellular glutathione and a relatively low concentration of glutathione (in the micromolar range) in the blood. This is especially true for tumor cells, in which low oxygen leads to increased reductase activity, thus leading to higher glutathione concentrations. The disulfide bond is thermodynamically stable and thus has better stability in blood plasma.

An enzymatically unstable linker, such as a peptide linker, has the ability to better control drug release. The peptide linker has the ability to be efficiently cleaved by a protease in lysosomes, such as cathepsin B or plasmin (the content of such an enzyme is increased in some tumor tissues). This peptide bond is believed to be very stable in blood plasma, since inappropriate extracellular pH as well as serum protease inhibitors tend to inactivate the protease outside the cell. Due to high plasma stability and good intracellular selectivity and cleavage efficiency, enzymatically labile linkers are widely used as cleavable linkers for antibody-drug conjugates. Typical enzymatically unstable linkers are vc linkers and the like.

The suicide linker is typically chimeric between the cleavable linker and the active drug, or is itself part of the cleavable linker. The suicide linker mechanism is that when the cleavable linker is degraded under the right conditions, the suicide linker can spontaneously rearrange itself to release its associated active drug. Common suicide linkers are such as para-aminobenzyl alcohols (PAB).

The term "active drug" in the description of the present invention broadly refers to any compound having the desired biological activity and reactive functional group to obtain a conjugate in accordance with the description of the present invention. Desired biological activity includes the ability to diagnose, heal, alleviate, cure, prevent disease in humans and other animals. Since new drugs are continuously being discovered and developed, these new drugs should also be included in the drugs in accordance with the description of the present invention. In particular, these drugs include, without limitation, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, an autoimmune disease drug, an anti-inflammatory drug, or an infectious disease drug. More specifically, these drugs include, without limitation, a tubulin or DNA inhibitor, an RNA damaging agent. Preferably, active drugs covered in the description of the present invention include, without limitation, the following:

(a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787/ZK222584, oxaliplatin, 5-fluorouracil, folinic acid, rapamycin, lapatinib, lonafanib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, improsulfan, busulfan , piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethyleneimine, altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide, trimethylol melamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callistatin, CC-1065, adozelesin, carzelesin, cryptophyselesin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, dichloromethyl diethylamine, melphalan, novembikhin, phenesterin, prednimucil and trofosfamistine, trofosfamidine , carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin γ1, calicheamicin ω1, dynemycin, dynemycin A, clodronate, esp eramycin, neokarzinostatin chromophore, aclacinomysin, actinomycin, autrarmycin (authrarmycin), azaserin, bleomycin, actinomycin C, carabitsin, carminomycin, carzinophyllin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin, morpholino adriamycin, cyanomorpholino adriamycin, 2-pyrrolino-adriamycin, liposomal adriamycin, deoxyadriamycin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptoxidin, streptonigrin, streptonigrin zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprin, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine, floxuridine , epitiostanol, mepitiostane, testolactone, aldophosphamide glycoside, mitotane, trilostane, folinic acid, aceglaton, Aldophosphamide Glycoside, Aminolevulinic Acid, Eniluracil, Amsacrine, Bestrabucil, Bisantrene, Edatraxate, Defofamine, Demecolcine, Diaziquone, Elformitin, Elliptinium Acetate, Ethoglucid, Gallium Nitrate, Hydroxyurea, Lentinan, Lonidamine, Maytansine, Ansamitocin, Mitoguazone, Mitoxantrone, Nitramopitanthrone, Nitramopitanthrone, Nitramopitanthrone, pentostatin, fenamet, pyrarubicin, losoxantrone, 2-ethyl hydrazide, procarbazine, polysaccharide-k, razoxane, rizomycin, sisofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, T-2 toxin, verracurin A , roridin A, and anguidin, urethane, vindesine, dacarbazine, mannomustine, dibromomannite, mitolactol, pipobroman, cytosine anhydrous, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-engineered paclitaxel nanoparticulate formulation, docetaxel, chlorambucil, gemcitabine, 6-thioguanine , mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, mitoxantrone, teniposide, edatraxate, downom icin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine, or any pharmaceutically acceptable salt, solvate or acid thereof;

(b) monokine, lymphokine, traditional polypeptide hormone, parathyroid hormone, thyroxin, relaxin, prorelaxin, glycoprotein hormone, follicle stimulating hormone, thyroid stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α , tumor necrosis factor-β, müllerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, trompopoetin, 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 thereof;

(c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, amanitin derivatives, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, ciguatoxin, ricin, toxin AM, microtubulin lysine, geldanamycin, maytansine compound , calicheamicin, daunorubicin, adriamycin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analogue, auristatin, cryptophycin, camptothecin, camptothecin derivative and metabolite, rizomycin, rizomycin derivative, CC-1065, CC-1065 analogue or derivative, duocarmycins, enediine antibiotics, esperamycin, epothilone, azonafid, aplidin, toxoid, or any combination thereof;

(d) an affinity ligand, where the affinity ligand is a substrate, inhibitor, stimulant, neurotransmitter, radioactive isotope, or any combination thereof;

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

(e) an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, and an antiparasitic agent, or any combination thereof;

(g) tamoxifen, raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, naloxifene, LY117018, onapristone, or toremifene;

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

(i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin or troxcitabine;

(j) an aromatase inhibitor;

(k) a protein kinase inhibitor;

(m) a lipid kinase inhibitor;

(i) an antisense oligonucleotide;

(o) ribozyme;

(o) a vaccine; and

(p) an anti-angiogenic agent.

In some embodiments of the description of the present invention, the "active drug" is selected from the group consisting of: maytansines, V-ATPase inhibitors, pro-apoptotic agents, Be12 inhibitors, McL1 inhibitors, HSP90 (heat shock protein 90) inhibitors, IAP inhibitors na), mTOr inhibitors (mammalian targets of rapamycin), microtubule stabilizers, microtubule destabilizers, auristatins, dolastatins, MetAP (methionine aminopeptidase), CRM1 core exit inhibitors, DPPIV (dipeptidyl peptidase IV) inhibitors, proteasome inhibitors, phosphoryl transfer reaction inhibitors in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 (cyclin-dependent kinase 2) inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC (histone deacetylase) inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, binding agents minor groove DNA, DHFR inhibitors (digid rofolate reductase), as well as dolastatin peptides, precursors of vitamin A and folic acid.

In some embodiments, as used herein, the "active drug" is a cytotoxic drug (eg, an antimetabolite, antineoplastic antibiotic, an alkaloid), an immunostimulant, or a radioisotope. Preferably, the drug may be selected from the group consisting of amanitins, anthracyclines, baccatins, camptothecins, cematotins, colchicines, colcemids, combretastatins, cryptophycins, discodermolides, docetaxel, doxorubicin, echinomycins, eleutherobins, epothilones, estramustines, lexytropsins, maytansines, methotrexate, netropsins, puromycins, rhizoxins, taxanes, tubulisins or vinca alkaloids. More preferably, the drug may be selected from the group consisting of MMAD (monomethylauristatin D) and its derivatives, MMAE (monomethylauristatin E) and its derivatives, MMAF (monomethylauristatin F) and its derivatives, merdensin derivative M1, mertansin derivative M4, duocarmycin and its derivatives, calicheamicin and its derivatives, PBDA (pyrrolobenzodiazepines), doxorubicin, vinca alkaloids, methotrexate, vinblastine, daunorubicin and its derivatives, tubulinsins and their derivatives.

In some specific embodiments, the "active drug" is maytansine or maytansinoids. Maytansine inhibits cell proliferation by inhibiting microtubule forming tubulin (Science 1975, 189, 1002-1005; US 5208020). Maytansinoids are derivatives of maytansine. Both maytansine and maytansinoids are highly cytotoxic, but they have significant limitations in clinical use in cancer therapy, mainly due to the low tumor selectivity of these molecules. However, this high cytotoxicity makes them the preferred drug moieties for antibody-drug conjugates. The following are maytansine, maytansinoids, and three maytansinoid molecular structures that are often used in antibody-drug conjugate applications.

The main starting material for the synthesis of maytansinoids is maytansinol, which is mainly obtained by hydrolysis of ansamitocins. Ansamitocins can be obtained by fermentation. Ansamitocin derivatives (WO 2012/061590) and alanyl maytansinol (US 2012/0121615) have also been reported as a drug for antibody-drug conjugates.

In some specific embodiments, the "active drug" is auristatins. Auristatins are analogues of dolastatin 10, which is a bioactive polypeptide isolated from the sea hare mollusk (US 7498298). Dolastatin 10 inhibits tubulin polymerization by binding to tubulin (same binding site as for vincristine). All of dolastatin 10, auristatin PE and auristatin E are linear polypeptides containing four amino acids (three are unique to dolastatin compounds) and a C-terminal amide group. Two exemplary auristatins, monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF) are preferred drug moieties for antibody-drug conjugates.

In some specific embodiments, the "active drug" is tubulizins. Tubulisins are a class of natural products extracted from myxobacteria that can effectively inhibit tubulin polymerization and thus have antimitotic activity. Among tubulizins, tubulisin D has the best activity. Tubulisin D is a tetrapeptide compound that contains O-acyl/N,O-acetal functional groups in its structure and thus is unstable under acidic and basic conditions. US 2011/0021568 and US 2013/0224228, respectively, disclose a series of tubulisin analogues in which the aforementioned labile functional groups are removed while maintaining high cell viability.

In some specific embodiments, the "active drug" is calicheamicins. Calicheamycins are anticancer antibiotics that induce apoptosis by binding to the minor groove of DNA and promote cleavage of double-stranded DNA at a specific site. Calicheamycins have high sub-picomolar activity in vitro with a low therapeutic index, which precludes their clinical applications. However, this high activity makes them ideal candidates for antibody-drug conjugates (such as gemguzumab and inotuzumab ozogamicin).

In some specific embodiments, the "active drug" is doxorubicins. Doxorubicin is used as a chemotherapeutic drug because it can intercalate into the DNA double helix structure to block DNA replication. However, due to the low cytotoxicity of doxorubicins (for human-derived cancer cell lines, the median inhibitory concentration is 0.1-0.2 micromoles, while cytotoxic drugs for antibody-drug conjugates usually have a subnanomolar level of activity), it is not widely used in antibody-drug conjugates.

In certain specific embodiments, the "active drug" is benzodipyrroles (duocarmycins, CC-1065, and the like) and other cyclopropapyrroloin-4-one (CPI) derivatives. These compounds are potent agents for binding and alkylating the minor groove of DNA. The cyclopropabenzindol-4-one (CBI) analogs have a more stable chemical structure, higher biological activity, and are easier to synthesize than parent compounds containing naturally occurring CPI alkylated subunits. An exemplary CBI derivative is a phenolic hydroxyl-protected CBI derivative (see figure below) with reduced prodrug toxicity and increased water solubility.

In some specific embodiments, the "active drug" is pyrrolo[2,1-c][1,4]benzodiazepines (PBD) or PBD dimers. PBDs are a class of natural products produced by Streptomycetes and are unique in their ability to form non-twisted covalent attachments in the minor groove of DNA, specifically in the purine-guanine-purine sequence. The use of PBD as a small molecule for targeting DNA sequences and as a new anticancer and antibacterial drug is attracting growing interest (Biochemistry 2008, 47, 11818-11829). A flexible carbon chain is used to link the C8/C8' hydroxyl groups of the two PBD units and the resulting dimer has enhanced biological activity (WO 2011/130616). It is believed that the PBD dimer can form sequence-selective DNA damage, such as reversible 5'-Pu-GATC-Py-3' interstrand cross-linking, resulting in its biological activity. These compounds have been shown to be very potent cytotoxic drugs and can be used as candidates for antibody-drug conjugates.

In other specific examples, "active drug" is not limited to the classes mentioned above, but also includes all drugs suitable for antibody-drug conjugates.

The term "pharmaceutical composition" in the description of the present invention means a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient, which are combined together to achieve a specific goal. In some embodiments, the pharmaceutical composition includes combinations that are separated in time and/or space, as long as they have the ability to work together to achieve the objectives in accordance with the description of the present invention. For example, the components of a pharmaceutical composition can be administered to a subject in combination or separately. When the components of a pharmaceutical composition are administered to a subject separately, these components may be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, buffered aqueous solution, isotonic saline 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, triglyceride, and the like. The type of pharmaceutically acceptable carrier used depends, among other things, on whether the composition is prepared in accordance with the description of the present invention for oral, intranasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition in accordance with the description of the present invention may contain a humectant, emulsifier or buffer agent as an additive.

A pharmaceutical composition, vaccine, or pharmaceutical formulation as described herein may be administered by any suitable route, such as orally, nasally, intradermally, subcutaneously, intramuscularly, or intravenously.

The term "effective amount" in the description of the present invention covers an amount sufficient to reduce the intensity or prevent the symptom or condition of a medical disease. An effective amount also means 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 being treated, the general health of the patient, the route and dose administered, 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.

Examples

The description of the present invention is further illustrated below in conjunction with specific examples. It should be understood that the examples are used solely to illustrate the description of the present invention, and are not intended to limit the scope of the present invention. The experimental methods in the following examples, where specific conditions are not specified, are generally carried out under normal conditions or under conditions recommended by the manufacturer, and reagents of unspecified origin are common commercially available reagents. Unless otherwise indicated, all percentages, parts, ratios, or parts are by weight.

The unit of mass by volume in the description of the present invention is well known to those skilled in the art and, for example, refers to the mass of a solute in 100 ml of a solution.

Unless otherwise defined, all disciplines and scientific terms used herein have the same meanings as known to those skilled in the art. In addition, in the methods in accordance with the description of the present invention, you can use any methods and substances similar to those set forth or equivalent to those set forth. The preferred examples and materials described herein are for illustrative purposes only.

Example 1 Preparation of Compound 1

145.4 mg of maleimidocaproic acid was dissolved in 10 ml of DMF (dimethylformamide), 244.7 mg of HATU (1-bis(dimethylamino)methylene-1H-1,2,3-triazolo-4,5-b-pyridinium-3-) was added oxide-hexafluorophosphate) and 226 μl of DIPEA (diisopropylethylamine) and stirred at room temperature. 201.9 mg of MMAF (monomethylauristatin F) was dissolved in 5 ml of DMF (dimethylformamide) and slowly added dropwise to the above reaction system, and the mixture was stirred at room temperature for 16 hours. The solvent was distilled off under reduced pressure and purification was carried out by preparative liquid chromatography to give 90.3 mg of product (maleimidocaproyl-MMAF, Mc-MMAF), in 36% yield. LC-MS (liquid chromatography/mass spectrometry): (M+H) ⁺ 924.8, (M-H) ^- 923.2.

36.1 mg maleimidocaproyl-MMAF was dissolved in 1.5 ml DMF, 5.6 mg Cys-Cys and 2.1 μl DIPEA were added and the mixture was stirred at room temperature for 3 h. stirred at room temperature for 2 hours. 17.7 mg of 6-(maleimido)caproic acid succinimidyl ester and 25 μl of DIPEA were added and the mixture was stirred at room temperature for 15 hours. The solvent was distilled off under reduced pressure and purification was carried out by preparative liquid chromatography to obtain 20.0 mg of compound 1 with a yield of 45%. LC-MS: (M+2H) ²⁺ 1134.1, (M-2H) ^2- 1132.2.

Example 2 Preparation of Compound 2

Maleimidocaproyl-MMAF was prepared according to the 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 μl of DIPEA were added and the mixture was stirred at room temperature for 2 hours 1.8 mg of Cys-Cys-Cys was added and the reaction was continued at room temperature for 2 hours. 0.9 mg of Cys-Cys-Cys was added and the reaction was continued at room temperature for 2 hours. 6-(maleimido)caproic acid succinimidyl ester and 21 μl of DIPEA and the mixture was stirred at room temperature for 15 hours. The solvent was distilled off under reduced pressure and purification was carried out by preparative liquid chromatography to obtain 23.1 mg of compound 2 with a yield of 49%. LC-MS: (M+3H) ³⁺ 1648.4, (M-3H) ^3-1646.3 .

Example 3 Preparation of Compound 3

Maleimidocaproyl-MMAF was prepared according to the method of Example 1. 31.6 mg of maleimidocaproyl-MMAF was dissolved in 2 ml of DMF and then 3.9 mg of Cys-Cys and 2.9 μl of DIPEA were added, the mixture was stirred at room temperature for 4 hours. 1.2 mg of Cys-Cys was added and the mixture was stirred at room temperature for 3 hours. 0.8 mg of Cys-Cys was added and the mixture was stirred at room temperature for 16 hours. 1,3,5-triazine-mercaptoacetic acid and 10.4 mg TSTU were dissolved in 1.5 ml DMF and 17 μl DIPEA was added, the mixture was stirred at room temperature for 2 hours. The mixture was then added to the above reaction system and stirred at room temperature. temperature for 5 hours. The solvent was distilled off under reduced pressure and purification was carried out by preparative liquid chromatography to obtain 18.4 mg of compound 3 with a yield of 29%. LC-MS: (M+2H) ²⁺ 1199.3, (M-2H) ^2-1197.4 .

Example 4 Preparation of Compound 4

Maleimidocaproyl-MMAF was prepared according to the 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 and 4.3 μl of N,N-diisopropylethylamine were added and mixed at room temperature for 3 hours. 36.1 mg of MP2-PNP (2-(2-maleimidoethoxy)ethyl(4-nitrophenyl)carbonate) was added to the reaction system, then 51 μl of N,N-diisopropylethylamine was added and the mixture was stirred at room temperature. temperature for 15 hours. The solvent was distilled off under reduced pressure and purification was carried out by preparative liquid chromatography to obtain 18.0 mg of compound 4 with a yield of 27%. LC-MS: (M+2H) ²⁺ 1142.3, (M-2H) ^2- 1141.1.

Example 5 Preparation of Compound 5

Maleimidocaproyl-MMAF was prepared according to the 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 and 4.3 μl of N,N-diisopropylethylamine were added and mixed at room temperature for 3 hours. 71.0 mg of Mal-propionamide-PEG8-propionate-Osu was added to the reaction system, then 51 μl of N,N-diisopropylethylamine was added and the mixture was stirred at room temperature for 15 hours. The solvent was distilled off under reduced pressure and purification was carried out by preparative liquid chromatography to obtain 19.3 mg of compound 5 with a yield of 28%, LC-MS: (M+2H) ²⁺ 1324.1, (M-2H) ^2- 1324.0.

Example 6 Preparation of Compound 6

Maleimidocaproyl-MMAF was prepared according to the 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 and 4.8 μl of N,N-diisopropylethylamine were added and the mixture stirred at room temperature for 3 hours. 86.6 mg of Mc-VC-PAB-PNP was added to the reaction system, and then 58 μl of N,N-diisopropylethylamine was added and the mixture was stirred at room temperature for 15 hours. The solvent was distilled off under reduced pressure and purification was carried out by preparative liquid chromatography to give 47.4 mg of compound 6 in 52% yield, LC-MS: (M+2H) ²⁺ 1336.0, (M-2H) ^2-1334.6 .

Example 7 Preparation of Compound 7

Maleimidocaproyl-MMAF was prepared according to the 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 and 3.6 μl of N,N-diisopropylethylamine were added and the mixture was stirred at room temperature for 3 hours. 84.4 mg of PY-MAA-VC-PAB-PNP was added to the reaction system and then 48 μl of N,N-diisopropylethylamine was added and the mixture was stirred at room temperature for 15 hours. The solvent was distilled off at reduced pressure and purification was carried out by preparative liquid chromatography to obtain 31.0 mg of compound 7 with a yield of 43%, LC-MS: (M+2H) ²⁺ 1401.2, (M-2H) ^2-1399.4 .

Example 8 Preparation of Antibody-Drug Conjugates (ADC)

General methods for the synthesis of antibody-drug conjugates are as follows.

Method A: Anti-Her-2 antibody was diluted to a 10 mg/ml solution using PBS buffer (pH=7.4), 2.4 molar equivalents of TCEP was added and the mixture was stirred by shaking for 1 hour. 5.0 molar equivalents of the linker-drug was added and the mixture was stirred by shaking and allowed to react for 1 hour. After completion of the reaction, the remaining small molecules were removed by ultrafiltration. The resulting solution was applied to a hydrophobic chromatography (HIC-HPLC) column for DAR analysis, drug distribution and free antibody percentage.

Method B: Anti-Her-2 antibody was diluted to a 10 mg/ml solution using boric acid-borax buffer (pH=9), 5 molar equivalents of TCEP was added and the mixture was stirred by shaking for 1 hour. 6.0 molar equivalents of the linker-drug was added and the mixture was stirred by shaking and allowed to react for 3 hours. After completion of the reaction, the remaining small molecules were removed by ultrafiltration. The resulting solution was applied to a hydrophobic chromatography (HIC-HPLC) column for DAR analysis, drug distribution and free antibody percentage.

The following compounds were obtained by the above general methods for obtaining ADC (A is an antibody or functional binding fragment, m is 1, 2, 3, 4, 5, 6, 7 or 8).

Example 9 Inhibitory effect of ADC on tumor cell line SK-BR-3

Tumor cells SK-BR-3 (human breast cancer cells) were destroyed and collected by centrifugation. Cells were counted and diluted to a cell suspension of 0.5-1.5×10 ⁵ cells/ml. 100 μl of cell suspension was added to each well of a 96-well plate. The tablet was incubated overnight in an incubator at 37°C with 5% CO ₂ . On the following day, ADC was added with 9 appropriate concentration gradients, cells without ADC treatment were used as a control group. After 72 hours of culture, a Cell Counting Kit-8 (CCK-8 for short) was used for activity analysis, and a 96-well plate after staining development was used to record the OD (optical density) value at 450 nm using a reader for microplates. Prism software was used to calculate IC ₅₀ (mean inhibitory concentration) based on OD values. When the smoothed curve is presented as "S-curve" and R ² ≥0.95, then the IC ₅₀ value is appropriate and taken into account.

The inventors selected ADC-0 with a DAR value of 3.87 (Comparative Example 1), ADC-0 with a DAR value of 7.18 (Comparative Example 2), ADC-10 with a DAR value of 4.3 (Comparative Example 3), and ADC- 34 with a DAR value of 4.29 (Comparative Example 4) for inhibition experiments performed on the SK-BR-3 tumor cell line. The IC ₅₀ values for them are presented in Table 2.

In Comparative Example 2, the DAR value was increased to 7.18 by increasing the number of MMAF sites associated with the antibody. Compared with Comparative Example 1, when the same effect was achieved, Comparative Example 2 can effectively reduce the use of the antibody (that is, the amount of antibody in the ADC, and if the loss of the antibody in the synthesis method is not taken into account, then the use of the antibody is equal to the concentration of the ADC), while the use MMAF increases by 19.3%.

In Comparative Example 3, two MMAFs were linked to one linker and the DAR value (4.3) was comparable to the DAR value for Comparative Example 1 (3.87). Compared with Comparative Example 1, when the same effect was achieved, Comparative Example 3 effectively reduced antibody usage by 65.9% and MMAF usage by 24.3%.

In Comparative Example 4, three MMAFs were linked to one linker, and the DAR value was comparable to the DAR value for Comparative Example 1. Compared with Comparative Example 1, Comparative Example 4 effectively reduced antibody usage by 85.5%, and MMAF usage by 51. 8%.

Based on the above data compared, ADC-10 and ADC-34 show a higher activity against cells. This effect is not due solely to the improved effect of ADC by increasing the amount of MMAF in the antibody. It also demonstrates that in the case where the ADC carries the same amount of MMAF, by binding multiple cytotoxic drugs to one site, the amount of antibody and the amount of drugs used are greatly reduced, providing an unexpected technical effect. And by having a single binding site with multiple drugs, the uniformity of ADC products can also be effectively improved, and this contributes to drug production and quality control process.

The description of the present invention is illustrated by specific examples. However, one skilled in the art will appreciate that the description of the present invention is not limited to specific examples, and those skilled in the art can make various modifications and changes within the scope of the description of the present invention, and various technical features mentioned in the description can be combined with each other. with the other depending on the essence and scope of the description of the present invention. Such modifications and variations are within the scope of the description of the present invention.

Claims (55)

1. An antibody-drug conjugate represented by formula (II) or formula (III):

where A is an antibody or a functional binding fragment; B ₁ , B ₂ and B ₃ may be the same or different; B ₁ and B ₂ , B ₂ and B ₃ are connected via a peptide bond as a result of a dehydrogenation reaction with condensation; each of L ₁ , L ₂ , L ₃ and L ₄ independently represents a linker and they may be the same or different; L ₁ is covalently bonded to the N-terminus of B ₁ , L ₂ and B ₁ , L ₃ and B ₂ , L ₄ and B ₃ are covalently bonded via a thiol group and L ₂ and D ₁ , L ₃ and D ₂ , L ₄ and D ₃ are covalently bonded; each of D ₁ , D ₂ and D ₃ independently represents an active drug and they may be the same or different; Z is covalently linked to a carbonyl group in B ₂ in formula (II) or a carbonyl group in B ₃ in formula (III); n is an integer equal to or greater than 4, which is the number of branches that bind to active drugs; and m is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8; structures B ₁ , B ₂ and B ₃ , respectively, are represented by formula (V):

where p is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8; and Z is selected from the group consisting of OH, SH and NH ₂ . 2. The antibody-drug conjugate of claim 1, having the structure represented by formula (VI-2), (VI-3) or (VI-5):

where A is an antibody or a functional binding fragment; each of L ₁ , L ₂ , L ₃ and L ₄ independently represents a linker and they may be the same or different; each of D ₁ , D ₂ and D ₃ independently represents an active drug and they may be the same or different; and m is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8. 3. Conjugate antibody-drug according to any one of paragraphs. 1, 2, where L ₁ is covalently linked to the amino residue or thiol residue of the antibody; preferably L ₁ is covalently linked to the thiol group of the antibody; more preferably, L ₁ is covalently linked to a thiol residue formed by breaking the interchain disulfide bond of the antibody. 4. Conjugate antibody-drug according to any one of paragraphs. 1-3, where L ₁ , L ₂ , L ₃ and L ₄ represent a cleavable linker, a combination of cleavable linkers, or a non-cleavable linker. 5. The antibody-drug conjugate of claim 4, wherein the cleavable linker comprises a peptide linker and a polysulfide bond, and wherein the peptide linker contains 2 to 20 amino acids, preferably the peptide linker is selected from the group consisting of -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; and the polysulfide bond contains 2 to 8 sulfur atoms, preferably the polysulfide bond is selected from the group consisting of a disulfide bond (-S-S-), a trisulfide bond (-S-S-S-) and a tetrasulfide bond (-S-S-S-S-). 6. Conjugate antibody-drug according to any one of paragraphs. 1-5, where L ₁ includes the following structures:

7. Conjugate antibody-drug according to any one of paragraphs. 1-6, where L ₂ , L ₃ and L ₄ include the following structures:

8. Conjugate antibody-drug according to any one of paragraphs. 1-7, wherein the antibody or functional binding fragment thereof comprises a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv ("scFv"), diabody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a fusion protein containing an antigen-binding fragment of an antibody; preferably the antibody is a humanized monoclonal antibody or a fully human antibody. 9. The antibody-drug conjugate of claim 8, wherein the antibody is an IgG (immunoglobulin G) antibody or a functional binding fragment thereof, preferably the antibody is selected from the group consisting of IgG ₁ , IgG ₂ , IgG ₃ and IgG ₄ antibodies. 10. Conjugate antibody-drug according to any one of paragraphs. 1-9, wherein the active drug is selected from the group consisting of a cytotoxic molecule, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for the treatment of an autoimmune disease, an anti-inflammatory drug, and a drug for the treatment of an infectious disease. 11. The antibody-drug conjugate of claim 10, wherein the cytotoxic molecule comprises a tubulin inhibitor and a DNA damaging agent; preferably the tubulin inhibitor comprises a cytotoxic molecule of dolastatins and auristatins, a cytotoxic molecule of maytansines; the DNA damaging agent includes calicheamicins, duocarmycins, pyrrolobenzodiazepine (PBD), an anthramycin derivative, camptothecins and derivatives thereof, and SN-38; more preferably, the auristatin cytokine molecule includes MMAE (monomethylauristatin E), MMAF (monomethylauristatin F) and a derivative thereof; and the maytansine cytotoxic molecule includes DM1, DM4 and a derivative thereof; and more preferably, the cytotoxic molecule comprises the following structures:

12. Conjugate antibody-drug according to any one of paragraphs. 1-11, having the following structure:

where A is an antibody or a functional binding fragment; and m is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8. 13. Pharmaceutical composition for the treatment of cancer, containing an effective amount of the conjugate of the antibody-drug according to any one of paragraphs. 1-12 or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable excipient. 14. The use of an antibody-drug conjugate according to any one of paragraphs. 1-12 for the manufacture of a drug for the treatment of cancer.