Intermediate for preparing antibody and drug conjugate as well as preparation method and application of intermediate

Abstract

The invention relates to an intermediate for preparing an antibody and drug conjugate as well as a preparation method and application of the intermediate. The intermediate for preparing the antibody and drug conjugate has a structure as shown in a formula I. The designed intermediate for preparing the antibody and drug conjugate can effectively couple compounds such as monomethyl auristatin to the antibody at a high DAR value. Compared with similar antibodies and drug conjugates in the prior art, the antibody and drug conjugate prepared by adopting the intermediate disclosed by the invention shows stronger killing activity, higher hydrophilicity and thermal stability, better pharmacokinetic performance and lower toxic and side effects on tumor cells, so that the antibody and drug conjugate can be applied to tumor treatment of tumor cells. The intermediate and the antibody and drug conjugate have wide application prospects. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine and biology, and specifically relates to an intermediate for preparing an antibody-drug conjugate (ADC), and a preparation method and application thereof. Background Art

[0002] Antibody-drug conjugates (ADCs) are usually a type of tumor targeted therapy, where antibodies targeting tumor antigens are covalently coupled to cytotoxic payloads through chemically synthesized linkers. The selection of drug linkers and payloads plays a key role in the therapeutic efficacy and safety of ADCs.

[0003] Currently, most ADC molecules entering clinical development use cleavable linkers, including acid-labile linkers, peptide linkers, and disulfide linkers. The cleavable dipeptide linker formed by valine-citrulline (Val-Cit, VC) connected to a self-cleaving spacer benzyl hydroxyl (PAB) is widely used in the clinical development of ADCs. However, due to the strong hydrophobicity of the VC linker, if combined with a medium-to-high hydrophobic payload, it will form a "surfactant-like" structure that is easy to aggregate with the more hydrophilic part of the antibody, especially when the DAR value is higher than 4. In vivo, the aggregation of ADCs can easily lead to rapid plasma clearance, reduced efficacy, and increased toxicity, especially for ADC molecules with high DAR values.

[0004] Monomethyl auristatin E (MMAE) can inhibit tubulin polymerization and play an effective mitotic inhibitory role. It is widely used as the payload of ADC drugs. Although it is effective in many refractory solid tumors, due to its poor water solubility, the existing linker has the best therapeutic effect when its DAR value is ~4 when coupled to MMAE. At a higher DAR, the molecules will aggregate and lead to rapid clearance. Due to the average DAR value of ~4, it also leads to problems in molecular uniformity and batch-to-batch stability, which is not conducive to quality control. The same problem also exists in other medium and high hydrophobic compounds.

[0005] In summary, there is an urgent need to develop new stable linkers and payloads in this field to obtain effectively targeted ADCs to achieve the highest therapeutic index.

[0006] Human trophoblast cell surface antigen 2 (Trop-2) is highly expressed in a variety of human solid tumors, such as breast cancer, lung cancer, colon cancer, bladder cancer and pancreatic cancer, but it is also expressed at different levels in key normal tissues such as skin and mucosa, which is a challenge for whether it can be drugged as an ADC target. However, the key issue of whether ADC technology can be successfully used in the targeted treatment of Trop-2 positive tumors is to screen the best combination of linker and payload to achieve the highest therapeutic index. Summary of the invention

[0007] The purpose of the present invention is to provide an ADC molecule containing a medium-to-high hydrophobic compound such as monomethyl auristatin as a payload and capable of being coupled to a high DAR value, as well as an intermediate (i.e., a compound of formula I) that can efficiently couple compounds such as monomethyl auristatin to an antibody.

[0008] In a first aspect of the present invention, a compound shown in the following formula I for preparing an antibody-drug conjugate is provided:

[0009]

[0010] In Formula I,

[0011] Z1 is a linker moiety for connecting to an antibody;

[0012] m1 is an integer from 1 to 10;

[0013] m2 is an integer from 10 to 50;

[0014] X represents a group derived from a small molecule cytotoxic drug.

[0015] In another preferred embodiment, the Z1 is a substituted or unsubstituted maleimide group.

[0016] In another preferred embodiment, the Z1 is linked to the antibody via position 1, position 3 or position 4 of the 5-membered ring of maleimide.

[0017] In another preferred embodiment, the connection includes: connection with a sulfhydryl group on the antibody, connection with an ε-amino group (such as the ε-amino group of lysine) on the antibody, connection with a selenocysteine ​​on the antibody, or a combination thereof.

[0018] In another preferred embodiment, the Z1 reacts with a naturally occurring active group on an antibody or an artificially introduced non-natural active group to generate a chemical connection structure.

[0019] In another preferred embodiment, after Z1 is linked to the antibody, a chemical linking structure selected from the following group is formed:

[0020] (a) a chemical connection structure formed by coupling reaction between the thiol group on the antibody and maleimide;

[0021] (b) a chemical connection structure formed by coupling reaction between the thiol group on the antibody and dibromomaleimide;

[0022] (c) the ε-amino group of lysine on the antibody reacts with the active amine group on the linker to form a chemical connection structure;

[0023] (d) a coupling reaction between lysine on the antibody and a maleimide-containing derivative to form a chemically linked structure;

[0024] (e) a coupling reaction between lysine on the antibody and iodoacetamide to form a chemical connection structure;

[0025] (f) a chemical connection structure formed by a coupling reaction of lysine on the antibody based on iminothiolane;

[0026] (g) a chemical connection structure formed by a pyridine disulfide coupling reaction of lysine on an antibody;

[0027] (h) site-specific coupling with selenocysteine ​​artificially introduced into the antibody to form a chemical connection structure;

[0028] (i) any combination of the above;

[0029]

[0030] In another preferred embodiment, in the above formulae (a) and (b), m3 is a positive integer of 1-8 (preferably 2-6).

[0031] In another preferred embodiment, in the above formulae (d) and (e), spacer is selected from: -(CH2) m4 -、-(CH2O) m4 -, wherein m4 is an integer of 0-10, preferably, an integer of 1-6.

[0032] In another preferred embodiment, the compound of formula I is a compound of formula Ia:

[0033]

[0034] In Formula Ia,

[0035] m1 is an integer from 1 to 10;

[0036] m2 is an integer from 10 to 50;

[0037] X is a hydrophobic compound such as monomethyl auristatin (ie, a group after a compound such as monomethyl auristatin loses an H atom).

[0038] In another preferred embodiment, m1 is 1-8, more preferably 4 or 5, and most preferably 5.

[0039] In another preferred embodiment, m2 is 12-40, more preferably 16-36, and most preferably 24.

[0040] In another preferred embodiment, X is a group derived from monomethyl auristatin E (MMAE), that is

[0041]

[0042] In another preferred embodiment, the structure of the compound of formula I is as shown in formula III:

[0043]

[0044] The definition of MMAE is as described above.

[0045] In a second aspect, the present invention provides an antibody-drug conjugate (ADC), wherein the antibody-drug conjugate is an antibody-drug conjugate (ADC) formed by coupling the compound of formula I described in the first aspect with an antibody.

[0046] In another preferred embodiment, the antibody-drug conjugate has a structure shown in Formula II:

[0047] Ab-(LX)n II

[0048] In Formula II,

[0049] Ab stands for antibody,

[0050] L is a divalent linker as shown below:

[0051]

[0052] Wherein Z1' is the group remaining after removing one hydrogen from the Z1 group;

[0053] X is as defined above; n is the average number of conjugated drugs conjugated to the antibody;

[0054] The definitions of m1 and m2 are as described above.

[0055] In another preferred embodiment, n is an integer or non-integer > 0, preferably 1≤n≤15, for example, n is an integer or non-integer of 5-10; preferably, an integer or non-integer of 6-9; more preferably, an integer or non-integer of 7-8.

[0056] In another preferred embodiment, the antibody is selected from the group consisting of a chimeric antibody, a bivalent or bispecific molecule, a diabody, a triabody and a tetrabody.

[0057] In another preferred embodiment, the antibody may be an antibody against any target, for example, the target is selected from the following group:

[0058] Trop-2, PD-L1, CTLA4, OX40, EpCAM, EGFR, EGFRVIII, HER2, PTPN2, VEGF, CD11a, CD19, CD20, CD22, CD25, CD30, CD33, CD40, CD56, CD64, CD70, CD73, CD74, CD79, CD105, CD138, CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphB2, Apo2, NMP179, p16INK4A, Nectin-4, B7H3, EMA, CEA, MIB-1, GD2, APRIL receptor, folate receptor, mesothelin inhibitor, MET, TM4SF1, IL13 receptor, etc.

[0059] In another preferred embodiment, in the antibody-drug conjugate, in the divalent linker L, m1 is an integer from 1 to 8.

[0060] In another preferred embodiment, in the antibody-drug conjugate, in the divalent linker L, m2 is an integer of 20, 21, 22, 23, 24, 25, or 26.

[0061] In another preferred embodiment, in the antibody-drug conjugate, in the divalent linker L, m1 is 4 and m2 is 24.

[0062] In another preferred embodiment, in the antibody-drug conjugate, in the divalent linker L, m1 is 5 and m2 is 24.

[0063] In another preferred embodiment, the antibody is hRS7.

[0064] In another preferred example, the amino acid sequence of the light chain variable region of the antibody hRS7 comprises the sequence shown in SEQ ID NO.: 1 or is the sequence shown in SEQ ID NO.: 1; and / or the amino acid sequence of the heavy chain variable region of the antibody hRS7 comprises the sequence shown in SEQ ID NO.: 2 or is the sequence shown in SEQ ID NO.: 2.

[0065] In another preferred embodiment, in the antibody-drug conjugate, the antibody is hRS7, and in L, Z1' is the group remaining after removing one hydrogen from maleimide, m1 is 5, m2 is 24, and n is an integer between 1-15.

[0066] The third aspect of the present invention provides a pharmaceutical composition, comprising: (a) the antibody-drug conjugate as described in the second aspect, and (b) a pharmaceutically acceptable carrier.

[0067] In a fourth aspect, the present invention provides a use of the antibody-drug conjugate as described in the second aspect or the pharmaceutical composition as described in the third aspect in the preparation of a drug for treating tumors or immune diseases.

[0068] In another preferred embodiment, the tumor is selected from Trop-2 targeted tumors, HER2 targeted tumors and EGFR targeted tumors.

[0069] In another preferred embodiment, the tumor is a Trop-2 targeted tumor and is selected from breast cancer, lung cancer, colon cancer, bladder cancer and pancreatic cancer.

[0070] In a fifth aspect, the present invention provides a method for preparing the antibody-drug conjugate according to the second aspect, the method comprising the steps of:

[0071] (1) providing a reaction system, wherein the reaction system comprises an antibody to be coupled and the compound of formula I described in the first aspect;

[0072] (2) In the reaction system, the antibody and the compound of formula I are subjected to a coupling reaction to obtain the antibody-drug conjugate of the second aspect.

[0073] In another preferred embodiment, the pH of the reaction system is 6.0-8.0; preferably, 7.0-8.0.

[0074] In another preferred embodiment, the molar ratio of the antibody to the compound of formula I (or hydrophobic compounds such as monomethyl auristatin) is 1:1 to 1:20; preferably, 1:6 to 1:15, and more preferably, 1:8 to 1:12.

[0075] In another preferred embodiment, the reaction time is 1 h to 48 h; preferably, 3 h to 36 h.

[0076] In another preferred embodiment, the coupling includes site-specific coupling and random coupling.

[0077] In another preferred embodiment, the site-specific coupling includes two coupling modes: K-Lock and C-Lock. In the K-Lock coupling mode, VK-X is coupled to a lysine (K) residue in the antibody sequence, and in the C-Lock coupling mode, VK-X is coupled to a cysteine ​​(C) residue in the antibody sequence.

[0078] In a sixth aspect of the present invention, there is provided a compound represented by formula IV

[0079]

[0080] Wherein, W1 is selected from: H, Fmoc-,

[0081] W2 is selected from: H, Trt-, Among them, when W1 is When W2 is not Or when W2 is When W1 is not

[0082] The definitions of X, m1, m2, and Z1 are as described above.

[0083] The seventh aspect of the present invention provides a method for preparing the compound of formula IV according to the sixth aspect, comprising the following steps:

[0084] (S1) Compound 1 reacts with XH in an inert solvent in the presence of a base to obtain a compound of formula IV;

[0085]

[0086] The definitions of X, W1, and W2 are as described above.

[0087] In another preferred embodiment, in step (S1), the inert solvent is DMF.

[0088] In another preferred embodiment, in step (S1), the base is DIEA.

[0089] In an eighth aspect, the present invention provides a use of the compound of formula I described in the first aspect in the preparation of an antibody-drug conjugate.

[0090] In a ninth aspect, the present invention provides a method for treating or preventing tumors, the method comprising the following steps: administering the antibody-drug conjugate or the pharmaceutical composition as described above to a subject in need thereof.

[0091] In another preferred embodiment, the subject is a mammal, including a human.

[0092] In another preferred embodiment, the dosage of the antibody-drug conjugate is 0.1-60 mg / kg; preferably, 0.5-15 mg / kg; more preferably, 1-10 mg / kg.

[0093] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0094] Beneficial Effects

[0095] Compared with the prior art, the main beneficial effects of the present invention include:

[0096] (1) The compound of formula I of the present invention can effectively couple compounds such as monomethyl auristatin to antibodies with a high DAR value, while the conventional Val-Cit-PAB linker cannot.

[0097] (2) The compound of formula I of the present invention has good hydrophilicity.

[0098] (3) The antibody-drug conjugate obtained from the compound of formula I of the present invention has an optimized DAR value and loading, which makes the therapeutic window of the ADC of the present invention larger.

[0099] (4) The antibody-drug conjugate provided by the present invention has significant tumor cell-killing activity.

[0100] (5) The present invention is the first to develop an ADC containing an X molecule that can be efficiently coupled to an antibody (such as hRS7 antibody) and stabilized. The ADC has a synergistic therapeutic effect and can significantly amplify the killing activity of X on tumor cells.

[0101] (6) The compound of formula I of the present invention can be conjugated with various antibodies, and the resulting antibody-drug conjugate has an optimized DAR value, thereby making the ADC of the present invention have higher hydrophilicity and thermal stability.

[0102] (7) The antibody conjugate of the present invention has higher pharmaceutical efficacy.

[0103] (8) The antibody-drug conjugate provided by the present invention has better pharmacokinetic properties and lower toxic side effects.

[0104] 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 only 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 usually performed under conventional conditions, such as those described in Sam brook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1:a. Molecular structures of VK1 (Val-Lys-PAB), VK2 (PEG4-Val-Lys-PAB), VC (Val-Cit-PAB) linkers and hRS7-VK1-MMAE linkers; b. Shows the preparation conditions and detection of anti-Trop-2-ADC; c. RP-HPLC analysis of drug-to-antibody ratio (DAR) in different ADC molecules; d. HIC-HPLC analysis of hydrophobicity of different ADC molecules; e. SEC-HPLC analysis of thermal stability of different ADC molecules; f. Summary of the analysis results of HIC-HPLC and SEC-HPLC.

[0106] Figure 2 : a. Body weight changes in mice treated with hRS7-VK-MMAE and hRS7-VC-MMAE; b. Hepatotoxicity of hRS7-VK-MMAE and hRS7-VC-MMAE; c and d. Efficacy of hRS7-VK-MMAE and hRS7-VC-MMAE on transplanted tumor models.

[0107] Figure 3 :a. Affinity of hRS7-VK-MMAE and hRS7-VC-MMAE for human, monkey and mouse TROP-2; b. Antigen expression levels in different tumor cell lines were detected by flow cytometry; c. The killing effects of hRS7-VK-MMAE and hRS7-VC-MMAE on different tumor cell lines; d. The internalization levels of hRS7-VK-MMAE and hRS7-VC-MMAE.

[0108] Figure 4 : a. The minimum effective dose of hRS7-VK1-MMAE for treating MDA-MB-468 tumor cell transplants, inhibiting tumor growth and mouse body weight; b. The minimum effective dose of hRS7-VK1-MMAE for treating BxPC-3 tumor cell transplants, inhibiting tumor growth and mouse body weight; c. The minimum effective dose of hRS7-VK1-MMAE for treating NCI-N87 tumor cell transplants, inhibiting tumor growth and mouse body weight.

[0109] Figure 5 : a. Drug metabolism of hRS7-VK1-MMAE in mice; b. Drug metabolism of hRS7-VK1-MMAE in crab-eating monkeys; c. Drug metabolism of different doses of hRS7-VK1-MMAE in mice; d. Total antibody concentration of different doses of hRS7-VK1-MMAE in mouse plasma. DETAILED DESCRIPTION

[0110] The inventors have obtained a highly efficient, stable and hydrophobic compound of formula I through extensive and in-depth research and a large number of screenings, which can be used to prepare a targeted antibody-drug conjugate (ADC). The introduction of the compound of formula I into the ADC can solve the problem of coupling X as an ADC payload with a high DAR value. The experimental results show that the ADC molecules using the compound of formula I and the antibody show good efficacy and safety both in vivo and in vitro under the condition of DAR8. The present invention was completed on this basis.

[0111] Experiments show that the compound of formula I of the present invention is a new type of stable linker and payload, which is particularly suitable for efficient coupling of drug molecules (such as MMAE, paclitaxel and other compounds) that are difficult to form high DAR value ADCs with antibodies, thereby forming stable and effective high DAR value ADCs. The ADC prepared based on the intermediate of the present invention has targeting, can achieve an excellent therapeutic index, and can significantly reduce the toxic side effects of ADC on normal cells or tissues.

[0112] the term

[0113] The "DAR value" refers to the drug-antibody ratio.

[0114] In the present invention, when "hydrophobic compounds such as monomethyl auristatin" are part of other compounds, they are groups of monomethyl auristatin compounds, that is, groups after monomethyl auristatin compounds lose H atoms.

[0115] Active ingredients

[0116] "Compound of Formula I", "antibody and drug conjugate intermediate", "linker and payload combination", "linker and payload" are used interchangeably.

[0117] The present invention provides a compound of formula I having the following structure

[0118]

[0119] In the formula,

[0120] Z1, m1, m2, and MMAE are as defined above.

[0121] In another preferred embodiment, m1 is an integer of 1-10;

[0122] X is a hydrophobic compound such as monomethyl auristatin.

[0123] In another preferred embodiment, m1 is an integer of 2-10, such as 2-8, more preferably 4 or 5, and most preferably 5.

[0124] In another preferred embodiment, m2 is 10-50, such as m2 is 12-40, more preferably 15-36, and most preferably 24.

[0125] Preferably, m1 is an integer from 2 to 10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10);

[0126] Preferably, m2 is an integer from 15 to 30 (i.e., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30);

[0127] Preferably, MMAE is a monomethyl auristatin compound.

[0128] Preferably, the compound of formula I is a compound of formula Ia:

[0129]

[0130] Wherein, m1, m2, and X are as defined above.

[0131] Preferably, the structure of the compound of formula I is as shown in formula III

[0132]

[0133] Wherein, MMAE is as defined above.

[0134] Antibody

[0135] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0136] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0137] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constants corresponding to the different classes of immunoglobulins are known as α, δ, ε, γ, and μ. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0138] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0139] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0140] In the present invention, the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0141] Table A

[0142] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser G1n(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0143] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0144] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0145] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0146] At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0147] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0148] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.

[0149] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.

[0150] The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0151] The antibodies of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0152] In another preferred embodiment, the antibody is antibody hRS7.

[0153] In another preferred example, the amino acid sequence of the light chain variable region (V-Kappa) of the antibody hRS7 is the amino acid sequence shown in SEQ ID NO.: 1 (DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGV PDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQ).

[0154] In another preferred example, the amino acid sequence of the heavy chain variable region (VH) of the antibody hRS7 is the amino acid sequence shown in SEQ ID NO.: 2 (QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTY TGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWG QGSLVTVSS).

[0155] Antibody-drug conjugates and their preparation

[0156] As used herein, the terms "antibody and drug conjugate of the present invention", "antibody and drug conjugate of the present invention", "conjugate of the present invention" or "ADC of the present invention" are used interchangeably to refer to an antibody-drug conjugate having the structure shown in Formula II.

[0157] In the present invention, the antibody-drug conjugate has a structure shown in Formula II:

[0158] Ab-(LX)n II

[0159] in,

[0160] Ab means an antibody, which may be any class of antibody as described above (such as a chimeric antibody (e.g., a humanized murine antibody), a bivalent or bispecific molecule (e.g., a bispecific antibody), a diabody, a triabody, and a tetrabody);

[0161] The definitions of X, L, n, m1, and m2 are as described above.

[0162] Preferably, in Formula II, n is an integer or non-integer of 4-12, preferably, n is an integer or non-integer of 5-10; preferably, it is an integer or non-integer of 6-9; more preferably, it is an integer or non-integer of 7-8.

[0163] Preferably, in the above formula II, m1 is an integer of 2, 3, 4, 5 or 6; m2 is an integer of 20, 21, 22, 23, 24 or 25.

[0164] Preferably, in the above formula II, m1 is 5; m2 is 24.

[0165] Coupling method

[0166] The present invention provides a coupling method for coupling the compound of formula I to an antibody, thereby greatly improving the killing ability of the antibody to tumor cells without changing the affinity of the antibody.

[0167] Typical coupling methods applicable to the present invention include C-Lock coupling methods. In the C-Lock coupling method, the drug molecule is coupled to the cysteine ​​(C) residue in the antibody sequence.

[0168] Preferably, the preparation method of the antibody-drug conjugate of the present invention is as follows:

[0169] A certain amount (e.g., 0.5 mg) of antibody is taken, and a certain amount (e.g., 8 times the substance equivalent) of tris (2-carboxyethyl) phosphine (TCEP) is added, and the reaction is carried out for a period of time (e.g., 2.5 hours) at a suitable temperature (e.g., 37° C.). Then, the compound of formula I (e.g., 15 times the substance equivalent) is directly added, and a coupling reaction is carried out (e.g., overnight reaction at 4° C., e.g., 17 hours), thereby obtaining the ADC of the present invention. Finally, the buffer solution is replaced with a PBS solution of pH 7.0 through an Amicon Ultra 4 10K ultrafiltration tube.

[0170] Preferably, the preparation method of the antibody-drug conjugate of the present invention is as follows:

[0171] A certain amount (e.g., 0.5 mg) of antibody is taken, and a certain amount (e.g., 2 times the substance equivalent) of tris(2-carboxyethyl)phosphine (TCEP) is added, and the reaction is carried out at a suitable temperature (e.g., 37° C.) for a period of time (e.g., 2.5 hours). After the reaction is completed, the reaction mixture is concentrated to less than 0.5 ml by centrifugal ultrafiltration, and then filled with coupling solution (75 mM NaAc, pH 6.5, 1 mM DTPA, 10% DMSO), and then centrifuged and concentrated to less than 0.5 ml, and repeated three times. Then, the compound of formula I (e.g., 8 times the substance equivalent) is directly added, and a coupling reaction is carried out (e.g., overnight reaction at 4° C., e.g., 17 hours), thereby obtaining the ADC of the present invention.

[0172] Preferably, the preparation method of the antibody-drug conjugate of the present invention is as follows:

[0173] A certain amount (e.g., 0.5 mg) of antibody is taken, a certain amount (e.g., 3 times the substance equivalent) of dithiothreitol (DTT) is added, and the reaction is carried out for a period of time (e.g., 2 hours) at a suitable temperature (e.g., 37° C.). After the reaction is completed, the reaction mixture is concentrated to less than 0.5 ml by centrifugal ultrafiltration, and the reaction mixture is filled with coupling solution (75 mM NaAc, pH 6.5, 1 mM DTPA, 10% DMSO), and then centrifuged and concentrated to less than 0.5 ml, and repeated three times. Then, the compound of formula I (e.g., 10 times the substance equivalent) is directly added, and a coupling reaction is carried out (e.g., overnight reaction at 4° C., e.g., 17 hours), thereby obtaining the ADC of the present invention.

[0174] Pharmaceutical compositions and methods of administration

[0175] The present invention also provides a pharmaceutical composition containing the ADC of the present invention, and a method for using the ADC of the present invention to treat a mammalian disease. Preferably, the disease is a tumor, including a tumor with a specific target, such as a Trop-2 targeted tumor, a Her2 targeted tumor, or an EGFR targeted tumor.

[0176] The present invention also provides the use of the antibody and drug conjugate in the preparation of anti-tumor drugs.

[0177] In the present invention, the pharmaceutical composition comprises an effective amount of ADC according to the present invention (as an active ingredient), and at least one pharmaceutically acceptable carrier, diluent or excipient. During preparation, the active ingredient is usually mixed with an excipient, or diluted with an excipient. When the excipient acts as a diluent, it can use solid, semisolid or liquid materials as a medium for the excipient, carrier or active ingredient. Therefore, the composition can be a solution, a sterile injection solution, etc.

[0178] Suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, etc. The preparation may also include: wetting agent, emulsifier, preservative (such as methyl and propyl hydroxybenzoate), etc. The anti-tumor drug can be prepared into unit or multiple dosage forms, each dosage form containing a predetermined amount of the ADC of the present invention calculated to produce the desired therapeutic effect, and a suitable pharmaceutical excipient.

[0179] The anti-tumor drug can be administered through conventional routes, including (but not limited to): intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, etc.

[0180] When using the drug, a safe and effective amount of the antibody and drug conjugate is administered to a human, wherein the safe and effective amount is preferably in the range of 0.5 to 50 mg / kg body weight, more preferably 1 to 10 mg / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0181] In addition, the conjugates of the present invention can also be used in combination with other therapeutic drugs, including (but not limited to): various cytokines, such as TNF, IFN, IL-2, etc.; various tumor chemotherapy drugs, such as 5-FU, methotrexate and other drugs that affect nucleic acid biosynthesis; alkylating agents such as nitrogen mustard and cyclophosphamide; drugs that interfere with the transcription process and prevent RNA synthesis, such as doxorubicin and actinomycin D; drugs that affect protein synthesis, such as vincristine and camptothecins, and certain hormone drugs, etc.

[0182] abbreviation:

[0183] Fmoc: 9-fluorenylmethoxycarbonyl

[0184] Trt: trityl

[0185] DIEA: N-ethyl-N-isopropyl-2-amine

[0186] DMF: dimethylformamide

[0187] TCEP: Tris(2-carboxyethyl)phosphine hydrochloride

[0188] Materials and Methods

[0189] (a) Cell lines and reagents

[0190] MDA-MB-468, BxPC-3, NCI-H2452, and HT-29 were provided by the Shanghai Cell Line Bank, China.

[0191] DMEM was supplemented with 10% newborn calf serum (Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. PE-labeled goat anti-human IgG marker was purchased from Biolegend (San Diego, USA). Humanized Trop-2 antibody hRS7 was prepared according to US Patent No. 9931417B2.

[0192] Cell Counting Kit-8 (CCK8) was purchased from Dojindo Laboratories (Tokyo, Japan). Matrigel (#354234) was purchased from BD Biosciences (San Jose, USA). MMAE, m-PEG24 acid and MC-OSu were provided by LevenaBiopharma (Suzhou, China). Anti-LAMP-1 (#9091), anti-Clathrin (#4796), anti-GM130 (#12480) and 555-anti-rabbit IgG F(ab')2 fragment were purchased from Cell Signaling Technology (Trask Lane Danvers, USA). IgG H&L was purchased from Abcam Trading (Shanghai) Company Ltd. Human Trop-2 recombinant protein (6×HisTag) was purchased from Sino Biological (Beijing, China). Formaldehyde was purchased from Polysciences, Inc. (Catalog No. 18814). Normal goat serum #5425 was purchased from Cell Signaling Technology (Trask Lane Danvers, USA). ProLongTM Live antifade reagent (#P36974) and TMB substrate were purchased from Thermo Fisher Scientific Inc. TCEP (BondBreakerTM) was purchased from Pierce (Rockford, IL, USA). Dimethyl sulfoxide (DMSO) and other chemicals were (Merck m-PEG4 acid and MC-OSu (structure as follows) were purchased from Lianning Biopharmaceutical Co., Ltd.

[0193]

[0194] (b) Preparation of ADC and detection of DAR value

[0195] By adding 6 molar equivalents of TCEP (1mg / mL) to 8mg / mL of an antibody solution containing 9% sucrose, 10mM acetate, 0.01% Tween-20, pH7.0 at 37°C for 2 hours of reduction reaction. After reduction, 20 molar equivalents of linker payload were added to each antibody sulfhydryl group and incubated at 4°C for more than 16 hours. The solution mixture was concentrated by centrifugation (4000rpm, 15 minutes, Thermo Fisher ST40RTX1) with an Amicon Ultracontainer (50,000MWCO, Millipore Corporation). The concentration of ADC was measured at 280nm absorption by a UV detector (Nanodrop 100, Thermo Fisher Scientific Inc.). The DAR value of the final product was confirmed by reverse phase (RP)-HPLC and QTOF mass spectrometer.

[0196] (c) RP-HPLC analysis of DAR values ​​of ADC

[0197] RP-HPLC analysis was performed on a 1260 Infinity UHPLC (Agilent Technologies) under the following conditions: 1) Agilent 8μm, 4.6*250mm; 2) Mobile phase A: 0.01% trifluoroacetic acid in water (TFA); 3) Mobile phase B: acetonitrile solution containing 0.01% TFA; 4) Gradient program: 0-3min (100% A), 3-25min (100% A-100% B), 25-30min (100% B); 5) Column temperature: 70°C; 6) Injection volume: 15ul (1mg / mL). The hydrophobicity of the ADC molecule is proportional to the amount of the coupled payload and can be reflected by its retention time. Since the drug linker has ultraviolet light absorption, the average number of conjugated drug molecules per antibody molecule in the ADC can be calculated according to the following expression: Average number of conjugated drug molecules = (L0 peak area ratio × 0 + L1 peak area ratio × 1 + H0 peak ratio × 0 + H1 peak ratio × 1 + H2 peak ratio × 2 + H3 peak ratio × 3) / 100 × 2.

[0198] (d) SEC-HPLC analysis of ADC aggregates

[0199] The SEC-HPLC method was used to monitor the aggregation and degradation of ADC. After incubation at 60°C for 1 hour, the samples were analyzed on a Thermo MAbPac SEC-1, 5 μm, (7.8×300 mm) P / N 088460, using a 1260 HPLC system (Agilent). Mobile phase: phosphate buffered saline (PBS), 50 mM sodium phosphate and 300 mM sodium chloride, pH 6.8. The column temperature was maintained at 26°C during separation. The detector was set at 280 nm. Flow rate: 0.7 mL / min. Sample injection volume: 15 μl.

[0200] (e)SDS-PAGE

[0201] Samples were boiled in 1× loading buffer for 10 min, and samples containing 5 μg of hRS7 or ADC were separated by SDS-PAGE (10% Bio-Rad Criterion Tris-HCl gel), then stained with Coomassie Brilliant Blue for 5 min and detected with BioRad ChemiDoc MP.

[0202] (f) Detection of Trop-2 expression level in tumor cells

[0203] Cells were incubated on ice with 5 μg / mL of hRS7 in ice-cold staining medium for 30 minutes and washed twice with ice-cold staining medium to remove unbound antibodies. Cells were then stained with PE goat anti-human IgG (5 μg / mL) for 30 minutes in ice-cold staining medium and washed twice. Cells were examined by flow cytometry.

[0204] (g) ELISA to detect the binding of antigen and antibody

[0205] 96-well immunoplates were coated with 2 μg / mL histidine-tagged antigen protein (Sino Biological Inc.) at 4°C overnight. The plates were washed, blocked with 1% casein, and then incubated with 10 nM antibody or ADC for 1 hour at room temperature. After washing, anti-human κ-HRP (Biolegend San Diego, USA) was added and incubated for 1 hour. After washing, TMB substrate was added and detected at 450 nm using a SpectraMax M5e (Molecular Devices) microplate reader.

[0206] (h) Detection of ADC endocytosis rate

[0207] To evaluate the ADC internalization rate, MDA-MB-231 or BxPC-3 or HT-29 cells (1×10 6) and ADC (3 μg / mL) samples were incubated on ice for 30 minutes in staining medium, washed twice, and then placed in fresh complete medium. Internalization was initiated in a 37°C humidified chamber. At the end of the incubation, the cells were washed twice with ice-cold staining medium and then stained with goat anti-human IgG-PE. Cells were analyzed by flow cytometry to detect the level of remaining surface ADC.

[0208] (i) Cell viability assay

[0209] Cell viability was determined using the Cell Counting Kit 8 (CCK-8). Cells were seeded into 96-well plates at 3,000-10,000 cells / well. After overnight incubation, drugs were added at different concentrations. The plates were incubated in a humidified chamber at 37°C, 5% CO2 for 96 hours. CCK8 reagent was added and returned to the incubator until the absorbance of the untreated control cells at 450nm was greater than 1.0. Growth inhibition was measured as a percentage of growth relative to untreated cells. Dose-response curves were generated from the average of two or three determinations, and the IC was calculated using Prism GraphPad software. 50 value.

[0210] (j) Detection of drug efficacy in mice

[0211] Five-week-old female athymic BALB / c nude mice were purchased from Charles River Company (Shanghai, China) and randomly divided into 5-6 mice per group. Animal handling and procedures were approved and performed in accordance with the requirements of the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. All models were inoculated subcutaneously in the flank of mice. BxPC-3 cells were injected by 5×10 6 By injecting 1×10 7 Cells were used to establish MDA-MB-468 cells. The tumor volume reached about 200mm 3 Afterwards, the tumor-bearing mice were randomly divided into treatment and control groups according to tumor volume. Each drug was administered intraperitoneally. It was injected into mice at a dose of 3 or 10 mg / kg (10 mL / kg). Tumor volume was defined as length × 1 / 2 × width × width. The inhibition percentage was calculated by the following formula: (1-average tumor volume of the treatment group / average tumor volume of the control group) × 100%. The toxicity associated with the different treatment groups was evaluated by monitoring weight loss.

[0212] (k) Dose tolerance determination

[0213] Dose tolerance was determined in BALB / c mice (5 mice per dose). The drug was administered as a single intravenous dose.

[0214] The injection doses of hRS7-VK-MMAE and hRS7-VC-MMAE were 60 and 40 mg / kg, respectively. Toxicity was evaluated by observing mouse behavior, weight loss, and survival. The weight change rate (%) was calculated as: (weight-pre-treatment weight) / (pre-treatment weight)×100%.

[0215] (l) Statistical analysis

[0216] Prism GraphPad version 6 was used for statistical analysis. The results are shown as mean ± SD. The statistical differences between the control and experimental groups were calculated by Student's t-test, and P < 0.05 was considered significant.

[0217] Example 1

[0218] Antibody preparation

[0219] In this example, hRS7 antibody is expressed and purified to obtain hRS7 antibody. The method is as follows:

[0220] The amino acid sequences of hRS7 light chain and heavy chain are from U.S. Patent 9931417B2, and then reverse transcribed into cDNA sequences by Vector NTI software. The DNA sequence was synthesized, subcloned into the pcDNA3.1 vector, and amplified in Escherichia coli. The purified plasmid was transfected into HEK293 cells by PEI. The cells were then suspended in FreeStyleTM293 expression medium for culture. After 5 days of culture, the cell culture supernatant was collected and the antibody was affinity purified by protein A. The amino acid sequence of its light chain variable region is SEQ ID NO.: 1, and the amino acid sequence of the heavy chain variable region is SEQ ID NO.: 2.

[0221] Example 2

[0222] Preparation of compound VK-MMAE

[0223] The structure of the compound VK1-MMAE is shown below:

[0224]

[0225] Monomethyl auristatin E is reacted with a VK1 linker to form a compound VK1-MMAE, namely

[0226] Mal-Val-Lys(PEG24)-PAB-monomethyl auristatin (VK1-MMAE), the method is as follows:

[0227]

[0228] Step 1: Synthesis of compound 3 (Fmoc-Val-Lys(Trt)-PAB-MMAE)

[0229] Fmoc-Val-Lys(Trt)-PAB-PNP (purchased from Levena Biopharma, USA) and MMAE (purchased from Levena Biopharma, USA) were dissolved in anhydrous DMF, and DIEA was added to the solution. The mixture was stirred at room temperature 22°C for 2 hours, and the target product Fmoc-Val-Lys(Trt)-PAB-MMAE was directly purified by reverse HPLC to form a white powder compound 3 after freeze-drying.

[0230] Step 2: Synthesis of compound 4 (Fmoc-Val-Lys-PAB-MMAE TFA salt)

[0231] The compound 3 obtained in step 1 was dissolved in 10% TFA / DCM solution and stirred at room temperature for 20 minutes. After the reaction, the mixture was concentrated under reduced pressure and purified by reverse phase HPLC to obtain compound 4, TFA salt of Fmoc-Val-Lys-PAB-MMAE.

[0232] Step 3: Synthesis of compound 5

[0233] Compound 4 obtained in step 2, m-PEG24 acid and HATU were dissolved in DMF, DIEA was added, and the mixture was stirred at room temperature for 30 minutes to obtain product 5a.

[0234] Diisopropylamine was added to the reaction mixture and stirred at room temperature for 3 hours. After the reaction, the mixture was concentrated and purified by reverse HPLC to obtain waxy compound 5 after freeze-drying.

[0235] Step 4: Synthesis of compound 6 (Mal-val-Lys(m-PEG24)-PAB-MMAE, VK1-MMAE)

[0236] The compound 5 obtained in step 3, MC-OSu or m-PEG4-acid and HATU were dissolved in DMF, DIEA was added, and stirred for 20 minutes. Afterwards, the desired compound 6, Mal-Val-Lys-PAB-MMAE (VK1-MMAE) or Mal-PEG4-Val-Lys-PAB-MMAE (VK2-MMAE) was finally obtained by reverse HPLC purification and freeze drying.

[0237] Example 3

[0238] Preparation of ADC

[0239] Through a coupling reaction, the VK-MMAE prepared in Example 2 was coupled with the antibody prepared in Example 1 to form a corresponding ADC.

[0240] Through the VK linker, a stable ADC molecule with a DAR value of 8 and MMAE as the payload was successfully prepared, named hRS7-VK1-MMAE and hRS7-VK2-MMAE ( Figure 1 (a)

[0241] For comparison, a Trop-2-ADC with a DAR value of 8 and a payload of MMAE, namely hRS7-VC-MMAE ( Figure 1 (a)

[0242] Under reducing conditions, SDS-PAGE of hRS7-VK2-MMAE with DAR values ​​ranging from 0 to 8 was as follows: Figure 1 As shown in b. RP-HPLC detection of three Trop-2-ADCs of DAR8 prepared with VK or VC linkers ( Figure 1 c) and HIC-HPLC hydrophobicity detection ( Figure 1 d), which proves that the product has good uniformity; even after incubation at 60°C for 1 hour ( Figure 1 In middle e), no serious aggregation and degradation were shown, indicating that PEGylation of the linker can significantly improve the hydrophobicity and stability of the ADC molecule.

[0243] Comparative Example 1

[0244] Preparation of control ADC molecules

[0245] Through the hydrophobic VC linker ( Figure 1 In a), a hRS7-VC-MMAE molecule targeting Trop-2 was prepared. However, the molecule partially precipitated out of the solution during the preparation process, so its therapeutic ability and safety could not be evaluated.

[0246] Therefore, it can be seen that conjugating a hydrophobic linker to a hydrophobic payload compromises the stability of the ADC molecule, leading to an increase in aggregates and precipitation.

[0247] Comparative Example 2: Tolerance Study of hRS7-VK-MMAE

[0248] The safety of relatively high doses of hRS7-VK1-MMAE and hRS7-VK2-MMAE in BALB / c mice was studied by acute toxicity experiments with a single intravenous administration in mice. Compared with hRS7-VK2-MMAE and hRS7-VC-MMAE, the maximum tolerated dose (MTD) of hRS7-VK1-MMAE. At a single dose of 60 mg / kg, hRS7-VK1-MMAE showed less toxicity or signs of weight loss, while hRS7-VK2-MMAE caused a certain amount of weight loss in mice, while hRS7-VC-MMAE caused serious adverse reactions and more than 15% weight loss ( Figure 2 Meanwhile, hRS7-VK1-MMAE and hRS7-VK2-MMAE did not show severe liver damage, while hRS7-VC-MMAE caused relatively severe liver damage ( Figure 2 (b)

[0249] Therefore, hRS7-VK1-MMAE has better safety and tolerability than hRS7-VK2-MMAE and hRS7-VC-MMAE.

[0250] Comparative Example 3: Inhibitory effects of hRS7-VK1-MMAE and hRS7-VK2-MMAE on tumor cells specifically expressing Trop-2 in vivo

[0251] On day 8, mice inoculated with BxPC-3 cells (n=5 per group) were treated with 1 mg / kg ADC or naked antibody, and tumors were measured every two days. On day 25, a two-tailed t-test was used to assess statistical significance between treatment and control groups. ****P<0.00001, hRS7-VK1-MMAE / hRS7-VK2-MMAE compared to hRS7 treatment groups; no significant difference (ns), hRS7-VK1-MMAE compared to hRS7-VK2-MMAE. Data = mean ± SD, results as Figure 2 As shown in c.

[0252] On day 8, mice inoculated with BxPC-3 cells (n=5 per group) were treated with ADC or control (PBS) at 0.5 mg / kg, and tumor volume was measured every two days. Two-tailed t-test was used to evaluate statistical significance between treatment and control groups. ****P<0.0001, comparing hRS7-VK1-MMAE / hRS7-VK2-MMAE with hRS7; no significant difference (ns), comparing hRS7-VK1-MMAE with hRS7-VK2-MMAE; ***P<0.0001, comparing hRS7-VK1-MMAE / hRS7-VK2-MMAE with hRS7-VC-MMAE. Data = mean ± SEM, results as shown Figure 2 As shown in Figure d, it is shown that hRS7-VK1-MMAE and hRS7-VK2-MMAE have comparable in vivo efficacy, but hRS7-VK1-MMAE has better tolerability ( Figure 2 (a)

[0253] Example 4: Inhibitory effect of hRS7-VK1-MMAE on tumor cells specifically expressing Trop-2 in vitro

[0254] (a) The hRS7-VK1-MMAE molecule with a DAR value of 8 has a good affinity to human and monkey Trop-2 proteins (6×His tag), but does not bind to mouse Trop-2, e.g. Figure 3 Middle a.

[0255] (b) Three tumor cell lines showed different expression levels of Trop-2 ( Figure 3 (b)

[0256] (c) The anti-tumor efficacy of hRS7-VK1-MMAE in different tumor cell lines was detected by CCK-8. Among the tumor cells with different Trop-2 expression levels, hRS7-VK1-MMAE had a significant killing effect on BxPC-3 and MDA-MB-468 cells with high Trop-2 expression, but had no killing effect on HT-29 cells with low TROP-2 expression. Figure 3 As shown in c.

[0257] (d) Cancer cells (1×10 6 Cells / mL) were incubated with 3 μg / mL hRS7 at 4°C for 30 min. Human IgG1 was used as an isotype control. The results are shown in Figure 3 These results indicate that hRS7-VK1-MMAE can trigger a faster internalization rate in TROP-2 high-expressing cells, which supports the higher cell killing efficacy.

[0258] Example 5: Minimum effective dose (MED) of hRS7-VK1-MMAE in different tumor cell lines

[0259] Mice inoculated with three highly Trop-2-expressing tumor cells MDA-MB-468, BxPC-3, and NCI-N87 were treated with different doses of hRS7-VK1-MMAE (n=5 per group), with the doses of 0.1 mg / kg, 0.2 mg / kg, and 0.4 mg / kg, respectively, once a week for a total of three times, and the tumor volume was measured every two days. The results of tumor volume, inhibition rate, and mouse weight are shown in Figure 2. Figure 4 shown.

[0260] In the BxPC-3 and NCI-N87 xenograft tumor models with higher Trop-2 expression, when the dose was 0.1 mg / kg, the tumor volume was significantly different from the blank control group. As the dose gradually increased, the tumor volume shrank more significantly, and the efficacy was proportional to the dose; in the MDA-MB-468 xenograft tumor model with high Trop-2 expression, when hRS7-VK1-MMAE was 0.1 mg / kg, it was slightly different from the blank control group at the beginning, but after stopping the administration, the difference gradually became smaller and even no difference in the end, but the 0.2 mg / kg group has always been significantly different from the blank control group. Combining the three models, it can be seen that the MED of hRS7-VK1-MMAE is 0.1-0.2 mg / kg, which is far below the tolerated dose and has a large therapeutic window.

[0261] Example 6: Pharmacokinetic (PK) study of hRS7-VK1-MMAE

[0262] (a) BALB / c mice (n=3) were intravenously administered with 3 mg / kg hRS7 and hRS7-VK1-MMAE. Blood was collected from the eye sockets of each mouse at 1, 4, 24, 72, and 180 hours after administration, and the total anti- and ADC concentrations in plasma were measured ( Figure 5 (a) hRS7-VK1-MMAE showed excellent PK characteristics comparable to the unconjugated parent hRS7.

[0263] (b) Male cynomolgus monkeys (n=5) were intravenously infused with 0.2, 0.5, and 1.0 mg / kg of hRS7-VK1-MMAE. Blood was collected from the vein at 0, 6, 24, 48, 72, 168, 336, and 504 hours after administration. The total antibody concentration was detected by ELISA, and the ADC and free MMAE were detected by mass spectrometry. Figure 5 hRS7-VK1-MMAE showed excellent PK characteristics comparable to the unconjugated parent hRS7. The metabolism in female cynomolgus monkeys was consistent.

[0264] (c) BALB / c mice bearing MBA-MB-468 (n=3) were intravenously administered with different doses of hRS7 and hRS7-VK1-MMAE. Blood was collected from the eye sockets of each mouse at 0, 1, 2, 4, 8, 24, 48, 120, 196, 264, 336, 408, and 504 hours after administration, and the ADC and total anti-antibody concentrations in plasma were measured (respectively). Figure 5 (c, d). hRS7-VK1-MMAE exhibited excellent PK characteristics comparable to the unconjugated parental hRS7.

[0265] Therefore, hRS7-VK1-MMAE has better tolerance than hRS7-VK2-MMAE, and its inhibitory effect on tumor cells specifically expressing Trop-2 is comparable to that of hRS7-VK2-MMAE. Both have better safety and tolerance than hRS7-VC-MMAE.

[0266] It is known in the prior art that inserting PEG into a molecule during ADC preparation will increase the hydrophilicity of the molecule. However, the inventors of the present application unexpectedly discovered that inserting PEG into the main chain of the molecule is not necessarily beneficial. As demonstrated above in the present application, hRS7-VK1-MMAE shows superior effects in terms of aggregation and tolerance compared to hRS7-VK2-MMAE (see Figure 1 and 2 ), the technical effect is difficult for technicians to foresee.

[0267] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound of formula I for preparing an antibody-drug conjugate, In Formula I, Z1 is a linker portion used to connect to the antibody; m1 is an integer from 1 to 10; m2 is an integer from 10 to 50; X represents a group derived from a small molecule cytotoxic drug.

2. The compound according to claim 1, wherein The Z1 is a substituted or unsubstituted maleimide group; For example, the Z1 is linked to the antibody via position 1, position 3 or position 4 of the 5-membered ring of maleimide. For example, the connection includes: connection to a sulfhydryl group on the antibody, connection to an ε-amino group on the antibody (such as the ε-amino group of lysine), connection to a selenocysteine ​​on the antibody, or a combination thereof. For example, the Z1 reacts with a naturally occurring active group on the antibody or an artificially introduced non-natural active group to generate a chemically linked structure. For example, after Z1 is connected to the antibody, a chemical connection structure selected from the following group is formed: (a) a chemical connection structure formed by coupling reaction between the thiol group on the antibody and maleimide; (b) a chemical connection structure formed by coupling reaction between the thiol group on the antibody and dibromomaleimide; (c) the ε-amino group of lysine on the antibody reacts with the active amine group on the linker to form a chemical connection structure; (d) a coupling reaction between lysine on the antibody and a maleimide-containing derivative to form a chemically linked structure; (e) a coupling reaction between lysine on the antibody and iodoacetamide to form a chemical connection structure; (f) a chemical connection structure formed by a coupling reaction of lysine on the antibody based on iminothiolane; (g) a chemical connection structure formed by a pyridine disulfide coupling reaction of lysine on an antibody; (h) site-specific coupling with selenocysteine ​​artificially introduced into the antibody to form a chemical connection structure; (i) any combination of the above; Wherein, in the above formulae (a) and (b), m3 is a positive integer of 1-8, and in the above formulae (d) and (e), spacer is selected from: -(CH2) m4 -、-(CH2O) m4 -, wherein m4 is an integer of 0-10, preferably, an integer of 1-6.

3. The compound according to claim 1, wherein The compound of formula I is a compound of the following formula Ia: In Formula Ia, m1 is an integer from 1 to 10; m2 is an integer from 10 to 50; X is a group after monomethyl auristatin and other compounds lose H atoms, Preferably, m1 is 1-8, more preferably 4, 5, and most preferably 5; m2 is 12-40, more preferably 16-36, and most preferably 24; X is MMAE shown in the following structure: Further preferably, the structure of the compound of formula Ia is shown in formula III below: The definition of MMAE is as described above.

4. An antibody-drug conjugate, wherein the antibody-drug conjugate is formed by coupling the compound of formula I according to claim 1 with an antibody, Preferably, the antibody-drug conjugate has a structure shown in the following formula II: Ab-(LX)n(II) In Formula II, Ab stands for antibody, L is a divalent linker as shown below: in, Z1' is the group remaining after removing one hydrogen from the Z1 group in the compound of formula I; n is the average number of conjugated drugs conjugated to the antibody; X, m1 and m2 are respectively as defined in claim 1.

5. The antibody-drug conjugate according to claim 4, wherein: n is an integer or non-integer > 0, preferably 1≤n≤15, for example, n is an integer or non-integer of 5-10; preferably, an integer or non-integer of 6-9; more preferably, an integer or non-integer of 7-8.

6. The antibody-drug conjugate according to claim 4, wherein: The antibody is selected from the group consisting of a chimeric antibody, a bivalent or bispecific molecule, a diabody, a triabody and a tetrabody, The antibody is an antibody of any target, for example, the target is selected from the following group: Trop-2, PD-L1, CTLA4, OX40, EpCAM, EGFR, EGFRVIII, HER2, PTPN2, VEGF, CD11a, CD19, CD20, CD22, CD25, CD30, CD33, CD40, CD56, CD64, CD70, CD73, CD74, CD79, CD105, CD138, CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphB2, Apo2, NMP179, p16INK4A, Nectin-4, B7H3, EMA, CEA, MIB-1, GD2, APRIL receptor, folate receptor, mesothelin inhibitor, MET, TM4SF1, IL13 receptor, Further preferably, the antibody is hRS7, More preferably, the amino acid sequence of the light chain variable region of the antibody hRS7 comprises the sequence shown in SEQ ID NO.: 1 or is the sequence shown in SEQ ID NO.: 1; and / or the amino acid sequence of the heavy chain variable region of the antibody hRS7 comprises the sequence shown in SEQ ID NO.: 2 or is the sequence shown in SEQ ID NO.:

2.

7. The antibody-drug conjugate according to claim 4, wherein: In the antibody-drug conjugate, the antibody is hRS7, and in L, Z1' is the group remaining after removing one hydrogen from maleimide, m1 is 5, m2 is 24, n is 7 or 8, and X is MMAE.

8. A pharmaceutical composition comprising: (a) the antibody-drug conjugate according to any one of claims 4 to 7, and (b) a pharmaceutically acceptable carrier.

9. Use of the antibody-drug conjugate according to any one of claims 4 to 7 or the pharmaceutical composition according to claim 8 in the preparation of a drug for treating tumors or immune diseases, Preferably, the tumor is selected from a Trop-2 targeted tumor, a HER2 targeted tumor and an EGFR targeted tumor, Further preferably, the tumor is a Trop-2 targeted tumor and is selected from breast cancer, lung cancer, colon cancer, bladder cancer and pancreatic cancer.

10. A method for preparing an antibody-drug conjugate according to any one of claims 4 to 7, the method comprising the following steps: (1) providing a reaction system, wherein the reaction system comprises an antibody to be coupled and a compound of formula I according to claim 1; (2) In the reaction system, the antibody and the compound of formula I are subjected to a coupling reaction to obtain the antibody-drug conjugate.

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