WO2023143319A1 - Antibody-drug conjugate, and pharmaceutical composition and use thereof - Google Patents

Abstract

Disclosed are an antibody-drug conjugate represented by formula (III), a pharmaceutical composition comprising the antibody-drug conjugate, and use of the antibody-drug conjugate and the pharmaceutical composition in the preparation of a drug for preventing and/or treating a tumor. The antibody-drug conjugate is formed by connecting a cytotoxic drug eribulin to an antibody or an antigen-binding fragment Ab by means of a linker. The antibody-drug conjugate can exert tumor cell-killing activity in vivo and in vitro, thereby significantly inhibiting the growth of different types of tumors.

Description

Antibody drug conjugate, pharmaceutical composition and use

This disclosure claims the priority of the following patent applications: the Chinese patent application submitted to the China Patent Office on January 28, 2022 with the application number 202210108108.7, and the Chinese patent application submitted to the China Patent Office on June 30, 2022 with the application number 202210771635.6 application; the entire content of the aforementioned patent application is incorporated by reference into this disclosure.

technical field

The present disclosure belongs to the field of medical technology, specifically, the present disclosure relates to an antibody-drug conjugate as shown in formula (III), a pharmaceutical composition comprising the antibody-drug conjugate, and an antibody-drug conjugate and a pharmaceutical composition. Uses, methods for treating or preventing tumors.

Background technique

Antibody-Drug Conjugates (ADCs) are new targeted drugs formed by covalently linking antibodies and cytotoxic drugs through specific linkers. Among them, after the antibody is used as a targeting carrier to transport cytotoxic drugs, that is, "effector molecules" or "warheads" to the target site of action, it specifically binds to the surface antigen of the target cell, enters the interior of the cell through endocytosis, and at the same time brings the small molecule drug into the cell. Internally, the cytotoxic drug is then released to kill the target cell via enzymatic cleavage or self-cleavage of the linker. Compared with chemotherapy and combination therapy, ADC drugs have high drug activity and high selectivity, which can significantly reduce off-target toxicity. In recent years, ADC drugs such as Brentuximab vedotin (Adcetris, SGN-35) and Trastuzumab emtansine (Kadcyla, T-DM1) have shown good safety and efficacy in clinical citations, and ADC drugs have become the mainstay in the field of disease-targeted therapy. Research hotspots and important development directions.

Eribulin is a tubulin polymerization inhibitor developed by Eisai. As a synthetic halichondrin B analogue, Eribulin has unique binding properties. The Eriblin structure is as follows:

Eribulin is thought to act by inhibiting the growth phase of microtubule dynamics that prevent cell division. In addition, non-clinical studies have shown Eribulin's unique effects in the tumor microenvironment, such as increasing vascular perfusion and permeability in the tumor core area, improving the state of epithelial cells, and reducing the migration ability of breast cancer cells, etc. At present, Eribulin has been approved in more than 70 countries and regions including Europe, America and Asia, and was approved for marketing in China in 2019.

In view of the high tumor killing and inhibitory activity of eribulin, how to stably couple eribulin to antibody molecules to achieve directional killing of tumor cells is an important problem that needs to be solved urgently in this field.

Contents of the invention

The problem to be solved by the invention

In view of the existing problems in the prior art, for example, it is necessary to develop more linkers suitable for linking antibodies and cytotoxic drugs, so as to improve the stability and drug activity of ADC drugs. To this end, the present disclosure provides an antibody-drug conjugate as represented by formula (III), which has both the targeting of antibodies and the high activity of eribulin in tumor cytotoxicity, and can specifically and efficiently kill tumor cells, It has important application prospects.

solutions to problems

In the first aspect, the present disclosure provides an antibody-drug conjugate, which has the structure shown in the following formula (III):

Wherein, q is any integer of 2-8, preferably 4; Ab is an antibody or an antigen-binding fragment;

Pep is -(X ₁ ) _e -Leu-Pro-X ₂ -Thr-, wherein e is any integer from 0-10, if present, each X ₁ is independently Gly or Ala; X ₂ is any species amino acid residues;

L ₁ is -(Gly) _p -(Z ₁ ) _a -, p is any integer of 1-10; a is any integer of 0-20, if present, each Z ₁ is independently any kind of amino acid residues;

L ₂ is -NH-R ₁ -(CH ₂ ) _c -C(=O)-, wherein R ₁ is -(CH ₂ -CH ₂ -X) _b -, b is any integer from 1 to 10, each Each X is independently -CH ₂ -, -NH-, -O- or -S-, and c is any integer from 1 to 5;

L ₃ is

Alternatively, _L3 is wherein m is any integer from 1 to 5, preferably 2; R ₂ is -(CH ₂ -CH ₂ -Y) _d -, d is any integer from 1 to 10, and each Y is independently -CH ₂ -, -NH-, -O- or -S-;

And, the amino terminus of Pep is connected to the carboxyl terminus of the heavy chain or light chain of Ab, the carboxyl terminus of Pep is connected to the amino terminus of _L1 , the carboxyl terminus of _L1 is connected to the amino terminus of _L2 , and the carboxyl terminus of _L2 is connected to the carboxy terminus of _L3 . amino terminus.

In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein,

Pep is -(Gly-Ala) _f -Leu-Pro-X ₂ -Thr-, wherein f is any integer of 1-5, preferably 1; X ₂ is selected from any kind of amino acid residues, preferably Glu residues ;

Preferably, Pep is -Gly-Ala-Leu-Pro-Glu-Thr-, wherein the amino terminal of Pep is connected to Ab, and the carboxyl terminal of Pep is connected to the amino terminal of _L1 .

In some embodiments, the antibody-drug conjugate according to the present disclosure, wherein the carboxyl terminus of the heavy chain of Ab is linked to Pep, and the carboxyl terminus of the light chain of Ab is linked to Pep;

Preferably, the carboxy terminus of the heavy chain of the Ab is linked to -Gly-Ala-Leu-Pro-Glu-Thr-, and the carboxy terminus of the light chain of the Ab is linked to -Gly-Ala-Leu-Pro-Glu-Thr-.

In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein, the antibody or antigen-binding fragment is specific for At least one protein in the HER protein family specifically binds; preferably, the antibody or antigen-binding fragment specifically binds to at least one of the following proteins: HER2, HER3.

In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein, the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein, the heavy chain variable region comprises such as SEQ ID NO: 9, SEQ ID NO: The amino acid sequence shown in at least one of ID NO: 11 and SEQ ID NO: 13; and/or,

The antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises amino acids as shown in at least one of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 sequence;

The light chain variable region and the heavy chain variable region are coded according to the analysis method of Kabat.

In some embodiments, the antibody drug conjugate according to the present disclosure, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3;

HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 13;

LCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 4, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein, the amino acid sequence of the heavy chain of the antigen or antigen-binding fragment is shown in SEQ ID NO: 18, and/or,

The amino acid sequence of the light chain of the antigen or antigen-binding fragment is shown in SEQ ID NO: 17.

In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein,

L ₁ is -(Gly) _p -, p is any integer of 1-10, preferably any integer of 3-5;

L ₂ , L ₃ are as defined in the first aspect.

In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein, _L3 is selected from any one of the following:

L ₁ , L ₂ are as defined in the first aspect.

In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein,

L ₂ is -NH-R ₁ -(CH ₂ ) _c -C(=O)-;

Wherein R ₁ is -(CH ₂ -CH ₂ -X) _b -, b is any integer of 1-10, preferably any integer of 1-5; each X is independently -CH ₂ - or - O-; c is any integer of 1-5, preferably any integer of 1-2;

L ₁ , L ₃ are as defined in the first aspect.

In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein,

-L ₁ -L ₂ -for

Wherein, w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2;

b is any integer of 1-5, preferably any integer of 2-4, each X is independently -CH ₂ - or -O-; and,
The structure is:

In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein,

-L ₁ -L ₂ -for

Wherein, w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2; b is any integer of 1-5 Integer, preferably any integer of 2-4, each X is independently -CH ₂ - or -O-; and,
The structure is:

In some embodiments, the present disclosure provides an antibody drug conjugate represented by any one of the following formulas (III-1) to (III-6):

A structure as shown in any of the following is preferred:

In the present disclosure, Ab may be an antibody or antigen-binding fragment that binds to any type of tumor antigen. Exemplary, tumor antigens include but are not limited to PD-1, PD-L1, PDL2, CTLA4, LAG3, TIM3, TIGIT, CD103, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, AFP, CEA, PSCA, GD2, NKG2D, BCMA, EGFR, HER2, HER3, EGFRvⅢ, CD171, FAP, IL13Rα2, VEGFR1, VEGFR2, GPC-3, Mesothelin, claudin 18.2, EpCAM, MUC1, MUC16, EPHA2, EPHA3, CD133, PSMA . The antibody or antigen-binding fragment can specifically deliver Eribulin to tumor cells by specifically binding to tumor surface antigens, thereby exerting an efficient and specific tumor cell killing effect.

Further, the antibody or antigen-binding fragment specifically binds to at least one protein in the HER protein family; preferably, the antibody or antigen-binding fragment specifically binds to at least one of the following proteins: HER2, HER3.

In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein, the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein, the heavy chain variable region comprises such as SEQ ID NO: 9, SEQ ID NO: The amino acid sequence shown in at least one of ID NO: 11 and SEQ ID NO: 13; and/or,

The antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises amino acids as shown in at least one of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 sequence;

The light chain variable region and the heavy chain variable region are coded according to the analysis method of Kabat.

In some embodiments, the antibody drug conjugate according to the present disclosure, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3;

HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 13;

LCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 4, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein, the amino acid sequence of the heavy chain of the antigen or antigen-binding fragment is shown in SEQ ID NO: 18, and/or, the antigen or The amino acid sequence of the light chain of the antigen-binding fragment is shown in SEQ ID NO: 17.

In the second aspect, the present disclosure provides a pharmaceutical composition, which includes a therapeutically effective amount of the antibody-drug conjugate according to the first aspect;

Optionally, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers.

In a third aspect, the present disclosure provides the use of the antibody drug conjugate according to the first aspect, or the pharmaceutical composition according to the second aspect in the preparation of a drug for preventing and/or treating tumors;

Optionally, the tumor is a tumor associated with the abnormal expression of one or two or more proteins in the HER protein family; preferably, the abnormally expressed protein is selected from at least one of HER2 and HER3;

Optionally, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, Colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.

In the fourth aspect, the present disclosure provides the antibody drug conjugate according to the first aspect, or the pharmaceutical composition according to the second aspect, which is used for preventing and/or treating tumors;

Optionally, the tumor is a tumor associated with the abnormal expression of one or two or more proteins in the HER protein family; preferably, the abnormally expressed protein is selected from at least one of HER2 and HER3;

Optionally, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.

In the fifth aspect, the present disclosure provides a method for preventing and/or treating tumors, which comprises administering the antibody-drug conjugate shown in the first aspect, or the pharmaceutical composition according to the second aspect;

Optionally, the tumor is a tumor associated with the abnormal expression of one or two or more proteins in the HER protein family; preferably, the abnormally expressed protein is selected from at least one of HER2 and HER3;

Optionally, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.

The effect of the invention

The antibody-drug conjugate represented by formula (III) provided by the present disclosure is formed by linking the cytotoxic drug eribulin with the antibody or antigen-binding fragment Ab through a linker, and is a novel antibody-drug conjugate. The antibody-drug conjugate represented by the formula (III) can have a specific structure with target cells such as tumor cells under the targeting effect of the antibody, and after entering the tumor cell, the linker part of the antibody-drug conjugate can be broken, Release the cytotoxic drug eribulin to exert high-efficiency tumor cell killing activity.

Furthermore, in the antibody-drug conjugates represented by formula (III), by linking antibodies or antigen-binding fragments that bind to different antigen targets, different types of cells can be specifically targeted, thereby achieving the goal of treating different types of tumors. The efficient and specific killing of the drug improves the effectiveness and safety of the drug.

In some embodiments, the present disclosure provides an antibody-drug conjugate as shown in any one of formula (III-1) to formula (III-6), and any one of formula (III-1) to formula (III-6) The antibody-drug conjugate shown is formed by linking an antibody to eribulin through a cleavable linker, which can kill tumor cells efficiently and at a specific location.

In some preferred embodiments, antibody-drug conjugates with the structures shown in formula (III-1) and formula (III-5). The present disclosure finds that the antibody drug conjugates represented by formula (III-1) and formula (III-5) can significantly reduce various types of tumor cells that are HER2 positive (for example, HCC1954, SK-BR-3 and NCI-N87) The in vitro survival rate and high tumor cell killing activity. Compared with the small molecule drug Dxd, Eribulin shows stronger anti-tumor activity; compared with the small molecule drug Eribulin, the antibodies represented by formula (III-1) and formula (III-5) in the present disclosure The drug conjugate has lower IC ₅₀ value and higher tumor cell killing activity. Moreover, in HER2-negative cells, the cytotoxicity of ADC drugs is lower than that of the small molecule drug eribulin; meanwhile, the antibody-drug conjugates shown in formula (III-1) and formula (III-5) are more effective in HER2-positive cells The cytotoxicity was higher than that in HER2-negative cells. The above results indicate that the antibody-drug conjugates in the present disclosure can target and bind tumor cells, and release the cytotoxic drug Eribulin inside the tumor cells to play a targeted and efficient role in killing tumor cells.

Further, in three tumor-bearing mouse models inoculated with different tumor cells of Capan-1, NCI-N87 and HCC1954, the in vivo tumor treatment effect of the compound represented by formula (III-1) and formula (III-5) was verified, The results show that the compounds represented by formula (III-1) and formula (III-5) can significantly inhibit the growth of Capan-1 tumors, NCI-N87 tumors and HCC1954 tumors, efficiently and specifically kill tumor cells in vivo, and have no It has obvious drug toxicity and has broad application prospects in the clinical treatment of tumors.

Description of drawings

Figure 1 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of HCC1954 cells with different drug concentration gradients;

Figure 2 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of SK-BR-3 cells with different drug concentration gradients;

Figure 3 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of NCI-N87 cells with different drug concentration gradients;

Figure 4 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of MDA-MB-468 cells with different drug concentration gradients;

Figure 5 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (Capan-1 tumor-bearing female BALB /c nude mice) body weight change detection results;

Figure 6 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (Capan-1 tumor-bearing female BALB /c nude mice) tumor growth curve;

Figure 7 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (NCI-N87 tumor-bearing female BALB /c nude mice) tumor growth curve;

Figure 8 shows vehicle (DPBS), Ab0100-H0037 (3mg/kg), Ab0100-H0038 (3mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (HCC1954 tumor-bearing female BALB/c Tumor growth curve of nude mice).

Detailed ways

definition

Unless stated to the contrary, the terms used in this disclosure have the following meanings.

In the claims and/or specification of the present disclosure, the word "one (a)" or "one (an)" or "one (the)" may mean "one", but may also mean "one or more", "at least one" and "one or more than one".

As used in the claims and specification, the words "comprising", "having", "comprising" or "containing" mean inclusive or open-ended and do not exclude additional, non-recited elements or means step. At the same time, "comprising", "having", "including" or "comprising" can also mean enclosing, excluding additional, unrecited elements or method steps.

Throughout this application, the term "about" means that a value includes the standard deviation of error of the apparatus or method used to determine the value.

In this disclosure, the use of "any integer from value A to value B" covers any type of natural number between value A and value B, and its range covers values A and B including the endpoints. Exemplarily, any integer of 1-10 is any integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

In this disclosure, "any kind of amino acid" can be common amino acids (amino acids shown in Table 1 in this disclosure), or uncommon amino acids (for example, 2-aminoadipic acid, 2-aminobutyric acid, 2-aminobutyric acid, 2 -aminopimelic acid, 2-carboxamide, etc.). Further, any kind of amino acid may be a modified amino acid (for example, acylation, ubiquitination, sugar chain modification, biotinylation, etc.), or an unmodified amino acid.

The term "independently" means that at least two groups (or ring systems) present in the structure with the same or similar value range may have the same or different meanings under specific circumstances. For example, the substituent X and the substituent Y are each independently hydrogen, hydroxyl, alkyl or aryl, then when the substituent X is hydrogen, the substituent Y can be either hydrogen or hydroxyl, alkyl or aryl ; Similarly, when the substituent Y is hydrogen, the substituent X can be either hydrogen, or hydroxyl, alkyl or aryl. Alternatively, the number of substituents X is two, and each X is independently hydrogen, hydroxyl, alkyl or aryl; then when one X is hydrogen, the other X can be either hydrogen or hydroxyl, Alkyl or aryl. Similarly, the number of substituents X is three or more, and each X is independently hydrogen, hydroxyl, alkyl or aryl, which means that any one of X can be selected from hydrogen, hydroxyl, alkyl or aryl .

Exemplarily, in -(X ₁ ) _e -, e is any integer of 0-10, and if present, each X ₁ is independently Gly or Ala. For example, when e is 2, -(X ₁ ) _e - may be -Gly-Gly-, -Ala-Ala-, -Ala-Gly- or -Gly-Ala- along the direction from the amino terminal to the carboxyl terminal.

The term "linker", "linker unit", "linker unit", "linker" or "linker fragment" refers to a chemical structural fragment or bond that is connected to a ligand/antibody at one end and a drug at the other end, and may also be connected to other The connector is then connected to the drug. In some embodiments, a linker of the present disclosure is a glycine-terminated amino acid fragment. Amino acid fragments with a glycine terminal can be protected by protecting groups such as Fmoc. As a preferred embodiment, the amino acid fragment at the end can be 3-5 glycines.

The term "antibody" is used herein in the broadest sense to refer to a protein comprising an antigen binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies ( For example, bispecific antibodies), Single chain antibodies, whole antibodies and antibody fragments.

The term "antigen-binding fragment" is a portion or segment of an intact or complete antibody having fewer amino acid residues than an intact or complete antibody, which is capable of binding antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) Binding antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single chain antibodies (e.g. scFv); single domain antibodies; bivalent or bispecific Antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and bispecific or multispecific antibodies formed from antibody fragments.

The term "complementarity determining region" or "CDR region" or "CDR" is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ("antigen contact point"). The CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus. The CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987) ), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr/), and affinity propagation clustering based on the use of a large number of crystal structures ) North CDR definition.

Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.

The term "murine antibody" in this disclosure refers to an antibody prepared using a mouse according to the knowledge and skill in the art. In preparation, test subjects are injected with the specified antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.

The term "chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the mouse variable region gene It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally expresses the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.

The term "humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), refers to the antibody variable region framework grafted with mouse CDR sequences to humans, that is, different types of human germline antibodies Antibodies generated in the framework sequences. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.

The term "fully human antibody", "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", the variable region and constant region of the antibody are all human, and the immunogen sex and side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The present disclosure is a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

The term "drug loading" can be expressed as the ratio of the amount of drug to the amount of antibody. The range of drug loading can be 1-20, preferably 1-10 cytotoxic drugs (D) attached to each antibody (Ab); more preferably 2-4.

In the present disclosure, "solvation" refers to the phenomenon that some solvent molecules are surrounded by solute molecules or ions to form a group.

The compounds of the invention may also be provided in the form of salts. These salts can be formed using commonly performed methods. Or, depending on the conditions of the production method of the present invention, the compound (for example, a substance derived from an additive) can be produced in the form of a salt.

The compounds of the invention may also be provided in the form of solvates. For example, a solvate with water is mentioned. Such solvates can be formed using commonly performed methods. Alternatively, depending on the conditions of the production method of the present disclosure, the compound can be produced in the form of a solvate. Furthermore, salts of the above-mentioned compounds may be produced and provided in the form of solvates.

The term "treatment" used in the context of the present disclosure refers to: after suffering from a disease, contacting (for example, administering) a compound of the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer thereof of a subject , tautomers, isotope labels, prodrugs, or pharmaceutical compositions containing them (hereinafter also referred to as "pharmaceutical compositions of the present disclosure"), thereby reducing the symptoms of the disease compared to when not in contact , does not mean that the symptoms of the disease must be completely suppressed. Suffering from a disease means that the body has symptoms of a disease.

The term "prevention" used in the context of the present disclosure refers to: prior to suffering from a disease, by making a subject contact (for example, administer) a compound of the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer isomer, tautomer, isotopic label, prodrug or pharmaceutical composition of the present disclosure, thereby reducing the symptoms after suffering from the disease compared with no contact, it does not mean that the disease must be completely suppressed.

The term "individual", "patient" or "subject" as used in the context of this disclosure includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., , mice and rats).

The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors. The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to herein.

The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal carcinoma , hepatocellular carcinoma, gastric cancer (including gastrointestinal and gastrointestinal stromal carcinoma), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colorectal cancer , endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic Myeloblastic leukemia, and post-transplantation lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome , brain tumors and cancers, and head and neck cancers, and related metastases.

The term "anti-tumor effect" refers to a biological effect that can be exhibited by various means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.

The term "effective amount" refers to an amount or dose of a compound or composition of the present disclosure which, when administered to a patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors: the species of the animal, such as a mammal; its size, age and general health; the particular disease involved; the extent or severity of the disease the individual patient's response; the particular antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.

The term "pharmaceutically acceptable salt" as used in the context of the present disclosure refers to salts prepared from compounds of the present disclosure with relatively non-toxic acids or bases. When the compounds of the present disclosure contain relatively acidic functional groups (such as carboxyl or sulfonic acid groups), base addition salts can be obtained by contacting their free forms with a sufficient amount of base in neat solution or in a suitable inert solvent . Non-limiting examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, ammonium, calcium, magnesium, organic amine, or similar salts. When the compounds of the present disclosure contain relatively basic functional groups (such as amino or guanidino), acid addition salts can be obtained by contacting their free forms with a sufficient amount of acid in neat solution or in a suitable inert solvent . Non-limiting examples of pharmaceutically acceptable acid addition salts include, but are not limited to, inorganic acid salts (e.g., hydrochloride, hydrobromide, hydroiodide, nitrate, carbonate, bicarbonate, phosphate , monohydrogen phosphate, dihydrogen phosphate, phosphite, sulfate, bisulfate, etc.), organic acid salts (such as acetate, propionate, isobutyrate, malonate, succinate , suberate, maleate, fumarate, citrate, tartrate, lactate, mandelate, benzoate, phthalate, methanesulfonate, benzenesulfonate salt, p-toluenesulfonate, glucuronic acid, etc.) and amino acid salts (such as arginine salt, etc.). Specific forms of pharmaceutically acceptable salts can also be found in Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66: 1-19).

The term "pharmaceutical composition" as used in the context of this disclosure refers to a pharmaceutically acceptable composition comprising one or more compounds or a pharmaceutically acceptable form thereof (e.g. salt, hydrate, solvate, stereoisomeric isomers, tautomers, metabolites, prodrugs, etc.), and other components (such as pharmaceutically acceptable excipients).

The term "pharmaceutically acceptable excipient" used in the context of the present disclosure refers to an auxiliary material widely used in the field of pharmaceutical production. The main purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature and/or has specific functionality, and also to provide a method so that after the drug is administered to the subject, the active ingredient can be used in the desired manner. The rate of dissolution, or the promotion of effective absorption of the active ingredient in the subject to which it is administered. Pharmaceutically acceptable excipients can be inert fillers, or functional ingredients that provide certain functions for the pharmaceutical composition (such as stabilizing the overall pH value of the composition or preventing the degradation of active ingredients in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifying agents, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adherents , glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, coloring agents, flavoring agents, sweeteners, etc.

The pharmaceutical compositions of the present disclosure can be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, milling, encapsulating, entrapping and/or lyophilizing processes.

In the present disclosure, the purpose of using the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients, and then exert biological activity. The pharmaceutical compositions of the present disclosure may be administered by any form, including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid formulation, oral liquid formulation), rectal, inhalation, implantation , topical (eg ocular) administration, and the like. Non-limiting examples of oral solid formulations include, but are not limited to, powders, capsules, lozenges, granules, tablets, and the like. Non-limiting examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, tinctures, elixirs, solutions, and the like. Non-limiting examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum formulations. Non-limiting examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry powder for injection, suspension for injection, emulsion for injection, and the like. The pharmaceutical compositions of the present disclosure can also be formulated as controlled or delayed release dosage forms (eg, liposomes or microspheres).

In this disclosure, the methods of administration can be varied or adjusted in any suitable manner to meet the needs of the nature of the drug, the convenience of the patient and medical staff, and other relevant factors.

The term "Dxd" used in the context of this disclosure, also known as Exatecan derivative, is a DNA topoisomerase I inhibitor, which has the following structure:

In the present disclosure, when L ₁ is located in the compound represented by formula (I), L ₁ is (Gly) _p -(Z ₁ ) _a -, and the glycine residue at the amino terminal in L ₁ is NH ₂ - _CH2 -C(=O)-.

In the present disclosure, when L ₁ is located in the antibody drug conjugate represented by formula (III), L ₁ is -(Gly) _p -(Z ₁ ) _a -, and the glycine residue at the amino terminal of L ₁ is The group is -NH-CH ₂ -C(=O)-.

Preparation method of antibody drug conjugate:

The antibody drug conjugates of the present disclosure can be prepared by any method known in the art. In some embodiments, the conjugate is prepared by enzyme-catalyzed specific binding of the antibody or antigen-binding fragment to the compound represented by formula (I), wherein the antibody or antigen-binding fragment contains a recognition sequence of the ligase. Exemplarily, the ligase is a transpeptidase, which includes but not limited to various natural Sortase enzymes (including Sortase of A, B, C, D, L. plantarum, etc.) and various novel transpeptidases that have been preferably modified. The reaction is realized by means of biological enzyme catalysis (refer to WO2015165413A1, which is incorporated herein by reference), the reaction conditions are mild, the physical and chemical damage to the antibody during the coupling process is reduced, the preparation process and process are more optimized, and it is easy to upgrade industrialization. Conducive to the quality control of ADC products.

In some embodiments, the present disclosure provides a compound of formula (I), which has the following structure:

Wherein, L ₁ is (Gly) _p -(Z ₁ ) _a -, p is any integer of 1-10; a is any integer of 0-20, if exists, each Z ₁ is independently any kind amino acid residues;

L ₂ is -NH-R ₁ -(CH ₂ ) _c -C(=O)-, wherein R ₁ is -(CH ₂ -CH ₂ -X) _b -, b is any integer from 1 to 10, each Each X is independently -CH ₂ -, -NH-, -O- or -S-, and c is any integer from 1 to 5;

L ₃ is

or

L ₃ is wherein m is any integer from 1 to 5, preferably 2; R ₂ is -(CH ₂ -CH ₂ -Y) _d -, d is any integer from 1 to 10, and each Y is independently -CH ₂ -, -NH-, -O- or -S-; the carboxyl terminal of _L1 is connected to the amino terminal of _L2 , and the carboxyl terminal of _L2 is connected to the amino terminal of _L3 .

Further, L ₁ is (Gly) _p -, p is any integer of 1-10, preferably any integer of 3-5.

Further, L ₂ is -NH-R ₁ -(CH ₂ ) _c -C(=O)-; wherein R ₁ is -(CH ₂ -CH ₂ -X) _b -, b is any of 1-10 Integer, preferably any integer of 1-5; each X is independently -CH ₂ - or -O-; c is any integer of 1-5, preferably any integer of 1-2.

Further, L ₃ is selected from any one of the following:
or

In some more specific embodiments,

L ₁ -L ₂ -for

Wherein, w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2; b is any integer of 1-5 Integer, preferably any integer of 2-4, each X is independently -CH ₂ - or -O-; and,
The structure is:

In some more specific embodiments,

L ₁ -L ₂ -for

Wherein, w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2; b is any integer of 1-5 Integer, preferably any integer of 2-4, each X is independently -CH ₂ - or -O-; and,
The structure is:

In some specific embodiments, the compound represented by formula (I) has the structure shown in any one of the following:

A structure as shown in any of the following is preferred:

The method includes step A and step B.

Step A: prepare the compound shown in formula (I)

In some preferred embodiments, in a preferred embodiment, the compound represented by formula (I) is formed by covalently linking a linker with eribulin.

The compound represented by formula (I) has a definite structure, definite composition and high purity, so when it is coupled with an antibody, less or no other impurities are introduced. Under the catalysis of ligase, when the compound represented by formula (I) is used to react with the antibody or antigen-binding fragment with the recognition sequence of ligase at a specific site, a highly controllable homogeneous ADC.

Step B: linking the target molecule with the compound represented by formula (I)

The target molecule in the present disclosure can be combined with the compound represented by formula (I) by any method known in the art to obtain the antibody drug conjugate represented by formula (III).

The target molecule and the compound represented by the formula (I) are connected to each other through the specific recognition sequence of the ligase of the substrate. The recognition sequence depends on the particular ligase used. In some embodiments, the target molecule is an antibody or an antigen-binding fragment, and a recognition-sequence-based terminal modification is introduced at the c-terminus (carboxyl-terminus) of the light chain and/or heavy chain, in wild-type or optimized engineered ligase or Under the catalysis of any combination thereof, the target molecule is combined with the compound represented by formula (I).

In some specific embodiments, the ligase is a Sortase enzyme. More specifically, the ligase is Sortase A enzyme.

Exemplary, the binding reaction can be represented by the following scheme:

The triangle represents a part of the antibody, and the carboxy-terminal of the antibody is connected to the recognition sequence LPXT(G) _r of the Sortase A enzyme; wherein, L is leucine, P is proline, X is any type of amino acid, T is threonine, (G) _r is glycine in quantity r, and r is any integer of 1-2. The pentagon represents a part of the compound of formula (II), (G) _p is glycine with a quantity of p, and p is any integer from 1 to 10. Exemplarily, p is 1, 2, 3, 4, 5, etc. .

Sortase A enzyme nucleophilic attack The peptide bond between threonine (T) and glycine (G) in the LPXT(G) _r sequence in A covalent thio intermediate is formed. This intermediate can capture Glycine linker (G) _p in , and re-form an amide bond between threonine (T) and glycine (G), release the Sortase A enzyme, and obtain an antibody drug in which the target molecule is combined with the compound represented by formula (I) Conjugate

The present disclosure will be described in detail below. However, this disclosure may be embodied in many different forms, and it should not be limited to the embodiments described herein, which are provided so that this disclosure will be more complete and comprehensive. All the reagents and raw materials used are commercially available except those provided for the preparation methods. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

Amino acids and their abbreviations and English abbreviations in this disclosure are shown in the following table:

Table 1

The laboratory reagents used in the present invention are all from commercial purchases, and the purity is all analytical pure. The abbreviations and full Chinese names of the reagents used in the synthesis process are shown in the table below:

Table 2

Example

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given for illustrative purposes only, since, upon reading this detailed description, all further changes will be made within the spirit and scope of the disclosure. Various changes and modifications will become apparent to those skilled in the art.

All reagents and materials used in this disclosure were purchased commercially unless otherwise noted.

The synthesis of embodiment 1 BP182a

Compound BP182a has the structure shown in the following formula (I-5):

The synthetic steps of compound BP182a are as follows:

Step 1, synthesis of 1186k: 1186k was synthesized by Fmoc solid-phase synthesis method, using Rink Amide MBHA Resin as a solid-phase carrier, using 20% piperidine/DMF (v/v) solution to remove Fmoc protection, and then using HOBT/DIC as Condensation system, DMF as the reaction solvent, sequentially coupled Fmoc-Cys(Trt)-OH, Fmoc-NH-PEG4-PA, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, where Fmoc-NH -PEG4-PA is condensed with HBTU/DIEA system, then cracked by TFA:TIS:H ₂ O=95:2.5:2.5, precipitated by methyl tert-butyl ether, filtered and dried to obtain 1186k crude product, which is finally subjected to high performance liquid chromatography Purification, desalination, concentration, freeze-drying, beating with methyl tert-butyl ether, and drying to obtain 1186k pure product.

Step 2, synthesis of BP102c05 and BP102c: for the synthesis of BP102c05 and BP102c, refer to the synthesis method of EP0624377A2, which is incorporated into this application by reference;

Step 3, the synthesis of compound BP182a11: add 687mg (1.2eq) BP102c, 565mg (1.0eq) eribulin, 6ml DMF to the flask, stir and dissolve, add 162ul (1.2eq) DIEA, TLC monitors the reaction after the completion of the reaction The solution was poured into TBME, the solid precipitated out, stirred and beaten, filtered, the filter cake was washed with TBME, the filter cake was collected, and vacuum-dried to obtain 1021 mg of off-white solid, namely BP182a11, the sample was directly carried out to the next step reaction without purification.

Step 4, the synthesis of compound BP182a: add 402mg (1.0eq) BP182a11, 16ml methanol to the flask, stir to dissolve; weigh 236mg (1.2eq) 1186k, dissolve it with 1ml purified water, and adjust the pH to 5 with sodium bicarbonate solution -6, and then added to the reaction bottle of BP182a11. After 30 minutes, the reaction was monitored by HPLC. The reaction solution was not treated, but was prepared and purified by reverse high performance liquid phase, and the pure product was collected and freeze-dried to obtain 166 mg of white solid, namely BP182a. LC/MS (m/z): calcd for C ₈₉ H ₁₃₅ N ₁₃ O ₂₈ S 1865.93; found 1867.00 [M+H] ⁺ , 934.10 [M+2H] ²⁺ , 623.30 [M+3H] ³⁺ .

The synthesis of embodiment 2 BP182d

Compound BP182d has the structure shown in the following formula (I-1):

The synthetic steps of compound BP182d are as follows:

Step 1, the synthesis of BP182d01: synthesized by Fmoc solid-phase synthesis method, using 2Cl Trt Resin as a solid-phase carrier, using 20% piperidine/DMF (v/v) solution to remove Fmoc protection, and then using HOBT/DIC as a condensation system , DMF as the reaction solvent, sequentially coupled Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-AEEA-OH, Fmoc-Gly-OH, Fmoc-Gly-OH , Fmoc-Gly-OH, and then cleaved by 2% TFA/DCM solution, precipitated by methyl tert-butyl ether, centrifuged, and dried to obtain BP182d01, which was directly carried out to the next step without purification.

Step 2, synthesis of BP182d02: add 501mg (1.0eq) BP182d01, 415mg (1.0eq) eribulin, 10ml DCM, 4ml DMF to the flask, stir to dissolve and cool to 5±5°C, add 237ul (2.5eq) DIEA, add 140ul (1.5eq) DEPC, rise to room temperature to react, after TLC monitors the reaction, concentrate the reaction solution in vacuo to remove DCM, then add TBME, stir to make a slurry, filter, wash the filter cake with TBME, collect the filter cake, and vacuum dry to obtain 1.023 g of off-white solid was directly carried out to the next reaction without further purification.

Step 3, synthesis of BP182d: add 1000mg BP182d02, 7ml methanol, 7ml THF to the flask, stir to dissolve; weigh 300mg LiOH.H ₂ O, dissolve it with 10ml purified water, add it dropwise to the reaction flask, adjust the pH to 12 After the reaction was monitored by TLC, acetic acid was added to quench the reaction, the reaction solution was concentrated in vacuo to remove the organic solvent, and the crude product of BP182d was obtained. The crude product was prepared and purified by forward high performance liquid phase, and the pure product was collected and concentrated to dryness to obtain 195 mg of off-white solid, namely BP182d . LC/MS (m/z): calcd for C ₆₇ H ₉₇ N ₉ O ₂₁ 1363.68; found 1364.70 [M+H] ⁺ , 682.85 [M+2H] ²⁺ , 1362.75 [MH] ⁻ .
Embodiment 4: Preparation of antibody drug conjugate

1. Production, purification and identification of anti-human ErbB2/Her2 antibody:

The carboxy-terminal of the heavy chain of antibody Ab0100 is connected with GALPETGG (hereinafter referred to as Ab0100-HCCT _L ), and its amino acid sequence is shown in SEQ ID NO: 16; the carboxy-terminal of the light chain of antibody Ab0100 is connected with GALPETGG (hereinafter referred to as Ab0100-LCCT _L ), the amino acid sequence of which is shown in SEQ ID NO:15. Therefore, a total of 4 ligase recognition sequences were introduced into antibody Ab0100. The synthesis of antibody Ab0100 can refer to WO1989006692A1, and WO1989006692 is incorporated into the present disclosure by reference.

Table 3 Sequence information of antibody Ab0100 light chain

Table 4 Sequence information of antibody Ab0100 heavy chain

2. To couple the antibody with the compound represented by formula (I-1) or formula (I-5), the method of coupling reaction can refer to the method in WO2015165413A1, as follows:

(1) Using the compound represented by BP182a as an intermediate and the above-mentioned anti-human ErbB2/Her2 as an antibody, the ADC prepared by enzymatic linking with Sortase A is named Ab0100-H0037. The specific structure of Ab0100-H0037 is shown in the following formula (III-5):

(2) Using the compound represented by BP182d as an intermediate and the above-mentioned anti-human ErbB2/Her2 as an antibody, the ADC prepared by enzymatic linking with Sortase a is named Ab0100-H0038. The specific structure of Ab0100-H0038 is shown in the following formula (III-1):

Embodiment 5: Synthesis of LB302-2-4

This example prepares the compound LB302-2-4 shown in the following structure:

The preparation steps of compound LB302-2-4 are as follows:

Step 1, preparation of linker HX20111:

HX20111 was synthesized by traditional solid-phase peptide synthesis method with Rink-amide-MBHA-resin. Amino acids in the linker unit were protected with Fmoc. Coupling reagents are selected from HOBT, HOAt/DIC, DCC, EDCI or HATU. After synthesis, the resin was cleaved with trifluoroacetic acid. The product was purified by high performance liquid chromatography and stored after freeze-drying. Theoretical mass: 1383.70, measured: [MH] ^- = 1382.6.

Step 2, preparation of compound LB302-2-4:

Weigh HX20111 and the intermediate MC-GGFG-Dxd (molar ratio ~1:2) and dissolve them in water and DMF respectively, mix thoroughly to obtain a mixture, and react at 0-40°C for 0.5-30h. After the reaction is completed, directly add an appropriate amount of Tris Base solution or other solutions that promote the ring-opening reaction. solution, and then react at 0-40°C for 0.2-20h. After the reaction was completed, the product was purified by semi-preparative/preparative HPLC and lyophilized to obtain the linker-payload intermediate LB302-2-4. Theoretical mass: 3486.52, measured: [(M+3H)/3] ⁺ = 1163.3.

Example 6: Preparation of Antibody Drug Conjugate BQ3

Using the method shown in Example 4, the compound LB302-2-4 was coupled to the anti-human ErbB2/Her2 antibody Ab0001, and the obtained ADC information is shown in the table below. Wherein, the amino acid sequence of the heavy chain of antibody Ab0001 is shown in SEQ ID NO: 19, and the carboxy-terminal of the light chain of antibody Ab0001 is connected with GALPETGG (hereinafter referred to as Ab0001-LCCT _L ), and its amino acid sequence is shown in SEQ ID NO: 15 .

table 5

Amino acid information of heavy chain and light chain of antibody Ab0001 of table 6

Example 7: In vitro experiments verify the cytotoxicity of ADC

1.1 Materials and equipment

The materials and equipment used in this experiment example are shown in the table below.

Table 7

1.2 Experimental steps

1.2.1 Cell plating

(1) Microscopic examination of cells;

(2) cleaning;

(3) digestion;

(3) centrifugal;

(4) cell count;

(5) Cell plating: 5000 cells/well for NCI-N87 and SK-BR-3, 4000 cells/well for HCC1954 and MDA-MB-468.

1.2.2 Cell administration

(1) Microscopic examination

(2) Drug preparation:

a. Sample configuration buffer configuration: configure the required amount of sample configuration buffer in the culture medium (10% FBS) used for the administered cells;

b. Sample configuration: Dilute the sample according to the ratio of the concentration required for the first concentration, and configure according to the following table in a 24-well plate or a 96-well plate;

Table 8

(3) Dosing: Drugs of different concentrations were added sequentially from low concentration to high concentration, with 3 replicate wells for each concentration, 100 μl/well, among which the puro group had 3 replicate wells, 100 μl/well.

(4) After adding the drug, the cells were moved to the incubator to continue culturing for 120 h. Culture conditions: 37 degrees, CO ₂ incubator.

1.2.3 Cell Viability Detection

(1) Detection reagent preparation: Equilibrate the CellTiter-Glo Luminescent Cell Viability Assay detection reagent to room temperature in the dark in advance.

(2) Cell preparation: take the cells to be tested out of the incubator, and equilibrate at 25° C. for 30 minutes.

(3) ATP detection: Discard the medium, add 100 μl/well DMEM, 50 μl/well CTG into a 96-well plate, store in the dark with aluminum foil, vortex shaker at 200 rpm for 15 minutes at room temperature (note: enzyme labeling can be performed at this time) instrument program settings).

(4) Detection procedure: the gain value is 135. After setting the procedure, remove the cover of the black wall bottom plate and place it in the way specified by the instrument.

(5) Data storage

1.3 Experimental results

Figure 1 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, and small molecule drugs Eribulin and Dxd on human breast cancer cell HCC1954. Among them, the _IC50 value of each drug molecule is shown in the table below:

Table 9

Figure 2 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, small molecule drugs Eribulin and Dxd on human breast cancer cell SK-BR-3. Among them, the _IC50 value of each drug molecule is shown in the table below:

Table 10

Figure 3 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, and small molecule drugs Eribulin and Dxd on human gastric cancer cell NCI-N87. Among them, the _IC50 value of each drug molecule is shown in the table below:

Table 11

Figure 4 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, and small molecule drugs Eribulin and Dxd on human breast cancer cell line MDA-MB-468. Among them, the _IC50 value of each drug molecule is shown in the table below:

Table 12

As shown in Figures 1-3, Ab0100-H0037 and Ab0100-H0038 can significantly reduce the in vitro survival rate of various types of tumor cells that are HER2 positive (for example, HCC1954, SK-BR-3 and NCI-N87), exerting high-efficiency tumor cell The killing activity indicates that the antibody-drug conjugate prepared in the present disclosure has an effective killing effect on HER2-positive tumor cells. As shown in Figure 4, in HER2-negative cells, the cytotoxicity of Ab0100-H0037 and Ab0100-H0038 was lower than that of the small molecule drug eribulin.

The cytotoxicity of Ab0100-H0037 and Ab0100-H0038 to HER2-positive cells HCC1954, SK-BR-3, and NCI-N87 is better than that of eribulin, and significantly better than that of Dxd, indicating that the linker coupling through a special structure in this disclosure The ADC drug obtained from the antibody and the cytotoxic drug eribulin has an improved killing effect on tumor cells. Further, Ab0100-H0037 and Ab0100-H0038 The cytotoxicity in HER2-positive cells is significantly better than that in HER2-negative cells, indicating that Ab0100-H0037 and Ab0100-H0038 can target cells with HER2-positive expression and enter the cells, releasing cytotoxicity in cells Eribulin, a drug, achieves specific and efficient killing of HER2-positive cells, and has good targeting and safety.

Example 8: In vivo experiments verify the activity and toxicity of ADC

2.1 Experimental animals:

Commercially purchased BALB/c nude mice were raised under constant temperature and humidity conditions.

2.2 Experimental methods and steps

2.2.1 Cell culture

Capan-1 tumor cells were preserved in vitro in IMDM medium supplemented with 20% fetal calf serum and 1% antibiotic-antimycotic at 37°C in an atmosphere of 5% _CO2 (ATCC, catalog number HTB-79 ). Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Cells in exponential growth phase were harvested and counted for tumor inoculation.

2.2.2 Tumor inoculation and animal grouping

Capan-1 tumor cells (5×10 ⁶ ) in a mixture of 0.2 mL of PBS and Matrigel (PBS:Matrigel=1:1) were inoculated subcutaneously on the right side of each mouse for tumor development. Treatment was initiated on day 5 after tumor inoculation when the average tumor volume reached 187 mm ³ . Animals were allocated into groups using Excel-based randomization software that stratified randomization based on the animals' tumor volume. Each group consists of 6 tumor-bearing mice, and the mice are given the test substance.

2.2.3 Preparation of the test article

The preparation method of the test sample is shown in the table below:

Table 13

Note: It is prepared and prepared before administration.

2.2.4 Tumor Measurements and Endpoints

The primary endpoint was to determine whether tumor growth could be delayed or whether the mice could be cured. Tumor volume measurements were performed in two dimensions using vernier calipers, twice a week, and the volume was expressed in mm ³ using the following formula: V=0.5a×b ² , where a and b are the long and short diameters of the tumor, respectively .

2.3 Experimental results and discussion

2.3.1 Mortality, morbidity and weight gain or loss

Animal body weights were monitored regularly as an indirect measure of toxicity.

Figure 5 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (Capan-1 tumor-bearing female BALB /c nude mice) body weight change detection results. Wherein, in Fig. 5, the calculated change based on the body weight of the animals on the first day of administration is shown in percentage change (%) of body weight; the data points represent the percentage group mean change of BW. Error bars represent standard error of the mean (SEM). Figure 5 shows that during the experiment, no significant weight loss, no death and no disease occurred in all groups of the model, and Ab0100-H0037 and Ab0100-H0038 did not show obvious drug toxicity in animal experiments.

2.3.2 Tumor volume

The mean tumor volume over time in Capan-1 xenograft tumor-bearing female BALB/c nude mice administered Ab0100-H0037, Ab0100-H0038 and BQ3 is shown in FIG. 6 . The entire study was terminated on day 42 under experimental conditions when the mean tumor volume in the vehicle group reached 1,420 mm ³ .

2.3.3 Tumor growth inhibition analysis

Based on the tumor volume measurements on day 42, the tumor growth inhibition of Ab0100-H0037, Ab0100-H0038 and BQ3 on mice inoculated with Capan-1 tumors was calculated, and the results are shown in Table 14 below:

Table 14

Note:

1. Mean ± SEM;

2. The relative T/C of each group was calculated using the following formula: T/C %=T _RTV /C _RTV ×100% (T _RTV : RTV of the treatment group; C _RTV : RTV of the control group on the same day). RTV=V ₄₂ /V ₀ , where V ₀ is the average tumor volume on the first day of treatment, and V ₄₂ is the average tumor volume on the 42nd day after the start of treatment;

3. TGI(%)=[1-(T ₄₂ -T ₀ )/(V ₄₂ -V ₀ )]×100%;

4. Calculation of p-values based on tumor volume.

From the results in Table 14, it can be seen that Ab0100-H0037, Ab0100-H0038 and BQ3 can significantly inhibit tumor growth in tumor-bearing mice, and achieve efficient and specific killing of tumor cells in vivo.

2.3.4 Tumor growth curve

Figure 6 shows the tumor growth curves of tumor-bearing mice after administration of vehicle (DPBS), Ab0100-H0037 (5 mg/kg), Ab0100-H0038 (5 mg/kg), and BQ3 (5 mg/kg).

In this experimental example, the drug toxicity and tumor therapeutic effect of Ab0100-H0037, Ab0100-H0038 and BQ3 were evaluated in a tumor-bearing mouse model subcutaneously inoculated with human pancreatic cancer cell Capan-1. The results showed that the average tumor volume of the vehicle control group reached 1,420 mm ³ on the 42nd day after the start of treatment. All treatments with 5 mg/kg Ab0100-H0037, Ab0100-H0038 and BQ3 significantly inhibited Capan-1 tumor growth. On day 42, Ab0100-H0037 5mg/kg (T/C=10.59%, TGI=102.98%, p=0.025), Ab0100-H0038 5mg/kg (T/C=0.75%, TGI=114.29%, p= 0.018) and BQ3 5 mg/kg (T/C=2.19%, TGI=112.62%, p=0.019) groups had mean tumor volumes of 150, 11 and 31 mm ³ , respectively. All tested products, including Ab0100-H0037, Ab0100-H0038 and BQ3, were well tolerated in Capan-1 tumor-bearing BALB/c nude mice. No significant mean body weight loss was observed in any treatment group.

Experimental Example 9: In vivo experiments to verify the activity and toxicity of ADC

3.1 Experimental methods and steps:

Using the same experimental conditions as in Experimental Example 2, the antitumor efficacy of Ab0100-H0037, Ab0100-H0038 and BQ3 in the subcutaneous NCI-N87 human gastric cancer xenograft model in female BALB/c nude mice was studied.

The subcutaneous NCI-N87 human gastric cancer xenograft model in female BALB/c nude mice was established as follows: in air containing 5% _CO2 at 37°C in RPMI supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic NCI-N87 tumor cells (ATCC, Manassas, VA, catalog number CRL-5822) were maintained in vitro as monolayer cultures in -1640 medium. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Cells in exponential growth phase were harvested and counted for tumor inoculation.

Tumor inoculation and animal grouping:

NCI-N87 tumor cells (10×10 ⁶ ) in 0.2 mL PBS supplemented with Matrigel (1:1) were inoculated subcutaneously on the right side of each mouse for tumor development. Treatment was initiated on day 8 after tumor inoculation when the mean tumor volume reached approximately 188 mm ³ . Animals were allocated into groups using Excel-based randomization software that stratified randomization based on the animals' tumor volume. Each group consists of 6 composed of tumor-bearing mice. Referring to Experimental Example 2, the test article was administered to tumor-bearing mice.

3.2 Experimental results:

3.2.1 Tumor growth curve

Figure 7 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (NCI-N87 tumor-bearing female BALB /c tumor growth curve of nude mice).

The results showed that: in the tumor-bearing mice administered with 5mg/kg Ab0100-H0037, 5mg/kg Ab0100-H0038 and 5mg/kg BQ3, the growth of NCI-N87 tumors was significantly inhibited; -N87 tumor-bearing BALB/c nude mice finally had the smallest tumor volume.

Experimental example 10: In vivo experiment to verify the activity and toxicity of ADC

4.1 Experimental method and steps:

Using the same experimental conditions as in Test Example 2, the anti-tumor efficacy of Ab0100-H0037, Ab0100-H0038 and BQ3 in the subcutaneous HCC1954 human breast cancer xenograft model in female BALB/c nude mice was studied.

The subcutaneous HCC1954 human breast cancer xenograft model in female BALB/c nude mice was established as follows: Culture in RPMI 1640 supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C and 5% _CO2 in the air HCC1954 tumor cells (ATCC, Manassas, VA, catalog number CRL-2338) were preserved in vitro. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Cells in exponential growth phase were harvested and counted for tumor inoculation.

Tumor inoculation and animal grouping: subcutaneously inoculate the right side of each mouse with HCC1954 tumor cells (5×10 ⁶ ) in a mixture of 0.2 mL of PBS and Matrigel (PBS:Matrigel=1:1) for tumor development. Treatment was initiated on day 13 after tumor inoculation, when the mean tumor volume reached 177 mm ³ . Animals were allocated into groups using Excel-based randomization software that stratified randomization based on the animals' tumor volume. Each group consisted of 6 tumor-bearing mice. Referring to Experimental Example 2, the test article was administered to tumor-bearing mice.

4.2 Experimental results:

Figure 8 shows vehicle (DPBS), Ab0100-H0037 (3mg/kg), Ab0100-H0038 (3mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (HCC1954 tumor-bearing female BALB/c Tumor growth curve of nude mice).

The results showed that: in the tumor-bearing mice administered with 3mg/kg Ab0100-H0037, 3mg/kg Ab0100-H0038 and 5mg/kg BQ3, the growth of HCC1954 tumors was significantly inhibited; and, 3mg/kg Ab0100-H0038 and 5mg/kg NCI-N87 tumor-bearing BALB/c nude mice administered with kg BQ3 finally showed complete inhibition of tumor growth.

All the technical features of the invention in this specification can be combined in any combination. Each feature of the invention described in this description may also be replaced by other features having the same, equivalent or similar effect. Thus, unless stated otherwise, each feature disclosed is only one example of a series of equivalent or similar features.

In addition, from the above description, those skilled in the art can easily understand the key features of the present disclosure from the present disclosure. Without departing from the spirit and scope of the present disclosure, many modifications can be made to the invention to adapt to various purposes and conditions of use, and therefore such modifications are also intended to fall within the scope of the appended claims.

Claims (15)

1. An antibody drug conjugate, which has the structure shown in the following formula (III):
   

   Wherein, q is any integer of 2-8, preferably 4; Ab is an antibody or an antigen-binding fragment;

   Pep is -(X ₁ ) _e -Leu-Pro-X ₂ -Thr-, wherein e is any integer from 0-10, if present, each X ₁ is independently Gly or Ala; X ₂ is any species amino acid residues;

   L ₁ is -(Gly) _p -(Z ₁ ) _a -, p is any integer of 1-10; a is any integer of 0-20, if present, each Z ₁ is independently any kind of amino acid residues;

   L ₂ is -NH-R ₁ -(CH ₂ ) _c -C(=O)-, wherein R ₁ is -(CH ₂ -CH ₂ -X) _b -, b is any integer from 1 to 10, each Each X is independently -CH ₂ -, -NH-, -O- or -S-, and c is any integer from 1 to 5;

   L ₃ is

   Alternatively, _L3 is wherein m is any integer from 1 to 5, preferably 2; R ₂ is -(CH ₂ -CH ₂ -Y) _d -, d is any integer from 1 to 10, and each Y is independently -CH ₂ -, -NH-, -O- or -S-;

   And, the amino terminus of Pep is connected to the carboxyl terminus of the heavy chain or light chain of Ab, the carboxyl terminus of Pep is connected to the amino terminus of _L1 , the carboxyl terminus of _L1 is connected to the amino terminus of _L2 , and the carboxyl terminus of _L2 is connected to the carboxy terminus of _L3 . amino terminus.
2. The antibody drug conjugate according to claim 1, wherein,

   Pep is -(Gly-Ala) _f -Leu-Pro-X ₂ -Thr-, wherein f is any integer of 1-5, preferably 1; X ₂ is selected from any kind of amino acid residues, preferably Glu residues ;

   Preferably, Pep is -Gly-Ala-Leu-Pro-Glu-Thr-, wherein the amino terminal of Pep is connected to Ab, and the carboxyl terminal of Pep is connected to the amino terminal of L1.
3. The antibody-drug conjugate according to claim 1 or 2, wherein the carboxy-terminal of the heavy chain of Ab is connected to Pep, and the carboxyl-terminal of the light chain of Ab is connected to Pep;

   Preferably, the carboxy terminus of the heavy chain of the Ab is linked to -Gly-Ala-Leu-Pro-Glu-Thr-, and the carboxy terminus of the light chain of the Ab is linked to -Gly-Ala-Leu-Pro-Glu-Thr-.
4. The antibody-drug conjugate according to any one of claims 1-3, wherein the antibody or antigen-binding fragment specifically binds to at least one protein in the HER protein family; preferably, the antibody or The antigen-binding fragment specifically binds to at least one of the following proteins: HER2, HER3.
5. The antibody-drug conjugate according to any one of claims 1-4, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises such as SEQ ID NO : 9. The amino acid sequence shown in at least one of SEQ ID NO: 11 and SEQ ID NO: 13; and/or, the antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain is variable The region comprises an amino acid sequence as shown in at least one of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; the light chain variable region and the heavy chain variable region are analyzed according to Kabat method encoding;

   Preferably, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3;

   HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 13;

   LCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 4, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 6;

   More preferably, the amino acid sequence of the heavy chain of the antigen or antigen-binding fragment is as shown in SEQ ID NO: 18, and/or, the amino acid sequence of the light chain of the antigen or antigen-binding fragment is as shown in SEQ ID NO: 17 Show.
6. The antibody drug conjugate according to any one of claims 1-5, characterized in that,

   L ₁ is -(Gly) _p -, p is any integer of 1-10, preferably any integer of 3-5;

   L ₂ , L ₃ are as defined in claim 1 .
7. The antibody-drug conjugate according to any one of claims 1-5, wherein _L3 is selected from any one of the following:
   

   L ₁ , L ₂ are as defined in claim 1 .
8. The antibody drug conjugate according to any one of claims 1-5, characterized in that,

   L ₂ is -NH-R ₁ -(CH ₂ ) _c -C(=O)-;

   Wherein R ₁ is -(CH ₂ -CH ₂ -X) _b -, b is any integer of 1-10, preferably any integer of 1-5; each X is independently -CH ₂ - or - O-; c is any integer of 1-5, preferably any integer of 1-2;

   L ₁ , L ₃ are as defined in claim 1 .
9. The antibody drug conjugate according to any one of claims 1-8, characterized in that,

   -L ₁ -L ₂ -for

   Wherein, w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2; b is any integer of 1-5 Integer, preferably any integer of 2-4, each X is independently -CH ₂ - or -O-; and,
   The structure is:
   
10. The antibody drug conjugate according to any one of claims 1-8, characterized in that,

    -L ₁ -L ₂ -for

    Wherein, w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2; b is any integer of 1-5 Integer, preferably any integer of 2-4, each X is independently -CH ₂ - or -O-; and,
    The structure is:
    
11. The antibody-drug conjugate shown in any one of the following formulas (III-1) to (III-6):
    
    

    A structure as shown in any of the following is preferred:
    
    

    Preferably, Ab is as defined in any one of claims 4-5.
12. A pharmaceutical composition comprising a therapeutically effective amount of the antibody-drug conjugate according to any one of claims 1-11;

    Optionally, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers.
13. Use of the antibody drug conjugate according to any one of claims 1-11, or the pharmaceutical composition according to claim 12 in the preparation of drugs for preventing and/or treating tumors;

    Optionally, the tumor is a tumor associated with the abnormal expression of one or two or more proteins in the HER protein family; preferably, the abnormally expressed protein is selected from at least one of HER2 and HER3;

    Optionally, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.
14. The antibody drug conjugate according to any one of claims 1-11, or the pharmaceutical composition according to claim 12, which is used for preventing and/or treating tumors;

    Optionally, the tumor is a tumor associated with the abnormal expression of one or two or more proteins in the HER protein family; preferably, the abnormally expressed protein is selected from at least one of HER2 and HER3;

    Optionally, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.
15. A method for preventing and/or treating tumors, comprising administering to a subject the antibody drug conjugate shown in any one of claims 1-11, or the pharmaceutical composition according to claim 12;

    Optionally, the tumor is a tumor associated with the abnormal expression of one or two or more proteins in the HER protein family; preferably, the abnormally expressed protein is selected from at least one of HER2 and HER3;

    Optionally, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.