Sacituzumab drug conjugate and its preparation

Abstract

The present invention provides an antibody-linker-drug conjugate in which the antibody is sacituzumab and the drug is an auristatin analog or a pharmaceutically acceptable salt thereof. The present invention further provides a method for preparing the antibody-linker conjugate of the present invention.

Description

[Technical Field]

[0001] The present invention provides an antibody-linker-drug conjugate in which the antibody targets a Trop-2 antigen, preferably the antibody is sacituzumab and the drug is an auristatin analog. [Background technology]

[0002] Antibody-drug conjugates (ADCs), composed of antibodies, linkers, and cytotoxic agents (drugs), are one of the most complex drug platforms in oncology medicine (armamentarium). [1] Antibody-drug conjugates are payload delivery systems and involve key variables influencing their success, including (i) the rate of payload internalization; (ii) the expression of the target antigen on tumor and normal tissues, which has implications for both patient selection and therapeutic index; (iii) the linker's chemical properties and the selection of chemical properties, potential in intracellular and extracellular stability; and (iv) the selection of the payload against tumor signs. [2] The mechanism of action of ADCs includes direct binding between the antibody and its targeted cell surface antigen, and intracellular or extracellular release of the cytotoxic agent. [3] The present invention provides a sacituzumab-derived antibody-drug conjugate that enables the specific delivery of a drug (aulistatin and / or its analog) to a target antigen on cancer cells. [Overview of the project] [Means for solving the problem]

[0003] The present invention provides an antibody-linker-drug conjugate in which the antibody is a trop-2 antigen-targeted antibody, preferably sacituzumab, and the drug is a suitable auristatin analog. The present invention provides higher efficacy than approved therapies in the field of trop-2 expressing cancers. The present invention further provides a method for preparing the antibody-linker-drug conjugate of the present invention. The present invention further targets antibody-linker-drug conjugates in the treatment of specific types of cancer, autoimmune diseases, or infections.

[0004] Abbreviated form: Ab: Antibody ADC: Antibody-drug conjugate or antibody-linker-drug conjugate HP-SEC: High-speed size exclusion chromatography HP-HIC: High-Speed ​​Hydrophobic Interaction Chromatography HMW: High molecular weight IRS: Internal Reference Standard LMW: low molecular weight MMAE: Monomethyl auristatin E MMAF: Monomethyl Auristatin F mc-vc-PABC: Maleimidocaproylvalinecitrulline paraaminobenzylcarbamate PAB: Para-aminobenzyl PABC: Para-aminobenzylcarbamate SDS PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis TCEP: Tris(2-carboxymethyl)phosphine

[0005] Embodiments of the present invention Embodiment 1: In one embodiment, the present invention is represented by formula (I): Ab-(Aa-Ww-Yy-D) p We provide an antibody-linker-drug conjugate (ADC) of the following formula, where the antibody (Ab) is a trop-2 targeted antibody, preferably sacituzumab; -Aa-Ww-Yy- is an enzymatically cleavable linker unit linking the drug unit and the antibody, where -A- is a stretcher unit; a is 1; each -W- is independently an amino acid unit; -Y- is a spacer unit; w is an integer ranging from 2 to 12; y is 1 or 2; p is ranging from 1 to about 20; and drug (D) has the following structure [ka] A suitable auristatin analog selected from MMAE and MMAF as defined by, In the formula, the wavy line represents the connection point to the linker.

[0006] In a further embodiment, the present invention relates to formula (I)Ab-(Aa-Ww-Yy-D) p The antibody-linker-drug conjugate is provided, wherein the antibody (Ab) is sacituzumab; -Aa-Ww-Yy- is an enzymatically cleavable linker unit linking the drug unit and the antibody, wherein -A- is a stretcher unit; a is 1; each -W- is an amino acid unit independently selected from -phenylalanine-lysine- or -valine-citrulline-; -Y- is a spacer unit selected from -glycine-glycine- or p-aminobenzyl alcohol (PAB) or p-aminobenzylcarbamate (PABC); w is an integer ranging from 2 to 12; y is 1 or 2; p is ranging from 1 to about 20; and drug (D) has the following structure [ka] Selected from MMAE and MMAF as defined by In the formula, the dashed line represents the connection point to the linker. A has the following structure [ka] Defined by, r is an integer ranging from 1 to 10; the carbonyl terminus of -A- forms a bond with the amino acid unit (W), and the succinimide terminus of -A- forms a bond with the antibody (Ab).

[0007] In a preferred embodiment, the present invention is [ka] We provide antibody-drug conjugates (ADCs) represented by, In the formula, mAb is a monoclonal antibody containing CDR1 (KASQDVSIAVA), CDR2 (SASYRYT), and CDR3 (QQHYITPLT) which are light chain variable region complementarity determining region (CDR) sequences, and CDR1 (NYGMN), CDR2 (WINTYTGEPTYTDDFKG), and CDR3 (GGFGSSYWYFDV) which are heavy chain variable region CDR sequences. In this embodiment, the antibody is trastuzumab.

[0008] In a preferred embodiment, the present invention provides an antibody-linker-drug conjugate (ADC) in which the antibody is a monoclonal antibody comprising light chain variable region complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA), CDR2(SASYRYT), and CDR3(QQHYITPLT), and heavy chain variable region CDR sequences CDR1(NYGMN), CDR2(WINTYTGEPTYTDDFKG), and CDR3(GGFGSSYWYFDV); the linker is an enzymatically cleavable linker unit mc-vc-PABC; the drug (D) is a suitable auristatin analog MMAE; and the monoclonal antibody (mAb) is attached to the drug portion between 4.2 and 4.4.

[0009] In a preferred embodiment, the present invention provides an ADC structure, [Chemical formula] Represented by, In the formula, mAb is a monoclonal antibody comprising light chain variable region complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA), CDR2(SASYRYT), and CDR3(QQHYITPLT), and heavy chain variable region CDR sequences CDR1(NYGMN), CDR2(WINTYTGEPTYTDDFKG), and CDR3(GGFGSSYWYFDV), wherein the monoclonal antibody (mAb) is attached to the drug portion between 4.2 and 4.4, providing an antibody-linker-drug conjugate (ADC).

[0010] In a preferred embodiment, the present invention provides an antibody-drug conjugate (ADC) in which the antibody (mAb) is attached to a drug portion of 4.2 (i.e., DAR4.2), a drug portion of about 4.3 (i.e., DARabout 4.3), or a drug portion of 4.4 (i.e., DAR4.4).

[0011] Further embodiments: In a second embodiment, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate embodied in Embodiment 1, preferably a trastuzumab-drug conjugate, and an acceptable carrier. The acceptable carrier is defined as any suitable pharmaceutical excipient known in the art 5,6 which is compatible with the active pharmaceutical ingredient (API), i.e., the antibody-drug conjugate of the present invention herein.

[0012] In a third embodiment, the present invention provides a method for preparing an antibody-drug conjugate embodied in Embodiment 1, preferably a trastuzumab-drug conjugate, comprising a) purification of the antibody trastuzumab, b) partial reduction of trastuzumab, and c) conjugation of the partially reduced trastuzumab with a vc-MMAE drug linker.

[0013] In a further embodiment, the complex according to the present invention, wherein the monoclonal antibody is reactive with an antigen or an epitope of the antigen, preferably trop-2, is associated with cancer, malignant cells, autoimmune diseases, or infectious organisms that express a Trop-2 cross-reactive epitope that binds to the antibody-linker-drug complex of the present invention.

[0014] In a further embodiment, the sacituzumab drug conjugate according to the present invention may be used in the treatment of cancer.

[0015] In a further embodiment, the sacituzumab drug conjugate according to the present invention may be used in the treatment of autoimmune diseases or infections.

[0016] In a further embodiment, the sacituzumab drug conjugate according to the present invention may be used alone as monotherapy in the treatment of cancer.

[0017] In a further embodiment, the sacituzumab drug conjugate according to the present invention may be used alone as monotherapy in the treatment of autoimmune diseases or infections.

[0018] In a further embodiment, the sacituzumab drug conjugate according to the present invention may be used in combination with other pharmaceuticals in the treatment of cancer, autoimmune diseases, or infections.

[0019] In further embodiments, the complex of the present invention may be administered intravenously (iv), intramuscularly (im), subcutaneously (sc), intraperitoneally (ip), or by any other suitable route of administration.

[0020] In a further embodiment, the dose of the antibody-drug conjugate (ADC) according to the present invention may be in the range of 0.1 mg / kg to 10 mg / kg.

[0021] Definition: The term "linker" refers to any chemical part of a compound, usually a drug, such as auristatin, that can be covalently linked to a cell-binding agent, such as sacituzumab. A linker is a compound of the formula... [ka] It includes a stretcher unit (-A-), an amino acid unit (-W-), and a spacer unit (-Y-), as defined by [the formula].

[0022] The linker may be sensitive to, or substantially resistant to, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions in which the compound or antibody maintains its active state. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-unstable groups, photo-dissociable groups, peptidase-unstable groups, and esterase-unstable groups. Linkers also include charged linkers and their hydrophilic forms, which are described herein and known in the art.

[0023] The term "pharmaceutical composition" refers to a preparation that is in a form that clearly enables the biological activity of the active ingredient and does not contain additional ingredients that are significantly toxic to the target to which the preparation will be administered. The terms "pharmaceutical preparation," "preparation," "pharmaceutical composition," or "composition" may be used interchangeably here.

[0024] The terms “patient” and “subject” are interchangeable and, in their conventional sense, refer to living organisms suffering from or susceptible to a condition that can be prevented or treated by administration of the compositions of the present invention, and include animals. The term “animal” refers to, but is not limited to, human or non-human animals, including, livestock such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals such as rodents such as mice, rats, and guinea pigs; birds such as poultry, wild birds, and game birds such as chickens, turkeys, and other gallinaceous birds, ducks, geese, and non-human primates such as monkeys, chimpanzees, and other apes and monkey species. This term does not imply a specific age. Therefore, adult individuals, juvenile individuals, and neonate individuals are of interest.

[0025] In relation to cancer, the term “treating” includes any or all of the following: killing tumor or cancer cells, inhibiting the growth of tumor or cancer cells, causing tumor or cancer cells to regress, inhibiting the replication of tumor or cancer cells, reducing the total tumor volume, and improving one or more symptoms associated with the disease.

[0026] In relation to autoimmune diseases, the term “treating” includes any or all of the following: direct killing of antibody-producing cells or killing of cells that promote the action of antibody-producing cells; interference with the replication of cells associated with the autoimmune disease state, including but not limited to cells capable of producing autoimmune antibodies; attenuation of autoimmune antibody levels; and improvement of one or more symptoms of the autoimmune disease.

[0027] In relation to infectious diseases, the term "treating" includes any or all of the following: inhibiting the growth, reproduction, or replication of the pathogen causing the infectious disease and / or the host cells infected with the pathogen, and improving one or more symptoms of the infectious disease.

[0028] As used herein, the expression “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable organic or inorganic salt of the drug (payload) of the present invention. Preferred salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, bisulfate, ascorbic acid, succinates, maleates, gentisinates, fumarates, glucons, glucarones, saccharates, formates, benzoates, glutamates, methanesulfons, ethanesulfons, and benzenesulfons.

[0029] Unless otherwise specified, "a" or "an" means "one or more."

[0030] An antibody-drug conjugate (ADC) is defined as an antibody component or fragment thereof conjugated with a therapeutic agent. The terms “antibody-drug conjugate,” “antibody-linker-drug,” “conjugate,” and “immune complex,” as used herein, are interchangeable. A preferred antibody-drug conjugate of the present invention is the sacituzumab-MMAE conjugate. “Sacituzumab-MMAE conjugate” and “sacituzumab vedotin” are interchangeable.

[0031] As used herein, the term "about" means that, when used in relation to a specific enumerated value, that value may differ from the enumerated value by ±0.1. For example, the expression "about 4.3" includes all integer and non-integer values ​​between 4.2 and 4.4, as well as all integer and non-integer values ​​between those values. Specific values ​​within the range between 4.2 and 4.4 include, but are not limited to, 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.30, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, or 4.40. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 shows the purity and size variant profiles of the sacituzumab-MMAE complex as evaluated by HP-SEC. [Figure 2] Figure 2 shows the drug-antibody ratio (DAR) of the sacituzumab-MMAE complex, as analyzed by HP-HIC. [Figure 3] Figure 3 shows the polypeptide profile of the sacituzumab-MMAE complex of the present invention, as determined by SDS-PAGE analysis. [Figure 4] Figure 4 shows the in vitro cytotoxic assay of the sacituzumab-MMAE complex using BxPC3 cells. [Figure 5] Figure 5 shows the in vitro cytotoxic assay of the sacituzumab-MMAE complex using NCI-N87 cells. [Figure 6] Figure 6 shows the in vitro cytotoxic assay of the sacituzumab-MMAE complex using HCC1806 cells. [Figure 7] Figure 7 shows the in vitro cytotoxic assay of the sacituzumab-MMAE complex using MDA-MB-468 cells. [Figure 8] Figure 8 shows the in vivo effect of the sacituzumab-MMAE complex in a gastric cancer xenograft disease model in SCID mice. [Figure 9]Figure 9 shows the in vivo effect of the sacituzumab-MMAE complex in a BRCA-mutated triple-negative breast cancer xenograft disease model in SCID mice. [Modes for carrying out the invention]

[0033] This invention relates to Ab-(Aa-Ww-Yy-D) p The present invention provides an antibody-linker-drug conjugate in which the antibody (Ab) is a trop-2 antigen-targeted antibody, preferably sacituzumab; -Aa-Ww-Yy- is an enzymatically cleavable linker unit linking the drug unit and the antibody, in which -A- is a stretcher unit; a is 1; each -W- is independently an amino acid unit; -Y- is a spacer unit; w is an integer ranging from 2 to 12; y is 1 or 2; p is ranging from 1 to about 20; and drug (D) is an auristatin analog or a pharmaceutically acceptable salt thereof.

[0034] Antibody (Ab): The antibody in the antibody-drug conjugate (ADC) of the present invention is a trop-2 targeted antibody, preferably sacituzumab, or a post-translational modification of the antibody or an antibody variant thereof. Post-translational modifications of the antibody include, but are not limited to, deamidated variants, acidic variants, basic variants, or oxidized variants, or any other variant of the antibody that does not affect the biological function or effect of the antibody. In one embodiment, the antibody in the antibody-drug conjugate (ADC) of the present invention is a sacituzumab variant. The sacituzumab variant of the present invention includes amino acid substitutions, insertions, and / or deletions in the polypeptide sequence of the antibody sacituzumab. In one embodiment, the trop-2 targeted antibody according to the present invention is an antibody comprising the light chain variable region complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA, SEQ ID NO: 1), CDR2(SASYRYT, SEQ ID NO: 2), and CDR3(QQHYITPLT, SEQ ID NO: 3), and the heavy chain variable region CDR sequences CDR1(NYGMN, SEQ ID NO: 4), CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO: 5), and CDR3(GGFGSSYWYFDV, SEQ ID NO: 6). In a preferred embodiment, the trop-2 targeted antibody according to the present invention is an antibody comprising the light chain sequence SEQ ID NO: 7, which represents the sacituzumab antibody. [ka] and heavy chain sequence number 8 [ka] The antibody contains the following sequence: The sequence is the International Generic Name of the Medicinal Product (INN, Vol. 29, No. 2, 2015) for sacituzumab products. [4]Disclosed in [publication name]. Sacituzumab is a humanized monoclonal antibody of immunoglobulin G1 kappa (IgG1κ) against the Trop-2 antigen, produced in a mammalian cell line (Chinese hamster ovary [CHO]) using recombinant DNA technology. In one embodiment, the antibody of the antibody-drug conjugate (ADC) of the present invention is a variant thereof that can improve the binding affinity of the ADC to sacituzumab or the Trop-2 antigen. In another embodiment, the antibody of the antibody-drug conjugate (ADC) of the present invention is a variant thereof that can improve the binding affinity of the ADC to sacituzumab or the Trop-2 antigen at a suitable pH. A preferred pH may be pH 6.0 or pH 7.0. In yet another embodiment, the antibody of the present invention is a variant thereof that can be used to extend the half-life of sacituzumab or the ADC. The half-life of the ADC of the present invention can be altered in the blood by mutations in the Fc domain of the antibody. Mutations in the Fc domain include, but are not limited to, one or more amino acid substitutions, insertions, and / or deletions in the amino acid sequence of the antibody.

[0035] Linker unit: The linker unit of the antibody-linker-drug conjugate connects the drug unit and the antibody unit, and is expressed by the formula: [ka] It has, During the ceremony, -A- is a stretcher unit linked to an antibody; a is either 0 or 1; Each -W- is independently an amino acid unit; w are independent integers ranging from 0 to 12; -Y- is a spacer unit connected to the drug / payload; Y is 0, 1, or 2.

[0036] Stretcher Unit: The stretcher unit (-A-), if present, links the antibody unit to the amino acid unit (-W-). In this regard, the antibody (Ab) has a functional group that can form a bond with the functional group of the stretcher. Useful functional groups that may be present in antibodies naturally or through chemical manipulation include, but are not limited to, sulfhydryl (-SH), amino, hydroxy, carboxy, anomeric hydroxyl groups of carbohydrates, and carboxyl. Preferred antibody functional groups are sulfhydryl and amino.

[0037] The stretcher unit (-A-) of the present invention has the following structure [ka] Defined by, r is an integer ranging from 1 to 10; the carbonyl terminus of -A- forms a bond with the amino acid unit (W), and the succinimide terminus of -A- forms a bond with the antibody (Ab).

[0038] In one embodiment, the stretcher unit forms a bond with the sulfur atom of the antibody unit. The sulfur atom may originate from the sulfhydryl group of the antibody. A typical stretcher unit of this embodiment is shown below. [ka]

[0039] Amino Acid Unit (-W-): If an amino acid unit (-W-) is present, it connects the stretcher unit to the spacer unit if a spacer unit is present, connects the stretcher unit to the drug unit if no spacer unit is present, and connects the antibody unit to the drug unit if neither a stretcher unit nor a spacer unit is present. The wavy line indicates the connection point.

[0040] -Ww- is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit. The amino acid unit of the ADC of the present invention can be enzymatically cleaved by one or more enzymes, including tumor-related proteases, to release the drug unit (-D). Exemplary Ww units are of the formula [Chemical formula] represented by wherein R 20 is isopropyl and R 21 is (CH2)3NHCONH2.

[0041] Spacer Unit: The spacer unit (-Y-), if present, links the amino acid unit to the drug unit if an amino acid unit is present. Alternatively, if an amino acid unit is absent, the spacer unit links the stretcher unit to the drug unit. The spacer unit further links the drug unit to the antibody unit if both an amino acid unit and a stretcher unit are absent. The spacer unit has two general types: self-immolative and non-self-destructive. In one embodiment, the spacer unit (-Y-) of the present invention may be self-immolative or non-self-destructive. In a non-self-destructive spacer unit, some or all of the spacer unit remains bound to the drug after cleavage from the antibody-linker-drug complex, particularly after enzymatic cleavage of the amino acid unit. Examples of non-self-destructive spacer units include, but are not limited to, (glycine-glycine) spacer units and glycine spacer units. When a glycine-glycine spacer unit or a compound of the present invention containing a glycine spacer unit is enzymatically cleaved via a tumor cell-associated protease, cancer cell-associated protease, or lymphocyte-associated protease, the glycine-glycine-drug moiety or glycine-drug moiety is cleaved from Ab-Aa-Ww-. In one embodiment, -Yy- is a p-aminobenzyl alcohol (PAB) unit. In one embodiment, the present invention provides a drug-linker compound or antibody-linker-drug conjugate in which the spacer unit is absent (=0). In one embodiment, -Y- is a p-aminobenzyl alcohol (PAB) group linked to -Ww- via its amino nitrogen atom and directly linked to -D via a carbonate, carbamate, or ether group.

[0042] Drug unit: In this invention, the antibody is sacituzumab, and the drug is of the following formula: [ka] The present invention provides an antibody-drug conjugate that is an auristatin analog selected from MMAE and MMAF as defined by, In the formula, the wavy line represents the connection point to the linker.

[0043] The antibody-drug complex of the present invention The present invention provides an antibody-drug conjugate in which the antibody is sacituzumab or a variant thereof that targets the trop-2 antigen, and the drug or payload is an auristatin analog, preferably MMAE. The preferred antibody-drug conjugate of the present invention is the sacituzumab-MMAE conjugate, also known as sacituzumab vedotin. The sacituzumab-MMAE conjugate of the present invention comprises sacituzumab or a variant thereof as the antibody, mc-vc-PABC as the linker, and MMAE as the toxin or drug or payload. The conjugate of the present invention further comprises the payload, i.e., a pharmaceutical salt of MMAE. The inventors of the present invention have found that the sacituzumab-MMAE conjugate of the present invention exhibits significantly superior biological activity compared to the approved drug conjugate therapy (Troderví®) for trop-2 expressing cancer in in vitro and in vivo studies in xenograft animal models. The biological activity of the sacituzumab-MMAE of the present invention is expected to be promising for clinical use in humans. The novel drug conjugate of the present invention is a PARP inhibitor to be administered as a second co-treatment, for example, in the case of Troderbi®, along with the primary treatment. 7 The novel drug conjugate of the present invention is superior to the approved drug conjugate Troderbi® in that it can treat tumor-bearing BRCA mutations alone without the need for other drugs (Example 8). Furthermore, the novel drug conjugate of the present invention is superior to the approved drug conjugate, i.e., Troderbi®, in that it is effective at significantly lower drug concentrations, which may make the drug less expensive and less toxic for the patient, while being as effective as or more effective than the approved drug conjugate.

[0044] Method for preparing antibody-drug conjugates according to the present invention: The antibody of the present invention, preferably sacituzumab, was expressed in CHO (Chinese hamster ovary) cells using recombinant technology. The cells were cultured in cell culture medium at appropriate pH and temperature for a specified period. After the completion of cell incubation, the cell culture was harvested, the antibody was isolated, and purified by appropriate chromatographic technique. The antibody of the present invention, preferably sacituzumab, was reduced with a reducing agent, for example, tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffer at a pH in the range of 5 to 8.5, at a temperature of about 37°C for 120 minutes. Alternatively, sacituzumab of the present invention may be reduced with dithiothreitol (DTT). The reduced sacituzumab was reacted with a linker drug conjugate of the present invention in an excess of about 5 to 10 molars. The linker drug conjugate of the present invention is vedotin, which is commercially available on the market. The vedotin of the present invention comprises an mc-vc-PABC linker conjugated with a monomethyl auristatin (MMAE) payload, and is also known as vc-MMAE or mc-vc-PABC-MMAE. The complex was purified by ultrafiltration-dialysis. The drug-antibody ratio (DAR) was determined by HPLC-HIC column chromatography. The drug-antibody ratio (DAR) obtained for sacituzumab vedotin of the present invention was: , book DAR of the invention of sacizumab vedotin Regarding this, typically it falls within the range of 1 to 8. range of 3 to 5 enclosure, or 3.5~4.5 Within the range there were.

[0045] In one embodiment, the present invention provides an antibody-drug conjugate (ADC) in which the antibody (mAb) is attached to a drug portion between 1 and 8, preferably between 4.2 and 4.4. Specific values ​​within the range of 4.2 to 4.4 include, but are not limited to, 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.30, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, or 4.40.

[0046] In a preferred embodiment, the present invention provides an antibody-linker-drug conjugate (ADC) in which the antibody is a monoclonal antibody comprising light chain variable region complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA), CDR2(SASYRYT), and CDR3(QQHYITPLT), and heavy chain variable region CDR sequences CDR1(NYGMN), CDR2(WINTYTGEPTYTDDFKG), and CDR3(GGFGSSYWYFDV); the linker is an enzymatically cleavable linker unit mc-vc-PABC; the drug (D) is a suitable auristatin analog MMAE; and the monoclonal antibody (mAb) is attached to the drug portion between 4.2 and 4.4. In a preferred embodiment, the present invention provides an ADC structure in which [ka] Represented by, In the formula, mAb is a monoclonal antibody comprising light chain variable region complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA), CDR2(SASYRYT), and CDR3(QQHYITPLT), and heavy chain variable region CDR sequences CDR1(NYGMN), CDR2(WINTYTGEPTYTDDFKG), and CDR3(GGFGSSYWYFDV), wherein the monoclonal antibody (mAb) is attached to the drug portion between 4.2 and 4.4, providing an antibody-linker-drug conjugate (ADC).

[0047] In a preferred embodiment, the present invention provides an antibody-drug conjugate (ADC) in which the antibody (mAb) is attached to a drug portion of 4.2, a drug portion of about 4.3, or a drug portion of 4.4. In a preferred embodiment, the drug-antibody ratio (DAR) of the ADC of the present invention is 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.30, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, or 4.40.

[0048] In one embodiment, the present invention provides an antibody-drug conjugate (ADC) in which the antibody (mAb) is attached to the drug portion of 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.30, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, or 4.40.

[0049] For long-term storage, a certain amount of the purified drug conjugate was stored under freezing conditions at -25±5°C.

[0050] Use of the composite of the present invention: In other embodiments, the antibody-drug conjugates of the present invention may be used in treatment of a target, comprising the step of administering a therapeutically effective dose of the therapeutic conjugate described herein, preferably the sacituzumab drug conjugate, to the target. The effective dose of the conjugate may be in the range of 0.1 mg / kg to 10 mg / kg. Diseases that can be treated with the sacituzumab drug conjugate include, but are not limited to, cancer, autoimmune diseases, and / or infections. In one embodiment, the conjugates of the present invention may be used in the treatment of pancreatic cancer, breast cancer, and / or gastric cancer. In one embodiment, the conjugates of the present invention may be used in the treatment of BRCA-mutated triple-negative breast cancer, cancer, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies. More specific examples of such cancers are described below and include squamous cell carcinoma (e.g., epithelial squamous cell cancer), Ewing's sarcoma, Wilms' tumor, astrocytoma, glioblastoma multiforme, and cervical cancer.Other examples of cancer or malignant tumors, though not limited to these, include: childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) adult acute lymphoblastic leukemia, adult acute myeloid leukemia, adult Hodgkin lymphoma, adult lymphocytic leukemia, adult non-Hodgkin lymphoma, AIDS-associated lymphoma, AIDS-associated malignant tumor, central nervous system (primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brainstem glioma, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, childhood extracranial germ cell tumor, childhood Hodgkin's disease, childhood Hodgkin lymphoma, childhood hypothalamic optic glioma, childhood lymph Blast cell leukemia, pediatric medulloblastoma, pediatric non-Hodgkin lymphoma, pediatric supratentorial endodermal neoplasm, pediatric rhabdomyosarcoma, pediatric soft tissue sarcoma, pediatric hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid leukemia, cutaneous T-cell lymphoma, endocrine islet cell carcinoma, endometrial cancer, ependymoma, epithelial carcinoma, Ewing's sarcoma-related tumor, extracranial germ cell tumor, extragonadal germ cell tumor, ocular cancer, Gaucher disease, Hodgkin lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, lymphoproliferative disorders, macroglobulinemia, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, multiple myeloma, multiple myeloma / plasmacytic neoplasm, myelodysplastic syndrome, myelogenous leukemia This includes leukemia, myeloid leukemia, myeloproliferative disorders, nasal cavity and paranasal sinus cancers, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-melanoma skin cancer, bone / malignant fibrosarcoma, osteosarcoma / malignant fibrous histiocytoma, osteosarcoma / malignant fibrosarcoma of bone, ovarian germ cell tumors, low-grade ovarian tumors, abnormal proteinemia, polycythemia vera, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumors, primary central nervous system lymphoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoidosis sarcoma, Sézary syndrome, skin cancer, testicular cancer, thymoma, thyroid cancer, Waldenström type macroglobulinemia, Wilms tumor, and any other hyperproliferative disorders, alongside neoplasia, located in the organ systems listed above.The ADC of the present invention may be administered once or repeatedly, depending on the disease state and the tolerability of the complex, and may be used alone as monotherapy or in combination with other therapies, such as surgical procedures, external radiation, radioimmunotherapy, immunotherapy, chemotherapy, antisense therapy, RNA interference therapy, gene therapy, etc. Each combination will be adapted to the tumor type, stage, patient condition, prior treatment, and other factors considered by the attending physician.

[0051] Formulation and administration Suitable routes of administration for the sacituzumab drug conjugate of the present invention include, but are not limited to, oral, parenteral, rectal, transmucosal, intestinal, intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection. The preferred route of administration is parenteral. Alternatively, the compound may be administered locally rather than systemically, for example, by direct injection of the compound into a solid tumor.

[0052] The antibody-drug conjugate of the present invention, preferably the sacituzumab drug conjugate, can be formulated by a method known for preparing pharmaceutically useful compositions, in which the antibody-drug conjugate is combined with a pharmaceutically appropriate excipient in a mixture. Sterile phosphate-buffered saline is an example of a pharmaceutically appropriate excipient. Other suitable excipients are well known to those skilled in the art. 5,6 .

[0053] In one embodiment, the present invention is represented by formula (I): Ab-(Aa-Ww-Yy-D)p We provide antibody-linker-drug conjugates (ADCs), In the formula, antibody (Ab) is a trop-2 targeted antibody; -Aa-Ww-Yy- is an enzymatically cleavable linker unit that links the drug unit (D) and the antibody, where -A- is a stretcher unit; a is 1; each -W- is independently an amino acid unit; -Y- is a spacer unit; w is an integer ranging from 2 to 12; y is 1 or 2; p is ranging from 1 to approximately 20; and the drug (D) has the following structure [ka] A suitable auristatin analog selected from MMAE and MMAF as defined by; In the formula, the wavy line represents the connection point to the linker.

[0054] In other embodiments, the present invention provides an antibody-linker-drug conjugate in which -A- is a stretcher unit; -W- is an amino acid unit independently selected from -phenylalanine-lysine- or -valine-citrulline-; and -Y- is a spacer unit selected from -glycine-glycine- or p-aminobenzyl alcohol (PAB) or p-aminobenzylcarbamate (PABC).

[0055] In other embodiments, the present invention has the following structure: -A- [ka] It provides an antibody-linker-drug conjugate (ADC), which is a stretcher unit as defined by In the formula, r is an integer ranging from 1 to 10; the carbonyl terminus of -A- forms a bond with the amino acid unit (W), and the succinimide terminus of -A- forms a bond with the antibody (Ab).

[0056] In yet another embodiment, the present invention provides an antibody-linker-drug conjugate (ADC) in which the mAb is a monoclonal antibody comprising the light chain variable region complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA), CDR2(SASYRYT), and CDR3(QQHYITPLT), and the heavy chain variable region CDR sequences CDR1(NYGMN), CDR2(WINTYTGEPTYTDDFKG), and CDR3(GGFGSSYWYFDV); the enzymatically cleavable linker unit is mc-vc-PABC, and the drug (D) is a suitable auristatin analog MMAE.

[0057] In yet another embodiment, the present invention provides an ADC structure, [ka] We provide antibody-linker-drug conjugates (ADCs) represented by, In the formula, mAb is a monoclonal antibody containing the light chain variable region complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA), CDR2(SASYRYT), and CDR3(QQHYITPLT), and the heavy chain variable region CDR sequences CDR1(NYGMN), CDR2(WINTYTGEPTYTDDFKG), and CDR3(GGFGSSYWYFDV).

[0058] In a further embodiment, the present invention provides an antibody-drug conjugate (ADC) in which the mAb is a monoclonal antibody comprising the constant domain and hinge domain of a human IgG1 antibody. In a further embodiment, the present invention provides an antibody-drug conjugate (ADC) in which the mAb is a monoclonal antibody comprising the constant domain and hinge domain of a human IgG1 antibody, wherein one or more amino acids in the Fc portion are mutated.

[0059] In one embodiment, the present invention provides an antibody-drug conjugate (ADC) in which the mAb is a monoclonal antibody, sacituzumab, or a variant thereof.

[0060] In one embodiment, the present invention provides that the mAb is a drug portion between 1 and 8. or Drug parts 3-5, or 3.5~4.5 range Drug portion Preferably in the range of 4.2 to 4.4 The attached antibody-drug conjugate (ADC) is provided.

[0061] In other embodiments, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate (ADC) according to any of the preceding claims and an acceptable carrier.

[0062] In yet another embodiment, the present invention provides a method for preparing an antibody-drug conjugate, comprising a) purification of the antibody sacituzumab, b) partial reduction of sacituzumab, and c) conjugation of the reduced sacituzumab with a vc-MMAE drug linker.

[0063] In yet another embodiment, the present invention provides an antibody-drug conjugate (ADC) in a form suitable for parenteral administration, for example, intravenous (iv), intramuscular (im), subcutaneous (sc), intraperitoneal (ip), or any other suitable route of administration.

[0064] In a further embodiment, the present invention provides an antibody-drug conjugate (ADC) used to treat cancer.

[0065] In a further embodiment, the present invention provides an antibody-drug conjugate (ADC) used to treat infections caused by pathogenic organisms.

[0066] In yet another embodiment, the present invention provides an antibody-drug conjugate (ADC) used to treat autoimmune diseases.

[0067] In a further embodiment, the present invention provides an antibody-drug conjugate (ADC) that is administered either as a monotherapy or in combination with one or more therapeutic agents selected from the group consisting of non-conjugate antibodies, radiolabeled antibodies, drug-conjugate antibodies, toxin-conjugate antibodies, gene therapy, chemotherapy, therapeutic peptides, oligonucleotides, local radiotherapy, surgical procedures, and RNA interference therapy.

[0068] In one embodiment, the present invention provides an antibody-drug conjugate (ADC) with a dose of approximately 0.1 mg / kg to 10 mg / kg.

[0069] In another embodiment, the present invention provides an antibody-drug conjugate (ADC) which is sacituzumab vedotin. [Examples]

[0070] (Example 1) Preparation of sacituzumab-linker-drug conjugates Sacituzumab was expressed in CHO cells and purified by conventional column chromatography, including r-protein A affinity column chromatography. The purity of sacituzumab after affinity column purification was evaluated by HP-SEC and found to be >98.5%. Drug conjugation to sacituzumab (at >1 mg / mL) was carried out under reducing conditions, on a thiol (-SH) group, with a valine-citrulline-linked MMAE drug molar excess (relative to the protein), at a temperature between 2°C and 30°C and a pH between 5.0 and 8.5. The linker-drug conjugate was dissolved in 5-20% acetonitrile and then mixed into the protein solution. The linker-drug conjugate can also be dissolved in other organic solvents such as dimethyl acetate or dimethyl sulfoxide. The conjugation reaction was carried out for approximately 60 minutes. After conjugation, the antibody-drug conjugate (sacituzumab-MMAE conjugate) was subjected to buffer exchange, filtered through a 0.2 μm filter, and stored at -25 ± 5°C under freezing conditions.

[0071] [Table 1]

[0072] As shown in Table 1, the antibody-drug conjugate was found to have a purity of approximately 94% by HP-SEC analysis.

[0073] The sacituzumab-MMAE complex may also be prepared by the method described in Example 2.

[0074] (Example 2) Method for preparing the sacituzumab-MMAE complex Sacituzumab monoclonal antibody was expressed in Chinese hamster ovary (CHO) cells. Purification was performed using conventional column chromatography steps, such as r-protein A affinity column chromatography, cation exchange chromatography, and mixed-mode column chromatography. After purification, the antibody purity was determined to be over 99% using high-speed size exclusion chromatography (HP-SEC).

[0075] Conjugation of vcMMAE drugs to sacituzumab: Purified sacituzumab monoclonal antibody was chemically conjugated with vc-MMAE. The vc-MMAE matrix, also known as vedotin, was purchased from the market. The process steps are described below.

[0076] Melting of the key intermediate (CI, i.e., sacituzumab) The CI (sacituzumab) was stored in a frozen state in a cryobag. Thawing was performed manually in the same cryobag. After thawing, the CI material was transferred to an appropriate container for the next reaction step.

[0077] Partial reduction of CI by TCEP Sacituzumab was partially reduced (interchain SS crosslinking) for approximately 300 minutes in the presence of a molar excess of tris(carboxyethyl)phosphine [TCEP]. For the reaction, the incubation temperature was maintained below 37°C under stirring conditions (200 rpm). At the end of the reaction, the protein mixture was passed through a 0.2 μm filter and collected in a clean, depyrogenic glass bottle (2 L) for the next reaction step.

[0078] Drug-linker conjugation to CI TCEP-treated sacituzumab was incubated with a molar excess of the drug-linker, vc-MMAE (valine-citrulline monomethyl auristatin E). Incubation was carried out at low temperatures with gentle stirring (200 rpm) for a maximum of 120 minutes (NMT). Conjugation occurred by the formation of a covalent thioether bond between the free SH group (interchain) of the partially reduced sacituzumab protein and the maleimide group of the drug-linker, vcMMAE. At the end of the conjugation reaction, to remove unreacted drug-linker molecules, the reaction mixture was passed through a 0.2 μm filter and exposed to UF / DF.

[0079] Excess drug-linker removal, and buffer exchange by UF / DF. After filtration, the crude reaction mixture containing sacituzumab vedotin was exposed to UF / DF to remove unreacted vcMMAE drug-linker, followed by final buffer exchange. A 30 kDa MWCO membrane filter was used for UF / DF. UF / DF was performed at a maximum transmembrane pressure difference (TMP) of 0.50 bar. Constant volume diafiltration was performed for a maximum of 30 dialysis volumes using 20 mM sodium citrate buffer at pH 6.6 ± 0.1 and a conductivity of 4.5 ± 0.5 mS / cm. Diafiltration was monitored and controlled to achieve target pH and conductivity of the retained solution. After achieving the target pH and conductivity, sacituzumab vedotin was recovered at approximately 7 mg / mL and passed through a 0.2 μm filter.

[0080] Preparation of formulation bulk The concentrated protein solution was collected in a clean, depyrogenic glass bottle. Trehalose was added to it from a freshly prepared 30% stock solution (filtered 0.2 μm) to achieve a final concentration of 7% (w / v). Subsequently, polysorbate 80 was added to a final concentration of 0.02%, and the final volume of the bulk was adjusted by adding the drug substance buffer to achieve a protein concentration of 5 mg / mL for sacituzumab vedotin. The formulated bulk of sacituzumab vedotin was passed through a 0.2 μm filter under a safety cabinet and collected in a disposable, sterile, non-pyrogenic polyethylene terephthalate glycol (PETG) bottle with a white high-density polyethylene (HDPE) screw cap. The filtered formulated bulk was referred to as the sacituzumab vedotin drug substance.

[0081] (Example 3) Purity of sacizumab vedotin by HP-SEC analysis To evaluate the purity level and the presence of product-related size variants (HMW and LMW species), the sacituzumab vedotin API sample from Example 2 was analyzed by analytical HP-SEC under native conditions. The sample was injected into a silica column (7.8 mm x 30 cm; 3 μm) used for HPLC analysis. The silica matrix used in this column exhibits surface activity of hydrophilic diol-type bonds. HP-SEC analysis was performed in isocratic mode using a pH 6.8 phosphate buffer containing 250 mM salt and polar solvent (15%) as the mobile phase. Separation of protein size variants was performed at a flow rate of 0.5 mL / min and a column temperature of 25°C, and recorded by UV detection at 214 nm.

[0082] The purity of sacituzumab vedotin in the sacituzumab active pharmaceutical ingredient sample was found to be >97.0%. As shown in Table 2 and Figure 1 below, the sample was found to exhibit <1.5% HMW variants and <2% LMW variants.

[0083] [Table 2]

[0084] (Example 4) Drug-antibody ratio (DAR) of the sacituzumab-MMAE complex To evaluate the distribution of drug-linkers (Dl) on sacituzumab, the sacituzumab vedotin API sample from Example 3 was analyzed by analytical hydrophobic interaction chromatography (HIC)-HPLC. Peaks are separated by HIC based on the relative hydrophobicity of drug conjugate antibodies (ADCs) with payloads (drug toxins) ranging from 0 to 8. This technique also allows for the determination of the mean drug-antibody ratio (DAR) in sacituzumab vedotin. Experiments were performed using a butyl (4.6 × 100 mm; 5 μm) column connected to an HPLC system. Samples containing the sacituzumab vedotin API were injected at 25°C into an HIC column saturated with mobile phase A (50 mM phosphate pH 7.0 and 1.2 M ammonium sulfate buffer containing 5% isopropyl alcohol). Sacituzumab vedotin with varying payloads was eluted from the column at a rate of 1.0 mL / min in a salt concentration reduction mode using mobile phase B (50 mM phosphate at pH 7.0 containing 20% ​​isopropyl alcohol), and elution was recorded by UV detection at 214 nm. Figure 2 shows the drug-bound antibody variants obtained using three independent batches of sacituzumab vedotin.

[0085] As shown in Figure 2, all three sacituzumab vedotin API samples were found to exhibit five prominent peaks corresponding to DAR0, DAR2, DAR4, DAR6, and DAR8. The retention time (RT) values ​​of the individual peaks obtained using batch samples were found to be nearly identical to each other. As presented in Table 3 and Figure 2 below, sacituzumab vedotin obtained from all derived batches exhibited an average DAR of 4±1, preferably about 4–4.5, on HP-HIC analysis. Or a DAR of approximately 3.5 to 4.5, preferably 4.2 to 4.4. Comfortably 4.3 This was shown in DAR.

[0086] [Table 3]

[0087] (Example 5) Polypeptide profiles of the sacituzumab-linker-drug conjugate using SDS-PAGE electrophoresis. Preparation of the sacituzumab-linker-drug conjugate: The sacituzumab-linker-drug conjugate was prepared by the steps described in Example 1.

[0088] SDS-PAGE electrophoresis: Equipment: BIO-RAD electrophoresis system. Electrophoresis buffer composition: 0.0247M Tris, 0.192M Glycine, 0.1% SDS, pH 8.3±0.1. Sample preparation: Each sample was diluted with Milli-Q water and 5X sample buffer, along with an internal reference standard, so that 10 μg of protein was loaded into each gel well during sample loading.

[0089] Polypeptide profile of sacituzumab-linker-drug conjugate (sacituzumab ADC): The polypeptide profile of sacituzumab ADC is shown in Figure 3. Figure 3 shows the results of SDS-PAGE analysis of various samples obtained during the conjugation reaction of sacituzumab ADC, including purified sacituzumab in lanes 4, 5, and 6. SDS-PAGE analysis of the samples was performed under non-reducing conditions. The internal reference standard, i.e., brentuximab vedotin (ADC), was also analyzed as a positive control under the same conditions (lane 3). After SDS-PAGE analysis, a single band was observed in lane 6, indicating that sacituzumab was intact in borate buffer after buffer exchange. After partial reduction with a 2.7 molar excess of TCEP, multiple bands of heterogeneous sacituzumab were observed, as seen in lane 7. The conjugated samples in lanes 8, 9, and 10 show successful conjugation. This is because each band containing the payload is shifted upwards, that is, towards higher molecular weights compared to the TCEP-treated sample (lane number 7).

[0090] (Example 6) In vitro cytotoxicity study of sacituzumab-linker-drug conjugate (Example 6A) In vitro cytotoxic assay of the sacituzumab-MMAE complex using BxPC3 cells. The sacituzumab-MMAE complex was prepared and purified as described in Example 1. Sacituzumab-MMAE was tested in an in vitro cytotoxicity assay as described below herein.

[0091] BxPC3 (ATCC CRL-1687, pancreatic cancer) cells were seeded in a 96-well black transparent bottom plate (Costar 3603) and incubated in a CO2 incubator for adhesion. The BxPC3 cell line was selected because it is known to express the trop-2 antigen. Adhering cells were treated with different concentrations of sacituzumab-MMAE conjugate and Troderbi® and incubated in a CO2 incubator for 2 hours. After incubation, excess or unbound drug conjugates were removed, fresh medium was added, and the plates were incubated at 5% CO2, 37°C for 4 days. At the end of incubation, Alamar Blue dye (Invitrogen, DAL-1100) was added to the cells and incubated in a CO2 incubator for 5 hours. After incubation, viable cells were quantified by measuring the fluorescence signal at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a SpectraMax M2E fluorescence plate reader. Figure 4 shows that the BxPC3 cytotoxic activity of the tested sacituzumab-MMAE complex was significantly superior to that of Troderbi®.

[0092] (Example 6B) In vitro cytotoxic assay of sacituzumab-MMAE using NCI-N87 cells. The sacituzumab-MMAE complex was prepared and purified as described in Example 1. Sacituzumab-MMAE was tested for in vitro cytotoxicity assays as described below herein. NCI-N87 (ATCC CRL-5822, gastric cancer) cells were seeded in a 96-well black transparent bottom plate (Costar 3603) and incubated in a CO2 incubator for adhesion. The NCI-N87 cell line was selected because it is known to express the trop-2 antigen. Adhering cells were treated with different concentrations of sacituzumab-MMAE and Troderbi® and incubated in a CO2 incubator for 2 hours. After incubation, excess or unbound complexes were removed, fresh medium was added, and the cells were incubated at 5% CO2, 37°C for 4 days. At the end of incubation, Alamar Blue dye (invitrogen, DAL-1100) was added to the cells and incubated in a CO2 incubator for 5 hours. After incubation, viable cells were quantified by measuring the fluorescence signal at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a SpectraMax M2E fluorescence plate reader. Figure 5 shows that the NCI-N87 cytotoxic activity of the tested sacituzumab-MMAE conjugate was significantly superior to that of Troderbi®.

[0093] (Example 6C) In vitro cytotoxic assay of sacituzumab-MMAE using HCC1806 cells. The sacituzumab-MMAE complex was prepared and purified as described in the examples above herein. Sacituzumab-MMAE was tested for in vitro cytotoxicity assays as described below herein.

[0094] HCC1806 (ATCC CRL-2335, triple-negative acantholytic squamous cell carcinoma of breast cancer) cells were seeded in a 96-well black transparent bottom plate (Costar 3603) and incubated in a CO2 incubator for adhesion. The HCC1806 cell line was selected because it is known to express the trop-2 antigen. Adherent cells were treated with different concentrations of sacituzumab-MMAE and Troderbi® and incubated in a CO2 incubator for 2 hours. After incubation, excess or unbound complexes were removed, fresh medium was added, and the cells were incubated at 5% CO2, 37°C for 4 days. At the end of incubation, Alamar Blue dye (invitrogen, DAL-1100) was added to the cells and incubated in a CO2 incubator for 5 hours. After incubation, viable cells were quantified by measuring the fluorescence signal at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a SpectraMax M2E fluorescence plate reader. Figure 6 shows that the HCC1806 cytotoxic activity of the tested sacituzumab-MMAE complex was significantly superior to that of Troderbi®.

[0095] (Example 6D) In vitro cytotoxic assay of sacituzumab-MMAE using MDA-MB-468 cells. The sacituzumab-MMAE complex was prepared and purified as described in the examples above herein. Sacituzumab-MMAE was tested for in vitro cytotoxicity assays as described below herein.

[0096] MDA-MB-468 (ATCC HTB-132, triple-negative mammary cancer) cells were seeded in 96-well black transparent bottom plates (Costar 3603) and incubated in a CO2 incubator for adhesion. The MDA-MB-468 cell line was selected because it is known to express the trop-2 antigen. Adherent cells were treated with different concentrations of sacituzumab-MMAE and Troderbi® and incubated in a CO2 incubator for 2 hours. After incubation, excess or unbound complexes were removed, fresh medium was added, and the cells were incubated at 5% CO2, 37°C for 4 days. At the end of incubation, Alamar Blue dye (Invitrogen, DAL-1100) was added to the cells and incubated in a CO2 incubator for 5 hours. After incubation, viable cells were quantified by measuring the fluorescence signal at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a SpectraMax M2E fluorescence plate reader. Figure 7 shows that the MDA-MB-468 cytotoxic activity of the tested sacituzumab-MMAE complex was significantly superior to that of Troderbi®.

[0097] (Example 7) In vivo effect of sacituzumab-MMAE complex in a gastric cancer xenograft disease model in SCID mice. In this study, the therapeutic potential of a novel sacituzumab-MMAE conjugate was evaluated compared to sacituzumab govitecan-hziy (Troderví®), a commercially available anti-trop 2 ADC molecule. To specifically induce gastric cancer xenografts, NCI-N87 cells (10 million cells / animal / 200 μL cell suspension and Matrigel in a 1:1 volume ratio) were subcutaneously injected into the right flank region of immunodeficient SCID mice on day 0. Animals were observed for palpable tumor development, and the average tumor volume of the animals was approximately 150-200 mm². 3Once the target was reached, the drug was administered on day 10. Sacituzumab-MMAE complex and Troderbi® were directly compared at a dose level of 15 mg / kg (IV route, monotherapy), and a further group of sacituzumab-MMAE complexes was tested at a much lower dose of 5 mg / kg (IV route, monotherapy). Figure 8 shows the effect of sacituzumab-MMAE complexes compared to Troderbi® in the NCI-N87 gastric cancer cell line. Troderbi® at a dose of 15 mg / kg was able to slow tumor progression compared to the placebo control group, but it could not substantially regress the tumor. On the other hand, the sacituzumab-MMAE conjugate substantially reduced tumor volume at both doses tested, and even at a dose one-third lower, the sacituzumab-MMAE conjugate demonstrated greater potential to reduce gastric cancer tumors with TROP2 overexpression compared to the commercially available drug Troderbi®.

[0098] (Example 8) In vivo efficacy of sacituzumab-MMAE complex in a BRCA-mutated triple-negative breast cancer xenograft disease model in SCID mice. This study evaluated the therapeutic potential of a novel sacituzumab-MMAE conjugate compared to Troderbi®, a commercially available anti-trop2 ADC drug, in a BRCA-mutated triple-negative breast cancer (TNBC) disease model. To specifically induce this TNBC xenograft, immunodeficient SCID mice were subcutaneously injected with HCC1806 cells (10 million cells / animal / 200 μL cell suspension and Matrigel in a 1:1 volume ratio) into the right flank region of the animals on day 0. Animals were observed for palpable tumor development, and the average tumor volume of the animals was approximately 200–250 mm². 3Once the target was reached, the drug was administered on day 10. Sacituzumab-MMAE conjugate and Trodervi® were directly compared at two different dose levels (IV route, monotherapy) of 2 mg / kg and 3 mg / kg. Figure 9 shows the effect of sacituzumab-MMAE conjugate compared to Trodervi® in a BRCA-mutated triple-negative breast cancer (HCC1806) model. Trodervi® was only able to slightly slow tumor progression at both doses tested and was unable to induce tumor regression at all. On the other hand, sacituzumab-MMAE conjugate was able to induce tumor regression up to day 17 and day 24 at dose levels of 2 mg / kg and 3 mg / kg, respectively, clearly demonstrating that sacituzumab-MMAE conjugate has greater potential than Trodervi® to regress and slow tumor progression as monotherapy in TROP2-overexpressing BRCA-mutated TNBC tumors. (References)

[0099] Embedding by reference The full disclosures of each patent document and scientific paper referenced herein are incorporated by reference for all purposes.

[0100] Equal parts The present invention may be implemented in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described above should be considered in all respects as illustrative rather than limiting the invention as described herein. Accordingly, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and any modifications occurring within the meaning and scope of the equivalents of the claims are intended to be encompassed therein.

Claims

1. Equation (I): Ab-(Aa-Ww-Yy-D)p an antibody-linker-drug conjugate (ADC), In the formula, antibody (Ab) is a trop-2 targeted antibody; -Aa-Ww-Yy- is an enzymatically cleavable linker unit that links the drug unit (D) and the antibody, where -A- is a stretcher unit; a is 1; each -W- is independently an amino acid unit; -Y- is a spacer unit; w is an integer ranging from 2 to 12; y is 1 or 2; p is ranging from 1 to approximately 20; and the drug (D) has the following structure 【Chemistry 1】 A suitable auristatin analog selected from MMAE and MMAF as defined by; In the formula, the wavy line represents the linkage point to the linker, indicating an antibody-linker-drug conjugate (ADC).

2. The antibody-linker-drug conjugate according to claim 1, wherein -A- is a stretcher unit; -W- is an amino acid unit independently selected from -phenylalanine-lysine- or -valine-citrulline-; and -Y- is a spacer unit selected from -glycine-glycine- or p-aminobenzyl alcohol (PAB) or p-aminobenzylcarbamate (PABC).

3. -A has the following structure 【Chemistry 2】 A stretcher unit as defined by; The antibody-linker-drug conjugate according to claim 2, wherein r is an integer ranging from 1 to 10; the carbonyl terminus of -A- forms a bond with an amino acid unit (W); and the succinimide terminus of -A- forms a bond with an antibody (Ab).

4. An antibody-linker-drug conjugate (ADC) according to claim 3, wherein the mAb is a monoclonal antibody comprising the light chain variable region complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA), CDR2(SASYRYT), and CDR3(QQHYITPLT), and the heavy chain variable region CDR sequences CDR1(NYGMN), CDR2(WINTYTGEPTYTDDFKG), and CDR3(GGFGSSYWYFDV); the enzymatically cleavable linker unit is mc-vc-PABC, and the drug (D) is a suitable auristatin analog MMAE.

5. The ADC structure is 【Transformation 3】 Represented by, The antibody-linker-drug conjugate (ADC) according to claim 4, wherein the mAb is a monoclonal antibody comprising the light chain variable region complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA), CDR2(SASYRYT), and CDR3(QQHYITPLT), and the heavy chain variable region CDR sequences CDR1(NYGMN), CDR2(WINTYTGEPTYTDDFKG), and CDR3(GGFGSSYWYFDV).

6. The antibody-drug conjugate (ADC) according to claim 5, wherein the mAb is a monoclonal antibody comprising the constant domain and hinge domain of a human IgG1 antibody.

7. The antibody-drug conjugate (ADC) according to claim 6, wherein the mAb is a monoclonal antibody comprising a constant domain and a hinge domain of a human IgG1 antibody, wherein one or more amino acids in the Fc portion are mutated.

8. The antibody-drug conjugate (ADC) according to claim 6 or 7, wherein the mAb is a monoclonal antibody, sacituzumab, or a variant thereof.

9. The antibody-drug conjugate (ADC) according to any one of claims 1 to 8, wherein the mAb is attached to drug portions between 1 and 8, preferably drug portions 3 to 5, and more preferably drug portions 3.5 to 4.

5.

10. A pharmaceutical composition comprising an antibody-drug conjugate (ADC) according to any one of claims 1 to 9, and an acceptable carrier.

11. A method for preparing an antibody-drug conjugate according to any one of claims 1 to 10, comprising: a) purification of the antibody sacituzumab; b) partial reduction of sacituzumab; and c) conjugation of the reduced sacituzumab with a vc-MMAE drug linker.

12. The antibody-drug conjugate (ADC) according to any one of claims 1 to 11, wherein the conjugate is in a form suitable for parenteral administration, for example, intravenous (iv), intramuscular (im), subcutaneous (sc), intraperitoneal (ip), or any other suitable route of administration.

13. An antibody-drug conjugate (ADC) according to any one of claims 1 to 12, used for treating cancer.

14. An antibody-drug conjugate (ADC) according to any one of claims 1 to 13, used to treat an infection caused by a pathogenic organism.

15. An antibody-drug conjugate (ADC) according to any one of claims 1 to 14, used for treating an autoimmune disease.

16. An antibody-drug conjugate (ADC) according to any one of claims 1 to 15, administered either as monotherapy or in combination with one or more therapeutic agents selected from the group consisting of a non-conjugate antibody, a radiolabeled antibody, a drug-conjugate antibody, a toxin-conjugate antibody, gene therapy, chemotherapy, therapeutic peptides, oligonucleotides, local radiotherapy, surgical procedures, and RNA interference therapy.

17. An antibody-drug conjugate (ADC) according to any one of claims 1 to 16, wherein the dose of the ADC is approximately 0.1 mg / kg to 10 mg / kg.

18. The antibody-drug conjugate (ADC) according to any one of claims 1 to 17, wherein the ADC is sacituzumab vedotin.

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